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The influence of K3O4 on the regulation of cholesterol side chain cleavage by ACTH and cycloheximide

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The influence of K3O4 on the regulation of cholesterol side chain cleavage by ACTH and cycloheximide

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Content THE INFLUENCE OF K3 PO4 O N THE REGULATION OF CHOLESTEROL SIDE CHAIN CLEAVAGE B Y ACTH AND CYCLOHEXIMIDE by Kathleen Sye Ts'ao A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillm ent of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) FEBRUARY 1981 UMI Number: DP21609 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI DP21609 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 81 0 6 -1 3 4 6 U N IVER SITY O F S O U TH E R N C A LIFO R N IA TH E GRADUATE S C HO O L U N IV E R S IT Y PARK LOS ANG ELES. C A LI FO R N IA 9 0 0 0 7 This dissertation, written by Kathleen Sye Ts'ao under the direction of Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of requirements of the degree of D O C T O R O F P H I L O S O P H Y Dean DISSERTATION COMMITTEE A C K N O W LE D G M E N T S I would lik e to thank m y advisor, Dr. Boyd W . Harding, fo r his valuable guidance and advice during m y research work and his assistance in w riting this thesis. I would also lik e to thank him for the financial aid to support m y graduate studies. I would Tike to thank Dr. Wayne Bidlack, Dr. Susan Oldham and Dr. Am y Lee for th e ir help as members of m y dissertation committee. I would lik e to express m y sincere gratitude to m y parents, Mr. and Mrs. P.T. Sye, m y husband, Jackson and m y daughter, Camille, for th e ir understanding and encouragement. Without th e ir support, m y graduate study would have been impossible. i i TABLE O F C O N T E N T S Page ACKNOWLEDGMENTS........................................... i i LIST O F TA B LES.......................... iv LIST OF FIGURES............................................................................................... . v Chapter I . INTRODUCTION ................. 1 I I . EXPERIMENTAL PROCEDURES ................... 12 A. Materials B. Preparation of Bovine Adrenocortical Mitochondria C. Animal Treatment D. Rat Adrenal Mitochondria E. Cytochrome P450 Assay F. Difference Spectroscopy G. Cytochrome P450<.££ A ctiv ity I I I . STATEMENT OF PROBLEM AND APPROACH . . . ............................ 23 IV, RESULTS...................... 25 V. DISCUSSION AND SUMMARY........................................ 92 REFERENCES.............................. . 104 LIST O F TABLES Table Page I . pH Effect on PII and Cholesterol SCC A c tiv ity Supported by Endogenous Electron Donors .......................... 29 I I . The Effect of Buffer Components on PI I ........................... 33 I I I . The Effect of Respiratory Inhibitors and K3 P0i,. on P II and Endogenous Electron Donor-Supported Cholesterol SCC A c t i v i t y ........................................................ 34 iv LIST O F FIGURES Figure Page 1. Type I Difference Spectrum Induced by K3 P0i» . ....................... 16 2. Pregnenolone-Induced Reverse Type I Difference Spectrum .............................. 18 3. Standard Curve of Pregnenolone Radioimmunoassay (RIA) . ..................... 21 4. The Effect of Preincubation of Bovine Adrenocortical Mitochondria at 27° C on Cholesterol SCC A ctivity Supported by Endogenous Electron Donors ................................... 26 5. The Effect of Preincubation of Bovine Adrenocortical Mitochondria at 27° C on the Pregnenolone-Induced Spectrum and on the Heat-Generated Type I Spectrum . . . 30 6 . The Effect of Salts on the Pregnenolone-Induced Spectrum in Bovine Adrenocortical Mitochondria .................. 36 7. Effect of K3 P0if on Cholesterol SCC A ctivity Supported by Endogenous Electron Donors from Mitochondria of ACTH-Treated Aerobic and Anaerobic Adrenals .. . . . . . . 39 8 . The Effect of MgCl2 and Na3 P0it on Cholesterol SCC A c tiv ity Supported by Endogenous Electron Donors From Mitochondria of ACTH-Treated Anaerobic Adrenals . . 41 9. Time Course of Cholesterol SCC A ctiv ity Supported by Endogenous Reducing Equivalents and Exogenous Iso citrate 43 10. The Effect of K3 PO4 on the pH P ro file of the Pregnenolone-Induced Spectrum in Mitochondria from ACTH plus Cycloheximi.de Adrenals 46 11. Time Course of the K^PO^-Induced Cholesterol-Bound P450scc Complex Measured as P II .................................................... 48 12. The K3 P0i*-Induced Pregnenolone Spectrum Measured in 2 to 3 Min or 10 to 15 Min in the Mitochondria from ACTH or ACTH plus Cycloheximide-Treated Adrenals .. .. 50 v LIST O F FIGURES (continued) Figure Page 13. The Effect of Aging on the K3 P0 i 4-Induced Pregnenolone Spectrum in the Mitochondria from ACTH and ACTH plus Cycloheximide-Treated Animals ........................................................... 53 14. K3 PO* Effect on P II (Measured in 2 to 3 Min A fter Exposure to K3 POO in Mitochondria from ACTH or ACTH plus Cycloheximide-Treated Aerobic or Anaerobic Adrenals . ................................. 55 15. Dual Wavelength Measurements of ;ther'.Reverse Type I Difference Spectra Induced by Mai ate or Pregnenolone at D ifferen t Concentrations of Bovine Adrenocortical Mitochondria P450 .................. 61 16. Rate of Formation of the Reverse Type I Difference Spectrum Induced by Mai ate .............................................................. 63 17, The Effect of K3 PO4 on In it ia l Rate of Malate-Induced Reverse Type I Spectrum in Bovine Adrenocortical Mi tochondri a Aged for Di ffe re n t Periods of'Time . . . . . 65 18, The Effect of Salts on In it ia l Rate of the Malate- Induced Reverse Type I Difference Spectrum in Bovine Adrenocortical Mitochondria 67 19. The Effect of K3 P0i» on the Ratio of the In it ia l Rate of Malate-Induced Reverse Type I Spectrum to the Pregnenolone-Induced Spectrum ( P I I ) yinsBd.vineoAdrenocortical Mitochondria Aged for D ifferent Periods: of T im e ................... 70 20. The Effect of Salts on the Ratio of the In it ia l Rate of Malate-Induced Reverse Type I Spectrum to the Pregnenolone-Induced Spectrum ( P I I ) in Bovine Adrenali, Corti cal "Mitochondria .......................... 72 21. The Effect of Aging on the K3 P0i*-Dependent Pregnenolone- Induced Spectrum in Bovine Adrenal Cortical Mitochondria 74 22, The Effect of K3 PO* o n th e Rate of isocitrate-Induced Reverse Type I Spectrum in ACTH-Treated .Rat ■ , Mitochondria . , ,,,, ., 77 vi LIST O F FIGURES (continued) Figure Page 23. The K3 P0it-Induced Pregnenolone Spectrum in Mitochondria from ACTH or ACTH plus Cycloheximide-Treated Aerobic qr Anaerobic Adrenals a fte r Exhaustion of Endogenous Electron Donors . . '...................................................... 80 24. The Effect of Variation in the P II Spectrum Induced by K3 P0i* on the Rate of Pregnenolone Formation in Mitochondria from ACTH or ACTH plus Cycloheximide- Treated Aerobic or Anaerobic Adrenals . ............................... 82 25. Correlation of the Pregnenolone-Induced Reverse Type I Spectrum Induced by K3 PO4 .............................. , . . 84 26. The Effect of Na3 P0n and MgCl2 on the Relative Rate Constant in the Mitochondria from ACTH-Treated Anaerobic Adrenals 87 27. The Effect of K3 PO4 on the Relative Rate Constant in the Mitochondria from ACTH plus- Cycloheximide- Treated Adrenals 89 vi i I . INTRODUCTION Cholesterol side chain cleavage (SCC) is the f i r s t step in the metabolism of cholesterol to steroid hormones. Exposure of rat adrenal cells to ACTH results in a stimulation of the rate of corticosterone formation within a period of minutes (Stone and Hechter, 1954). This response appears-to be due e n tire ly to the stimulation of cholesterol SCC a c tiv ity leading to the formation of pregnenolone (Simpson et a l ., 1972; Brownie et a l ., 1972; Jefcoate et a l., 1973; Jefcoate et a l ., 1974). The function of cholesterol side chain cleavage enzyme in the control of steroidogenesis has recently been reviewed by Simpson (1979) and Mitani (1979). Protein synthesis in hibitors such as cycloheximide and puromycin cause a rapid in hib ition of the ACTH stimulation (Ferguson, 1963; Davis et al., 1968). Recently, three other protein synthesis in h ib ito rs --b la s to c id in , anisomycin and trichodermin-hhave also been shown to in h ib it steroidogenesis (Hanukoglu and Jefcoate, 1980). I t is concluded that the synthesis of a la b ile protein and steroi dogenes i s are i imti.mateTycoappTed. In common with other mixed function oxidases, cholesterol SCC requires molecular oxygen and reduced pyridine nucleotides (Shikita and Hall, 1974). The scission of cholesterol side chain occurs via an in it ia l hydroxylation at the 2 2 position of cholesterol to y ield (22R)-22 hydroxycholesterol followed by a second hydroxylation at the 20 position to y ie ld the dihydroxy intermediate (20R,22R)-20,22 dihydroxycholesterol, and fin a lly a th ird hydroxylation which then yields pregnenolone and isocaproic acid (Burstein et al., 1972; 4 Burstein et al., 1974; Burstein et a l ., 1975; Hume and Boyd, 1978). Further hydroxylation occurs at the 113-, 17- and 21- positions leading to a corticosteroid hormone formation. Both mitochondrial and microsomal cytochrome P450 systems are involved in these hydroxylations. Cytochrome P450 serves as the terminal oxidase catalyzing the hydroxylation reactions in steroidogenesis. Mitochondrial and micro somal enzyme systems are characterized by d iffe re n t functions and also by d iffe re n t enzyme components in th e ir P450 reductase system. The mitochondrial enzyme system u tiliz e s adrenodoxin (ADX), an iron sulfur protein, to mediate the transfer of reducing equivalents from NADPH- 1 inked flavoprotein dehydrogenase (FLP) called adrenodoxin reductase to cytochrome P450. On the other hand, the microsomal system transfers electrons d ire c tly from the flavoprotein to the P450 oxidase. Mitochondriarcytochrome P450s called P450-|^^ and P450scc catalyze 118 -hydroxylation of 11-deoxycortisol to cortisol (Harding et a l., 1964; Cooper et a l ., 1964; Wilson et a l., 1965; Wilson et a l ., 1970) 18- hydroxyl ati on of deoxycorticosterone or corticosterone to 18-hydroxy deoxycorticosterone or 18-hydroxy-corticosterone (Greengard and Psychoyos et a l., 1967) and side chain cleavage of cholesterol to pregnenolone (Simpson and Boyd, 1967; Wilson and Harding, 1967), respectively. These two forms of cytochrome P450 can be distinguished, not only on the basis of th e ir d iffe re n t behavior with regard to pu rificatio n techniques (Takemori and Suhara et al., 1975a; Wang anid Kimura, 1976), but also by virtue of th e ir d iffe re n t electron 2 paramagnetic resonance (EPR) properties (Simpson and WiTliams-Smith, 1969; Brownie et a l ., 1973). They are also distinguished by differences in temperature dependence of th e ir optical spectra (Paul et al., 1976). No evidence exists for heterogeneity between preparations of the com ponents of the mitochondrial P450 reductase system. FLP, ADX and cytochrome P450 are localized inside the inner mitochondrial membrane (Rydstrom and Gustafsson et a l ., 1976). ADX can be extracted by s a lt solutions from the in tact mitochondria. Ultrasonic treatment results in a soluble FLP and a particulate P450. Further solu bilization of P450 is performed by use of the non-ionic detergent, Triton N101, or cholate, or deoxycholate (Mitani and Horie, 1969; Jefcoate et al.,,,1970a; Schleyer et a l ., 1972; Ramseyer and Harding, 1973; Takemori et al., 1975a and 1975b; Simpson and William-Smith, 1976; Akhrem et a l ., 1977a). Cytochrome P450, like most other hemoproteins, possesses iron protoporphyrin IX as a prosthetic group (Maines and Anders, 1973), which in d iffe re n t hemoproteins shows sim ilar physical and chemical behavior. Because of specific interactions between the prosthetic group and protein, however, peculiar spectral, magnetic, chemical and functional properties are induced by d iffe re n t proteins. Chemically or enzymat ic a lly reduced cytochrome P450 can form a complex with carbon monoxide which gives an absorption spectrum at 450 nm (Omura and Sato, 1964). Along with several hemoproteins including liv e r microsomal P450 and adrenocortical mitochondrial P450 (Schleyer and Cooper et a l ., 1973), spin e q u ilib ria between the high spin state with a total spin of S = 5/2 and the low spin state of S = 1/2 have been described. The spin state equilibrium in hemoproteins is caused not only by changes of the 3 ligand fie ld strength s u ffic ie n tly to correspond to the so-called spin pairing energy around the iron (G riffith and Orgel, 1957), but also by changes of the temperature which overcome the small energy gap between the two magnetic states. Changes in pH also affect the magnetic measurement. ADX binding or cholesterol binding to cytochrome P450^^ increases the binding of the other by a factor of 2 0 , causing a con version from low spin to high spin (Lambeth and Seybert et al., 1980). With increasing magnetic susceptibility the Soret band is shifted to shorter wavelength (high spin) and, conversely, with decreasing mag netic s u scep tib ility, a red s h ift of the Soret band (low spin) is observed. The heme spin state can be estimated by optical measurements at room temperature or by EPR spectrum at low temperature because both optical and EPR properties are linked with the spin state (Scheler et al., 1957; Rein et al., 1976; Rein and Ruckpaul, 1977). By recording temperature difference spectra, the Soret bands of the high spin form and the low spin form, respectively, can be resolved. Because the soret band can be shifted reversibly in opposite directions, an equilibrium of mixed spin states o f the heme iron in P450 is assumed to e xis t. EPR spectrometry of in tact ra t adrenal mitochondria is also indicative of a mixture of low spin (substrate free) and high spin (substrate bound) forms of the enzymes (Brownie and Alfano et a l., 1973). The position of the observed Soret band is determined by the proportion of the enzyme in a given spin state. A blue s h ift of the Soret band indicates forma tion of the high spin state. Conversely, a red s h ift of the Soret band results from an increase in the amount of the low spin. Estimates of the amount of P450SC£ bound cholesterol based on optical or EPR spectrum are complicated by reversible changes which may be induced in this enzyme by either changes in pH (Jefcoate et a l., 1973; Simpson and William-Smith, 1976; Hume and Boyd, 1978) or changes in temperature (Paul and Gallant et a l., 1976). Also, the a v a ila b ility of free ADX seems to be a major effector which also varies the level of cholesterol- bound P450scc (Lambeth and Seybert et a l ., 1980). In adrenal cortical preparations the absorbance changes seen in d iffe re n t spectra have been classified into three general types (Scheckman and Reonmer et al., 1967): Type I spectra are characterized by a 385 nm peak and an associated 420 nm trough which are produced by a ll of the steroid hydroxylase substrates; Type I I spectra with a maximum at 427-430 nm and a trough at 380-390 nm which are in general produced by nitrogen-containing agents, including such steroid bio synthetic inhibitors as metyrapone, aminoglutethimide and cyanoketone (Wilson et a l ., 1969; Jefcoate and Gay!or, 1970b); modified Type I I , or the reverse Type I spectra with an absorption peak at 420 nm and a trough at 385 nm, is seen under conditions in which the substrate is depleted from the sample cuvette by hydroxylating conditions, or in which 2 0 2 hydroxycholesterol or pregnenolone has been added to sample cuvettes containing mitochondria with cholesterol-bound P450<*££ (Whysner et a l ., 1968; Whysner et a l ., 1970; Wilson and Harding, 1973; Alfano et al., 1973). There is no correlation between the binding type being induced and the chemical structure of the steroid substrate. The high spin form of the enzyme is characterized by an absolute absorption spectrum having a maximum at 394 and a low spin absolute absorption spectrum having a maximum at 418 (Horie and Watanabe, 1975). The high 5 spin forms of both cytochrome P450<.££ and P450 "lift are characterized by EPR features at g = 8.0, 3.7 and 1.8 (Whysner et a l ., 1970; Alfano et a l ., 1973), and the low spin form of these enzymes is characterized by EPR features at g = 2.4, 2.25 and 1.0 (Whysner et a l ., 1968; Whysner et a l ., 1970; Tsai and Yu et a l ., 1970; Alfano et a l ., 1973; Jefcoate et a l ., 1976). The high spin forms of cytochrome P450 and P450^-j^ can be distinguished from each other by EPR spectrometry. p450S£C has a 9 value of 8.1 and 3.5, whereas P450-|-|g has a g value of 7.8 and 4.0 (Simpson and William-Smith, 1969; Brownie et a l ., 1973). Substrate binding and formation of the high spin form allow the transition from the fe rric high spin state (S = 5/2) to the ferrous high spin state (S = 2) and raise its rate of reduction (Kadish and Davis, 1973). Further evidence for acceleration of the reduction of high spin hemoprotein is provided by the accelerated reduction rate of methemoglobin seen in the presence of organic phosphates which are known to increase the high spin state (Champion and Munek et al., 1975). Due to the unfavored reductive reaction of the low spin s ta te, its reduction rate is slow. The importance of the spin state of P450 for its enzymatic mechanism has been discussed recently by Rein and Ristau (1978), Therefore, the concentration of the high spin state determines the reduction rate of the cytochrome (Gunsalus and Meek et al., 1973). Cytochrome P450, a fte r being reduced, and in the substrate-bound s ta te, is capable of binding molecular oxygen. In the presence of reduced ADX, a postulated unstable fe rric enzyme-hydroperoxo complex is formed and is decomposed to water and a highly reactive fe r r ic enzyme mono-oxygen species. The reactive species rapidly hydroxylates substrates by a two electron oxidation to form H20 and hydroxylated product and regenerates the low spin fe rr ic enzyme. The reaction cycle of P450 is postulated (Gustafsson and Itrycay et a l., 1976) as follows. AH e- Fe3+------------Fe.3 + --------------- * Fe2+ > ! j < ■ AO H 2H+ - H20 AH AH r Fe3+ *02 <— • Fe2+*02“ 4— ^ — Fe2 + -(T2 0 2 . f t , * * 1 AH AH AH For the above reaction cycle, donation of the second electron leading to the subsequent breakdown of the AH • P450 Fe2+*022“ ternary complex is the slow step (Akhrem and M etelitza et a l., 1977b). There fore, the cytochrome P450^^^ catalyzed hydroxylation rate depends on the electron being transferred e ffic ie n tly in the system. The process of cholesterol side chain cleavage is further complicated by the need for three c a talytic cycles for formation of pregnenolone (Shikita and Hall, 1974). However, in certain reconstituted side chain cleavage systems, the in it ia l mixed function oxidation step 22R-hydroxylation appears to be the ra te -lim itin g step (Burstein and Gut, 1976). The electron transfer process between NADPH and FLP and also be tween FLP and ADX has been extensively studied (Lambeth and Kamin, 1976a; Lambeth et a l., 1976b). ADX forms a 1:1 complex with FLP and P450, respectively (Chu and Kimura, 1973; Katagir et a l ., 1977; Seyber et al.),. There is no ternary FLP-ADX-P450 complex detected in the reconstituted system (Seyber et a l ., 1978; Hanukoglu and Jefcoate, 1980). Due to its high a ffin ity for ADX, FLP is an effective competitor fo r ADX in P450 mediated hydroxylation reactions (K A/F = 1.8 x 10" 8 M, K A/P = 8 x 10" 8 M) where A/F and A/P denote the dissociation constants fo r ADX and FLP and ADX and P450, respectively (Hanukoglu and Jefcoate, 1980). This competition is demonstrated by the finding of a sigmoid binding curve instead of a hyperbolic binding curve when the ADX*P450*cholesterol high spin complex is plotted against ADX in the presence of FLP. Although the FLP-ADX complex is the active species for cytochrome c reduction, the complex is not s u ffic ie n t to allow cytochrome P450 mediated hydroxylation (Lambeth and McCaslin et al., 1976B). In v it r o , the c a talytic a c tiv ity appears only when ADX is in excess of FLP (Seyber and Lambeth et al., 1978), a situation which corresponds to the in tra mitochondrial concentration of these components. In mitochondria the molar ra tio of IFLP];IADXJ:'[P450J appears to be approximately 1:10:10 (Estabrook et a l ,, 1973; Kimura et a l ., 1978; Okashi and Omura, 1978). Since FLP«ADX is not an active species in the electron transport system and the dissociation of reduced ADX from FLP is required fo r catalytic a c tiv ity , factors alterin g the K^ for ADX-FLP or ADX*P450 will affect the rate of reduction of P450 and therefore the hydroxylation rate. Reduction of ADX shifts the potential which becomes less negative than oxidized ADX, thus favoring dissociation of the complex. Increasing s a lt also causes a s h ift of redox potential to a less negative value and also drops the apparent dissociation constant (pK^) of FLP and ADX 8 phase followed by a slower phase. Both phases are increased by c o rti cotropin stimulation (Simpson et a l., 1972; Alfano et al., 1973; Brownie et al., 1973; Arther and Boyd, 1976a; Simpson et a l ., 1978). Product in h ib itio n by pregnenolone is regarded as being of minor sig nificance in explaining the biphasic rate of'pregnenolone formation because, in the presence of isomerase in h ib ito r which in hibits preg nenolone metabolism, cholesterol SCC a c tiv ity is not inhibited. I t seems lik e ly that depletion of an active mitochondrial substrate pool which is in equilibrium with P450scc and the cholesterol-P450scc high spin complex is the best explanation fo r the biphasic rates (Simpson and Jefcoate et a l ., 1972). Free cholesterol is known to accumulate in adrenal mitochondria from ACTH.plus CHI-treated animals (Brownie, et a l., 1973; Mahaffee et a l ., 1974). I t is assumed that this occurs because the cholesterol ester hydrolase, which is activated by ACTH (Trzeciak and Boyd, 1973; Beckett and Boyd, 1977; Naghshimaah et al., 1978), is unaffected by cycloheximide. Therefore, i t seems lik e ly that the transport of the mitochondrial free cholesterol into an active pool which can gain access to p450scc or the a ffin ity of substrate binding by P450< ;q£ to form the high spin cholesterol•P450<»££ complex is inhibited by cycloheximide. This is supported by the evidence that pretreatment of animals with cycloheximide in h ib its the ACTH-induced increase in both the reverse Type I spectrum and the height of the g = 8.1 signal in mitochondria from these anoxic tissues (Simpson et a l ,, 1972; Bell et a l.., 1973; Brownie e t a l ., 1973; Jefcoate et a l,, 1974), Evidence fo r the s ite ACTH stimulation and CHI-inhibition has been derived from the observation that exogenous cholesterol either dissolved 1 o. in alcohol or acetone, or contained in adrenal lipoproteins, does not supersede the e ffe c t of in vivo administration of ACTH. ACTH increased the rate of metabolism of a ll these forms of exogenous cholesterol (Simpson et a l ., 1978; Farese, 1978). O n the other hand, cycloheximide has li ttle e ffec t on the metabolism of hydroxylated cholesterol derivatives which are known to gain rapid access to cytochrome (Jefcoate et a l ., 1974; Arther et a l ., 1976b). Apparently these derivatives bypass the s ite of CHI in h ib itio n . Pregnenolone induced difference spectrum and EPR spectroscopy have been used to probe the concentration of cholesterol bound P450scc (Simpson et a l ., 1972; Brownie et al., 1973; Alfano et a l., 1973; Jefcoate et a l ., 1974). The modified Type IT spectrum produced by pregnenolone is e ith er due to the pregnenolone binding to the enzyme resulting in a conformational change and loss of bound cholesterol or a lte ratio n of the ligand fie ld strength of cholesterol-bound P450^cc. In order to observe an ACTH induced increase in this complex in isolated mitochondria, i t is necessary that the glands become anaerobic prio r to homogenization (Jefcoate and Orme-Johnson,'1975), EPR spectrum of whole glands isolated aerobically show little or no difference in the amount of low spin cytochrome P450 in glands from ACTH and from ACTH plus cycloheximide-treated rats (Williamr-Smith and Simpson et a l ., 1976), This implies that in the normal operation of the cycle, when oxygen is present, the bound substrate is metabolized as quickly as the enzyme substrate complex forms, This is further evidence that the rate- lim itin g step in steroidogenesis may be the binding of cholesterol to cytochrome P450. 11 Disruption of the mitochondrial membranes, eith e r by vigorous homogenization (Farese and Prudente, 1977), hypoosmotic shock, or by addition of high concentrations of calcium (Koritz and Kumar, 1970) overcomes the in hib itory e ffe c t of CHI on ACTH-stimulated pregnenolone formation or cholesterol SCC a c tiv ity . Apparently these procedures bypass the need fo r the la b ile protein factors synthesized during ACTH action. These experiments provide evidence that the primary event in ACTH action is an intra-mitochondrial step which is dependent on the in te g rity of the mitochondrial membrane. Regardless of the source of supply of cholesterol, ACTH treatment causes a stimulation of steroidogenesis, but a greater rate can be obtained when cholesterol is supplied from lipoprotein uptake rather than de novo biosynthesis (Faust and Goldstein et a l., 1977). Calcium appears to be involved in steroidogenesis at the level of the interaction of ACTH with its plasma membrane receptor and activa tion of the adenylate cyclase system. Since Ca2+ uptake by ra t adrenal slices incubated in the presence of ACTH was enhanced and found to accumulate prim arily in mitochondria and microsomes, a further action at the level of mitochondria and microsomes remains a p o ssib ility (Leier and Jungmann, 1973). Neher and Milam (1978) have presented data indicating that calcium can replace ACTH in triggering steroido genesis in isolated rat adrenal cortical cells, provided the calcium was presented to the cells under conditions favoring the formation of colloidal calcium. Moreover, the e ffe c t of calcium to stimulate steroidogenesis was inhibited by CHI, Additional studies are necessary to establish the role of Ca2+ in mitochondrial steroid hydroxylation reaction. I I . EXPERIMENTAL PROCEDURES A. Materials [H3] pregnenolone (62.5 mCi/mg) and Formula-963 was purchased from New England Nuclear Corp. ACTHi-2 m . (cortrosyn) was purchased from Organon Inc. Pregnenolone and bovine serum albumin were obtained from Sigma Chemical. KH2 PO4 , K2 H B O .1t, NaHaPOu, NaaHPOi,, K2 SO4 , NaCl, acetone, methanol and anhydrous ether were obtained from Mallinckrodt. MgCl2 and Na2 S2 0 it were obtained from J.T. Baker Chemical Co. Cycloheximide, NADP and NADPH were obtained from Calbiochem. Sucrose, g e latin , KC1., activated charcoal and dextrane were obtained from MCB. Anti serum of pregnenolone was purchased from Dr. G. Albraham at Harbor General Hospital. Win 24, 540 was a g if t from Sterling-Winthrop Research In s titu te . Aminoglutethimide was a g i f t from Ciba-Geigy Co. Bovine adrenal glands were obtained from Gold Pak Meat Co., Inc. B. Preparation of Bovine Adrenocortical Mitochondria Bovine adrenal glands were collected from Gold Pak Meat Company at Vernon, C alifo rn ia. The glands were kept at 4° C in saline. All subsequent procedures were done at 4° C. Medulla were removed from the defatted glands. Cortical tissue was scraped o ff and homogenized in 0.25 M sucrose with 1% BSA solution (pH = 7.4). F irs t, the cortical tissue was homogenized with a few passes in a loose f it t in g , reground Ten Brock homogenizer, followed by several passes using a standard Ten Brock homogenizer. The homogenate was centrifuged a t 900 x g fo r 10 min 13 to remove whole cells and connective tissue. The supernatant was cen trifuged at 8,700 g for 10 min. The sedimented mitochondria were washed twice in 0.25 M sucrose. The isolated mitochondria were resus pended in 0,25 M sucrose to a fin a l concentration of 4.25 yM cytochrome F450 and kept at 4° C until used, C. Animal Treatment Female Sprague-Dawley rats weighing 180-200 g were used in a ll animal experiments. Aminoglutethimide (a-(p-Aminophenyl)-2 - ethylgluarimide (AG))-treated rats received 20 m g A G subcutaneously 45 min prio r to ACTH or ACTH plus cycloheximide in jec tio n . Cortrosyn, 1-24 corticotropin, is a synthetic subunit of ACTH, Cortrosyn (i.m., 0.025 mg/rat) and cycloheximide (i.p,, 10 mg/rat) were injected into ra t, A fter in je c tio n , an interval of 15 min was given before decapita tion for anaerobic glands and before removal of glands fo r aerobic study. D. Rat Adrenal Mltochondria Adrenal glands were removed from etherFanesthesiizedlTiving liv iii ' 1 rats a fte r various treatments and homogenized in ice-cold 0,25 M sucrose-1% BSA solution (pH = 7,4) immediately, to obtain "mitochondria from aerobic adrenals", The glands from decapitated rats were removed 5 to 25 min a fte r the decapitation. The pooled adrenals were homo genized in ice-cold 0,25 M sucrose-1% BSA solution (pH = 7,4) to obtain "mitochondria from anaerobic adrenals", Several passes of a te flo n - coated homogenizer were used in a ll the homogenizing processes, The homogenate was centrifuged at 400 x g fo r 10 min to remove unbroken 14 cells and connective tissue. The supernatant was centrifuged at 8,700 x g fo r 10 min to obtain the mitochondria. Isolated mitochondria were washed once in 0.25 M sucrose. This washed mitochondria p e lle t was resuspended in 0.25 M sucrose to a fin a l concentration of 4.25 yM cytochrome P450 and kept at 4P C u n til used. E. Cytochrome P450 Assay The mitochondrial preparations were diluted in a solution (pH = 7.4) containing 16 m M KC1, 13 m M Na2 HPQi,, 3 m M KHjjPO*, 6 m M MgCl2 (C + W buffer) to a volume of 1.7 ml. A few small crystals of Na2 S2 02 were added to the mitochondrial suspension. The suspension was divided equally (0.85 ml) between the sample and reference cuvettes and a base lin e was recorded. The sample cuvette was gently gassed with C O for 1 min. The difference in absorbance was measured between 450 and 490 nm. The concentration of P450 was calculated using the mi H i molar extinction of 91 cm - 1 m M - 1 (Omura and Sato, 1964). Spectra were recorded with an American Instruments DW-2 spectrophotometer using the split-beam mode. E. Difference Spectroscopy Absorption spectra was measured at 27° C with an Aminco DW-2 spectrophotometer in eith e r the split-beam or dual wavelength mode. Mitochondria were suspended in titr a tio n buffer (pH = 7.0) containing 0.25 M sucrose, 15 m M triethanolamine, 20 m M KC1, 5 m M MgCl2 , 100 yM EDTA and 0,2% BSA (unless i t is s p e c ific a lly stated otherwise) at a P450 concentration of 0.2 to 0.25 yM. Pregnenolone in alcohol was added to the sample cuvette to obtain the pregnenolone-induced modified 1 5 Type I I spectrum (PII), as shown in Figs. 2 and 15. Mai ate or iso c itr a te , at the concentration of 5 to 10 mM, was used to obtain the re- verse Type I spectrum, as shown in Fig. 15. Type I spectrum is in Fig. 1. G. Cytochrome P450^£ A ctivity: Incubation was performed at 27° C in a Dubnoff shaker. Mitochon dria equivalent to 0.2 yM P450 were made up in the above titr a tio n buffer (pH = 7.0), with 0.2 m M NADP, 6 yM rotenone and 10 yM Win-24,540 (standard incubation b u ffe r). Mitochondria were preincubated for 10 min before the reaction was in itia te d with 5 m M D, L -is o c itra te . Aliquots, 0.2 ml, were taken and added to 6 ml of anhydrous ether to terminate the reaction. A fter extraction, the ether layer was removed, evaporated to dryness under a nitrogen stream, and the residue dissolved in 2 ml of methanol. Aliquots were taken fo r pregnenolone determination by radioimmunoassay, according to the procedure of Abraham and Buster et a l . (1973). The methanol aliquots were taken to dryness under nitrogen. 0.35 Ml of the mixture of pregnenolone a n ti body plus 3 H-pregnenolone (which was made up in 0,1 M sodium phosphate, 0.9% NaCl, 0.02% sodium azide and 1% gelatin (PBS-G b u ffer, pH = 7.4)) were added to the tubes, mixed well and incubated at 37° C for 10 min, followed by incubation at 4° C overnight. 0.1 ml of a cold charcoal suspension (0.625% N itra A, 0,0625% dextrane) was added and mixed well. This mixture was incubated for 20 min, followed by centrifuging at 2,500 x g fo r 10 min to sediment charcoal and charcoal-adsorbed free 3 H-pregnenolone, To 0,35 ml of supernatant, 8 ml of a s c in tilla tio n FIGURE 1 TYPE I DIFFERENCE SPECTRUM INDUCED B Y K3 P0^ Mitochondria equivalent to 0.25 yM P450 from ACTH-treated rats were used. The mitochondrial suspension in the reference cuvette contained no-I^PO* (buffer B) and the sample cuvette contained 100 m M K3 PO*. An Aminco DW-2 spectrophotometer was used. A, absorbance scale, 0.02; scanning speed, 1.2 nm/sec; s lit width, 3,0 nm bandpass. 17 A A= 0.002 390 420 Wavelength nm FIGURE 2 PREGNENOLONE-INDUCED REVERSE TYPE I DIFFERENCE SPECTRUM Frozen and thawed mitochondria equivalent to 0.25 yM P450 from ACTH- treated rats were suspended in buffer B containing no K3 PO1, (A) or containing 78 m M K3 P0it (B) and the absorbancy plots were adjusted to obtain a flat baseline. Saturating level of pregnenolone (about 20 yM) in alcohol was added to the sample cuvette and the same volume of alcohol was added to the reference cuvette. The Aminco DW-2 spectro photometer setting was the same as Fig. 1. 19 .... -2J 2 0 I_________ I_________ 1 _________ I________ I---------------J --------------- 1 --------------- 1 390 420 Wavelength nm mixture (formula 936) was added, mixed and counted in a Beckman LS 7500 s c in tilla tio n system. Standard curve for the RIA is shown in Fig. 3. 2 1 FIGURE 3 STANDARD CURVE OF PREGNENOLONE RADIOIMMUNOASSAY (RIA) Pregnenolone at a concentration of 0, 0,05, 0 ,1 , 0 .2, 0 .4 , 0 .8 , 1.5, 3.2 and 5.0 ;n g per tube was used. 4,500 to 5,000 cpm of H3-pregnenolone (specific a c tiv ity , 62.5 mCi/mg) and pregnenolone antibody in 0,35 ml PBS-G buffer was added as described in Procedures. % Bound 95 90 80 70 - 60 - 50 - 40 * 30 - 20 - 10 • 0.05 0. 10 0.20 0.40 0.80 1.50 5.00 Pregnenolone ng/tube ro yj I I I . STATEMENT OF PROBLEM AND APPROACH A number of investigations performed on mitochondria isolated from anaerobic adrenal glands are in agreement that ACTH treatment increases and cycloheximide treatment reduces the cholesterol-bound cytochrome P450<j££, and the rate of pregnenolone formation. On the other hand, measurement of the e ffe c t of cycloheximide and ACTH on levels of cholesterol-bound P450^qq in mitochondria isolated from aerobic glands has produced co n flicting results. B e ll, Cheng and Harding (1973) have reported that in mitochondria obtained from aerobic glands of ACTH or ACTH plus cycloheximide treated ra ts , cycloheximide increases the cholesterol-bound P450<-££ le v e l, suggesting that the s ite of cyclo heximide in h ib itio n was not on the formation of high spin P450scc, but on enzyme turnover. In a sim ilar study, Brownie and Alfane e t a l . (1973) found that cycloheximide reduced the cholesterol-bound P450^q^ and, consequently, claimed that cycloheximide affected substrate binding only. Since Brownie et a l . collected th e ir adrenals from decapitated ra ts , one of the major differences in the two studies was the degree of anoxia of the adrenals. In addition, these investigators used a triethanol amine buffer for thei r EPR and opti cal studies of steroi d binding, while Bell and Harding employed a phosphate buffer. Sub sequently, Jefcoate and Orme-Johnson (1975) investigated the e ffe c t of anoxia on such mitochondrial preparations and found low cholesterol- bound P450scc levels in adrenal mitochondria from both ether-stressed and ether-stressed plus cycloheximide-treated animals when adrenals were 2 k ' removed under aerobic conditions. However, again, Jefcoate et a l. used a buffer fo r his steroid binding studies which was basically sim ilar to Brownie's group. Presumably, both the difference in buffer components and the state of anoxia have contributed to these d iffe re n t results. Consequently, at this point there is no agreement upon whether or not cycloheximide in h ib its an ACTH-mediated increase in cholesterol a v a ila b ility or binding to or in h ib its an ACTH-mediated increase in c a talytic a c tiv ity of P450^^^ or both. In order to establish the s ite (s ) of action of ACTH and cyclohexi mide on the cholesterol side chain cleavage system, independent measure ments of the active pool of cholesterol and substrate binding and the c a ta ly tic rate of the side chain cleavage enzyme w ill be required. The active pool of cholesterol cannot be established except as i t is reflected by the level of cholesterol-bound cytochrome P450<.qq. Con sequently, this parameter can only be inferred in d ire c tly by accurately determining the steady-state level of high spin P450<.££. The in trin s ic c a ta ly tic a c tiv ity of the enzyme can be determined by estimating a re la tiv e rate constant fo r the conversion of cholesterol-bound P450scc to P4 5 O c - 0 0 plus pregnenolone. 25 IV. RESULTS Freshly isolated mitochondria have active cholesterol side chain cleavage (SCC) a c tiv ity supported by endogenous electron donors. Fig. 4 shows mitochondrial SCC a c tiv ity during 5 minutes 27° C incubation from isolated bovine adrenal co rtical mitochondria. The mitochondria at a concentration of 4.25 pM P450 were e ith er kept at 4° C or pre incubated fo r the indicated time period and then returned to 4° C prio r to d ilu tio n to 0.25 pM P450 in the buffer containing 10 pM of Win-24, 540. As can be seen, a dramatic drop in endogenous electron donor supported pregnenolone formation was produced by preincubation. A sim ilar decrease in endogenous energy-supported cholesterol SCC was also observed i f the isolated mitochondria were aged at 4° C for several hours (results not shown). Cholesterol substrate levels in isolated mitochondria a ffe c t the spin state equilibrium of cytochrome P450 by binding to P450j Cq and forming cholesterol‘-P450g£Q high spin cytochrome. Since the cholesterol*P450^^ level measured at room temperature by pregnenolone- induced spectra CPU) in fresh mitochondria reflects enzyme‘ substrate complex remaining a fte r the exhaustion of endogenous electron donors, any condition or buffer ingredient which affects cholesterol SCC a c tiv ity or the level of endogenous electron donors w ill obviously change the cholesterol level and consequently the P II value. Increased H + concentration changes the spin state equilibrium by favoring high spin P450 (Jefcoate et a l , , 1973; Simpson and Wi11iam-Smith, 1976; 2 6 FIGURE 4 THE EFFECT OF PREINCUBATION OF BOVINE ADRENOCOTICAL MITOCHONDRIA AT 27° C O N CHOLESTEROL SCC ACTIVITY SUPPORTED BY ENDOGENOUS ELECTRON DO NO RS Bovine mitochondria at a concentration of 4,25 uM P450 were e ith e r kept at 4° C or preincubated at 27° C fo r the time period indicated. They were then diluted to 0.25 pM P450 in cold buffer A containing 10 pM of Win-24,540 and 6 pM of rotenone. Pregnenolone was measured following a 5 min 27° C incubation in a 0,2 ml aliquot. n moles of Pregnenolone/n mole P 4 5 0 2 — ■ 3 (A ^ 3 o ° u “ 1 C D 3 o c cr O ) » -► IB o 3 O ' to : 0 0 Hume and Boyd, 1978), but lowering the buffer pH below the optimum for enzyme a c tiv ity w ill resu lt in decreased SCC a c tiv ity . Incubation buffers containing Win and rotenone at pH 5 .0 , 6 .0 , 6.3 and 7.0 were used to d ilu te the mitochondria to fin a l concentration of 0.25 pM P450. P II was measured at room temperature; 0.2 ml of the diluted mitochondria was used to estimate the pregnenolone formed while warming to room temperature in each pH buffer. As shown in Table I , the net resu lt of these two effects is a higher level of cholesterol-bound P450SC£. Aging the bovine mitochondria by preincubation at 27° C decreases cholesterol SCC a c tiv ity which results in an irre v e rs ib le increase in P I I . Temperature dependence of cholesterol binding to cytochrome P450S££ of the ra t adrenal mitochondria was demonstrated by the type I absorbance change observed as the temperature is increased from 0° C to 22° C (Paul and Gallant et a l . , 1976). This heat generated type I (HGI) spectrum, o rig in a lly described in ra t mitochondria, has been suggested by Paul et a l . (1976) as another approach to measure cholesterol-bound P450$cc high spin complex. However, HGI is measurable in bovine adrenal cortical mitochondria only when they are reasonably fresh. Results shown in Fig. 5 indicate that aging the beef mitochondria at 27° C w ill increase HGI at f i r s t and then reduce i t as preincubation is prolonged. In contrast to the irre ve rs ib le increase in P II produced by aging, the HGI spectrum apparently does not represent the same cholesterol-bound P450< .qq detected by P II. Apparently, there is a species difference between ra t and bovine mitochondria. Chance and W illiam 's phosphate buffer (C & W buffer) at pH = 7.2 has generally been used in this laboratory for steroid binding studies. 29 TABLE I pH Effect on P II and Cholesterol SCC A c tiv ity Supported by Endogenous Electron Donors pH of buffer A 7.0 6.3 6.0 5.0 P II/O ,2 pM P450 (x 10_lf) 22 34 65 93 nmoles pregnenolone /nmole P450 (x 10"2) 146 101 67 14 FIGURE 5 THE EFFECT OF PREINCUBATION OF BOVINE ADRENAL CORTICAL MITOCHONDRIA AT 27° C O N THE PREGNENOLONE-INDUCED SPECTRUM AND O N THE HEAT-GENERATED TYPE I SPECTRUM The heat-generated type I spectrum (HGI) (4° C to 27° C) was generated in a mitochondrial suspension equivalent to 0.25 pM P450 in buffer A (pH = 6.3) and measured by dual wavelength spectroscopy. Depending on the size of the HGI, d iffe re n t time periods are required to complete the formation. The pregnenolone-induced spectrum (P II) is obtained a fte r the complete formation of the HGI, x P E 200 - m 3 3 wo / O N X Q . MCI o 20 40 27°C Preincubation C min) 3 2 In this bu ffer, aerobic mitochondria from animals treated with ACTH plus cycloheximide constantly show higher P II value than mitochondria from animals treated with ACTH alone. This re s u lt, f i r s t reported by B e ll, Cheng and Harding (1973) was not confirmed by several more recent studies (Simpson et a l . , 1972; Alfano et a ! , , 1973; Brownie et a l ., 1973; Jefcoate et a l . , 1974). I t appears that differences were probably due to the d iffe re n t buffers employed in the steroid binding studies. In Table I I , P II levels obtained in C & W buffer and triethanol amine buffer in mitochondria from aminoglutethimide plus ACTH-treated adrenals are shown. The e ffe c t of triethanolamine buffer (buffer A) and its components on the P II level from ACTH mitochondria is also shown. C & W buffer gives a higher P II value than the triethanolamine buffer (buffer A) which is basically the buffer used by Brownie, Jefcoate and Simpson's group, except that 0,2% instead of 0.5% BSA is used in buffer A. The e ffe c t of d iffe re n t components of buffer 4 on P II was studied. Eliminating 10 m M K3 P0i, (buffer B) gives a lower P II value, Inorganic phosphate has been reported to accelerate the reduction rate of iron in methemoglobin (Champion and Mlinck et a l ,, 1975), A possible explanation fo r this K ,3 P0i* e ffe c t is discussed la te r, I t is known that BSA and MgCl* are required for optimal cholesterol SCC a c tiv ity . Removal of BSA (buffer C) or BSA and MgCl2 (buffer D) from buffer B reduces SCC a c tiv ity while increasing the value'of P II, Comparison of buffer D and buffer E reveals that 20 m M KC 1 has no effect,on P II, In Table I I I the respiratory in h ib ito rs , rotenone ( 6 pM) and antimycin A (4 y g /m l), show only minor effects on the P II value in adrenal mitochondria from ACTH and ACTH plus cycloheximide-treated _______________________________________________________33. TABLE I I The Effect of Buffer Components on PII Component pH Pregnenolone- induced spectrum (x 1 0 V 0 . 2 yM) Treatment C + W Buffer K C 1 (96 jnM)MgCl2 * 6 H20 ( 6 mM), KHzPO* (3 m M ) NazHPO* (13 m M ) 7.2 51 AG-ACTH Buffer A Sucrose (250 mM), triethanol amine/HCl (15 m M ) K C 1 (20 mM), KHzPO* (10 m M ) MgCl2 (5 m M ) BSA (0.2%) 7.0 27 25 AG-ACTH ACTH Buffer B Sucrose (250 mM), triethanolamine HC1 (15 m M ) K C 1 (20 mM), MgCl2 (5 m M ) BSA (0.2%) 7,0 1 2 :ACTH Buffer C Sucrose (250 mM), tfiethanolamine/HCl (15 m M ) K C 1 (20 mM), MgCl2 (5 m M ) 7.0 33 ACTH Buffer D Sucrose (250 mM), triethanolamine/HCl (15 m M ) K C 1 (20 m M ) 7.0 43 "'ACTH Buffer E Sucrose (250 mM), triethanol amine/HCl (15 m M ) 7.0 44 ACTH ^ aminoglutethimide v *> TABLE I I I The Effect of Respiratory Inhibitors and ^POi, on PII and Endogenous Electron Donor-Supported Cholesterol SCC A ctivity Treatment ACTH ACTH + CHIa ACTH ACTH + CHI ACTH ACTH + CHI (A) Buffer (7.0) with no in hibitor P II/O .2 yM .0017 P450 .0012 .0055 .0032 .0107 .0080 A5 preg^nmoles/ 8.9 nmole P450 3.6 2.4 - 0 . 2 2 < 0.05 (B) Buffer (7.0) with 6 yM rotenone P II/O .2 yM .0025 P450 .0012 .0055 .0025 .0105 .0072 A5 preg nmoles/ 8 . 8 nmole P450 3.3 - 0 . 1 1 < 0.05 . ( c ) Buffer (7.0) with 6 yM rotenone and 2 m M antimycin A P I1/0.2 yM .0035 P450 .0015 .0052 . 0 0 2 2 . 0 1 0 0 .0070 A5 preg nmoles/ 8.5 nmole P450 2.4 2.4 - 0.05 < 0.05 xni aCycloheximide; ^Pregnenolone animals. In h ib itio n of SCC a c tiv ity supported by endogenous reducing equivalents also was observed. The in tra c e llu la r concentrations of potassium and phosphate are 157 m M and 113 mM, respectively. Accordingly, mitochondria in vivo are normally exposed to high concentration of these ions. Because most studies have employed approximately 10 m M potassium phosphate, the e ffe c t of these ions on cholesterol SCC was examined. The in tra c e llu la r concentration of other ions are: Na+ , 14 m M; M g+2, 26 m M; HC03~, 10 mM. The e ffe c t of several other salts such as NaCl, KC1, Mg02, Na2 S0it, Na3 P0i, and K3 P0^ was examined in order to test the s p e c ific ity of the K ^PO -i, e ffe c t. Using fresh beef adrenocortical mitochondria, the P II values induced by the above salts are shown in Fig. 6 . I t is seen th a t, except in the case of MgCl 2 » increasing s a lt concentration increases P II value.. This e ffe c t is much more marked in the case of K3 P0!*, In the case of MgCl2> there is an in it ia l increase followed by decrease in P II. A s im ilar bell shaped curve was obtained with MgCl2 using ra t adrenal mitochondria (results, not shown). In fresh mitochondria,' addition of K3 P0i* to buffer B in h ib its cholesterol SCC supported by endogenous reducing equivalents, In addition to the a v a ila b ility of endogenous reducing equivalents, the level of cholesterol-bound P450 and free, cholesterol determines the amount of pregnenolone formed in a given mitochondrial preparation, In the ACTH-stimulated animals, s ig n ific a n t cholesterol accumulation in mitochondria depends, upon the adrenals being allowed to become anaerobic prio r to preparation of the mitochondria (Oefcoate and Orme^Johnson, 1975). I f the adrenals are collected aero bically, the mitochondrial 3 , 6 , . FIGURE 6 THE EFFECT OF SALTS O N THE PREGNENOLONE-INDUCED SPECTRUM IN BOVINE ADRENOCORTICAL MITOCHONDRIA Fresh mitochondria equivalent to 0.2 pM P450 was suspended in buffer B which is sucrose (.25 M), t r i ethanol ami ne/HCl (.15 mM), K C 1 (20 mM), MgCli (5 mM), BSA (0,2%) and various salts of indicated fin a l concentration. pH of 7.0 is used for a ll the studies. Potassium phosphate and sodium phosphate are made up from th e ir monobasic and dibasic sa lts . Diluted HC1 and K O H or NaOH are used to adjust the pH of the rest of salts before use. 3 7 PJX ( A A42 0 -3 9 0 ) o - ti o 3 2 8 . a 10 o cholesterol-bound levels are as low as those in hypophysec- tomized or ACTH plus cycloheximide-treated animals (Jefcoate and Orme-Johnson, 1975). Fig. 7 shows, in a 27° C, 10 min incubation, pregnenolone formation is much greater in mitochondria from ACTH-treated anaerobic mitochondria than from ACTH-treated aerobic mitochondria. This is p a rtic u la rly true i f there is no K3 P0i» in the incubation media (buffer B) where the mitochondria isolated from anaerobic glands formed 10 nmoles pregnenolone/nmole P450 and the mitochondria isolated from aerobic glands formed 2 nmoles pregnenolone/nmole P450. Even though the cholesterol SCC a c tiv ity of anaerobic mitochondria forms preg nenolone at a higher ra te , P II measurement at room temperature of these mitochondria shows higher values than that of aerobic mitochondria. The remaining cholesterol-P450 high spin complex can be converted to pregnenolone i f Krebs cycle intermediates are added to r e in itia te the SCC a c tiv ity , as shown in Fig. 9. Therefore, the lim itin g facto r for SCC in the low s a lt range fo r anaerobic mitochondria is the level of endogenous reducing equivalents; fo r aerobic mitochondria, i t is the level of the high spin cholesterol P450 complex. As the K3 P0t* concen tra tio n increases, SCC a c tiv ity of anaerobic mitochondria drops rapidly to a level comparable to that present in aerobic mitochondria, even though a large difference in cholesterol abound P450<,££ levels between the two mitochondria remains (as shown in Fig, 23). The e ffe c t of MgCl2 and NaaPOi, on the cholesterol SCC a c tiv ity supported by endogenous electron donors in mitochondria from ACTH-treated anaerobic adrenals is shown in Fig. 8 . Increasing MgCl2 concentration drops this SCC a c tiv ity in the same manner as K3 P0ij (as shown in Fig, 7), In the case of 3.9 FIGURE 7 THE EFFECT OF KaPO* O N CHOLESTEROL SCC ACTIVITY SUPPORTED BY ENDOGENOUS ELECTRON DO NO RS FRO M MITOCHONDRIA OF ACTH-TREATED AEROBIC AND ANAEROBIC ADRENALS. K3 POi, was added to mitochondria and diluted with buffer B to obtain the fin a l concentration indicated. Pregnenolone formation by mitochondria from ACTH-treated aerobic adrenals or anaerobic adrenals,was performed in incubation buffer containing Win (10 yM), rotenone ( 6 yM) and NADP (0.2 m M) during a 1,0 mi'n 27° C incubation. 0.2 ml of mitochondria equivalent to 0.2 yM P450 was used fo r the assay. anaerobic mitochondria aerobic mitochondria O or 8 . o o E c < 0 c o o c o c 05 0 CL 4 - o (A o E c 120 80 40 K3P04 mM FIGURE 8 THE EFFECT OF MgCl2 AND Na3 PO^ O N CHOLESTEROL SCC ACTIVITY SUPPORTED BY ENDOGENOUS ELECTRON DONORS FRO M MITOCHONDRIA OF ACTH-TREATED ANAEROBIC ADRENALS MgCl2 and Na3 P0i, was added to the cold mitochondria and diluted with incubation buffer B containing Win (10 yM), NADP (0,2 m M) and rotenone ( 6 liM) to obtain the fin a l concentration indicated. Buffer B consists of sucrose (250 mM), triethanolamine/HCl (15 mM), K C 1 (20 mM) MgCl2 (5 m M ) and BSA (0.2%). Measurement of pregnenolone is the same as Fig. 7. nmoles of Pregnenolone/nmole P 450 to v * > - FIGURE 9 TIME COURSE OF CHOLESTEROL SCC ACTIVITY SUPPORTED BY ENDOGENOUS REDUCING EQUIVALENTS AND EXOGENOUS ISOCITRATE, Pregnenolone formation by mitochondria from ACTH-treated anaerobic adrenals incubated at 27° C, Prior to and a fte r addition of is o c itra te (5 mM), pregnenolone was measured in 0.2 ml of mitochondria equivalent to 0.2 yM P450. Mitochondria were suspended in incubation buffer B containing e ith e r no K3 P0* or 1 0 m M or TOO m M K3 P04, The PIT value for mitochondria suspended in buffer B without K3 P0it was 0.0026; with 10 m M K3 P0i* , 0.0041; w ith 100 m M K3 P0*, 0. 0085. Isocitrate 9 - o p _ LO O o r 7 - U i C L 4 - 0 mM K3 PO 4 1 0 mM K3 PO 4 100 mM K0 PQ4 3 - o 1 2 4 8 16 24 20 (mins) 45 Na3 P0it, the SCC a c tiv ity is enhanced i n i t i a l l y by Na3 P0it up to 20 m M . Further increase in Na3 P0t, causes a decrease in this SCC a c tiv ity . These studies indicated that unlike K 3 P O 1 , and MgCl2 the concentration of Na3 P0i, in buffer B is not optimum fo r SCC a c tiv ity . The e ffe c t of K3 P0i» on the SCC system is not only to in h ib it SCC a c tiv ity supported by endogenous reducing equivalents, but also to increase the P II of fresh mitochondria which have exhausted th e ir reducing equivalents. In addition, KsPOj , further increases the P II induced by low pH, as shown in Fig. 10 and also the P II of frozen and thawed mitochondria, as shown in Fig. 2. I t appears that the K3 P0i, e ffe c t of increasing P II and cholesterol SCC a c tiv ity results from the conservation of endogenous cholesterol by in h ib itin g endogenous energy-supported SCC a c tiv ity and a s h ift in the spin state equilibrium of P450<~££ since there is no evidence that K3 P0i, increases the pool size of cholesterol. The conclusion that pool size is not increased by K3 P(K is supported by the data shown in Fig. 8 . I t is seen that the to tal pregnenolone formation from endogenous and exogenous reducing equivalents supported SCC a c tiv ity is the same in both high and low K3 P0t, buffers, Fig, 11 shows that this K3 P0«, e ffec t on the spin state equilibrium is a time-dependent process. This is d iffe re n t from the low pH-induced high spin state where the maximal PII is achieved promptly. Cycloheximide treatment slows this K3 P0i, time-dependent process on inducing P II. Fig. 12 shows that because of the difference of time dependence in the ACTH and ACTH plus cyclo- heximide-treated animals, the pattern of the d iffe re n t K3 P0i,-induced PII fo r a given preparation is dependent upon the time of measurement. 46 FIGURE 10 THE EFFECT OF K3 P04 O N THE PH PROFILE OF THE PREGNENOLONE-INDUCED SPECTRUM IN MITOCHONDRIA FRO M ACTH PLUS CYCLOHEXIMIDE ADRENALS The P II of mitochondria equivalent to 0.2 yM P450 was measured in buffer B at d iffe re n t pH's in the presence of e ith e r 10 mM, 31 m M or 78 m M K3 P0i* . A 7 120 80 - 0 4 • 10 mM K3PO, * 31 mM K3PQ x 78 mM KoPQ pH FIGURE 11 TIME COURSE OF THE KaPO^-INDUCED CHOLESTEROL-BOUND P450«.rr COMPLEX MEASURED AS P II. ^ Pregnenolone-induced spectrum in adrenal mitochondria from ACTH and ACTH plus cycloheximide-treated animals measured at the various time intervals a fte r exposure to buffer B containing 25 m M of K3 P0i,. 49 20 " i t i o Y “ X E c 0 o > C O 1 o CM < <1 • ACTH Mitochondria ± ACTH-CHI Mitochondria N Q_ 10 8 6 4 mins ui :GX FIGURE 12 THE K3 PO4 -INDUCED PREGNENOLONE SPECTRUM MEASURED IN 2 TO 3 MIN O R 10 TO 15 MIN IN THE MITOCHONDRIA FROM ACTH O R ACTH PLUS CYCLOHEXIMIDE- TREATED ADRENALS P II is expressed as percent of the maximal P II induced by 80 m M K3 P0it buffer in mitochondria equivalent to 0,2 uM P450 incubated 5-6 hr a fte r isolation at 4P C. The PIT was measured in adrenal mitochondria from animals treated with ACTH and with ACTH plus cycloheximide a fte r exposure to various KsPOi* concentrations for 2 to 3 min ( 0 A) or 10 to 15 min (• A ) . 100 - § C O S 5 0 - o * ACTH Mitochondria a a ACTH-CHI Mitochondria 25 The difference in P II levels in ACTH and ACTH plus cycloheximide-treated mitochondria seen when the P II is measured 2-3 min a fte r exposure to K3 P0n decreases i f P II is measured a fte r 10 to 15 min a fte r exposure of mitochondria to K3 P0i». The e ffe c t of aging mitochondria on K3 P0t,-induced P II from both ACTH and ACTH plus CHI is shown in Fig. 13 where P II was measured a few minutes a fte r exposure to K3 P0.+ . Aging of mitochondria from cyclo heximide-treated animals eventually releases the in h ib itio n of substrate binding to P450<.C£, which then approaches that seen in mitochondria from ACTH-treated animals. The P II induced by K3 P0^ in mitochondria from ACTH and ACTH plus cycloheximide-treated animals isolated under aerobic or anaerobic con ditions is shown in Fig. 14. P II was measured a few minutes a fte r mitochondria were suspended in the d iffe re n t K3 P0i, buffer. Therefore, the difference between the treatments is more profound. In general, P II values in buffer B are only a fraction of the maxi mal P II values obtained by addition of K3 P0i,. to buffer B. Anaerobic" mitochondria were obtained from animals•'subjected to th e ' following treatments: aminoglutethimide (AG) plus ACTH, A G plus ACTH plus cyclo- heximide, ACTH, ACTH plus cycloheximide gave sim ilar maximal P II values; that is 100-120 x 10-1* P II per 0.2 yM P450. Those mitochondria a ll have a high free cholesterol content which permits achievement of maximal P II values in the presence of high K3 P0i*. In the range of no KsPOi* to low K3 P0lt, mitochondria from CHI-treated animals show decreased substrate binding to P 450^(,. In vivo cholesterol SCC a c tiv ity is highly active in the ACTH-treated animals. Therefore, l i t t l e free cholesterol is accumu- 5 3 .; FIGURE 13 THE EFFECT OF AGING O N THE KgPO^-INDUCED PREGNENOLONE SPECTRUM IN THE MITOCHONDRIA FRO M . ACTH AND ACTH PLUS CYCLOHEXIMIDE-TREATED ADRENALS The P II measurement was performed 10 to 15 min a fte r mitochondria equivalent to 0.2 yM P450 were exposed to K3 P0 4 -containing buffer. 1 0 0 - o p o E c o < y > co | 5 0 - <3 > C L o • ACTH Mitochondria ^ ACTH-CHI Mitochondria 100 5 0 25 75 K3 P04 mM Vi Vr FIGURE 14 K3 PO4 EFFECT O N PI I (MEASURED IN 2 TO 3 MIN AFTER EXPOSURE TO K3 P0O IN MITOCHONDRIA FRO M ACTH O R ACTH PLUS CYCLOHEXIMIDE-TREATED AEROBIC O R ANAEROBIC ADRENALS Rat mitochondria equivalent to 0,2 yM P450 from ACTH-treated aerobic (A( ) or anaerobic (A) glands and ACTH plus cycloheximide aerobic (B1) or anaerobic (B) glands were suspended in buffer B with d iffe re n t KsPOij concentrations. 56, P II (A A 420-390 n m X " I 0 120 'S t I A ----- 80 - °A 40 - o • ACTH Mitochondria A a ACTH-CHI Mitochondria 25 50 100 125 150 VI L - - <Tat;edei®;;tfeeaniAio'thQndwiai;ieve.g<vtBqughii.S6Ci'*4i5a iratf^^it^agilstepniim the steroidogenesis. This is reflected in the low free cholesterol found in aerobic mitochondria from ACTH-treated animals. In addition, optical or EFR spjecf-r.aii of these mitochondria indicate that they have low levels of high spin cholesterol-P450< .qq (Simpson e t a l . , 1972; Brownie et a l . , 1973; Jefcoate et a l . , 1974). In the mitochondria from ACTH plus cycloheximide-treated animals, P II is low, although free cholesterol in the mitochondria is high. Free cholesterol content in this mitochondria obtained from aerobic adrenals is 40.8 ± 1.5 nmoles/mg protein, while that ;fro m '' ACTH-treated aerobic adrenals is 20.8 ± 1.2 nmoles/mg protein. The maximal free cholesterol content in those mitochondria from animals pretreated with AG is 53,7 ± 2.4 nmoles/mg protein from ACTH plus cycloheximide-treated adrenals, and 51.4 ± 2.4 nmoles/mg protein from ACTH-treated adrenals, respectively (unpublished data). The cholesterol accumulation is due to cAMP mediated stimulation of cholesterol ester hydrolase which is not inhibited by cycloheximide and to the decrease in the cholesterol SCC a c tiv ity which is in h ib ited . At the low K3 P0i, range (.0-70 mM), mitochondria from CHI-treated animals show a blunted response, as indicated by a low P II value as compared to the control, Amtnoglutethimide (AG) is an in h ib ito r of cholesterol SCC and causes the free cholesterol accumulation when the animal is pretreated with AG before ACTH stim ulation, AG also is a potent in h ib ito r for cholesterol SCC i f i t is added d ire c tly to the isolated mitochondria, because i t is able to form an iron ligand which inducesaa t.typeX 1 1 • ' iow spin cytochrome P450 spectra. No P II spectra is detectable unless A G 58' is washed out o f mitochondria. This in v itro e ffe c t of AG is not observed in the case of cycloheximide. Only in vivo treatment with this protein synthesis in h ib ito r w ill produce an in h ib itio n of steroido genesis. Therefore, i t has been proposed that the action of cyclo heximide is mediated through an in h ib itio n synthesis of a la b ile protein (Ferguson, 1963; David et a l . , 1968). In h ib itio n by an adrenal metabolite of CHI is another p o s s ib ility . The K3 P0^ e ffe c t on P II, as indicated e a r lie r , is p a rtia lly due to in h ib itio n of transfer of endogenous reducing equivalents to the cholesterol SCC electron transport pathway and to a s h ift of the equilibrium in the high spin state P450<-££. This cholesterol SCC a c tiv ity supported by endogenous reducing equivalents is much lower in CHI-treated mitochondria (Table I ) ; therefore, a smaller e ffe c t of K3 P0it on P II would be expected. As a matter of fa c t, the portion of P II which can be induced by K3 P0it is much higher in cycloheximide mitochondria (Figs. 11, 13, 14). A possible explanation is that in the absence of K3 P0i», the high s p in — ^low spin equilibrium is shifted to the rig h t of CHI. This then permits a larger change in the spin state. Mai ate , is o c itra te , as well as other Krebs cycle intermediates added to the isolated mitochondria w ill in itia te cholesterol SCC. The conversion of cholesterol to pregnenolone results in a high spin to low spin change in P450s^c . Therefore, in a difference spectrum in which Krebs cycle intermediates are added to the sample cuvette, the size of the reverse type I difference spectrum indicates the tran sitio n of high spin cholesterol-P450<jQ£ to low spin cholesterol free cytochrome P450 in the given mitochondria preparation, In the case of aging 59 mitochondria the a^ea-under the descending arms remains, the same,-."g! whi les that of the'.ri si ng arms ‘ increases. ---The1 KaPO^ concenira-O'csAtfa- tion required to produce the maximal rate in each mitochondrial preparation decreases with aging of the mitochondria, changing from 70 m M in the fresh mitochondria (curve A) to 20 m M in the most aged mitochondria (curve D). These increasing reaction rates and reduction in K3 PO4 concentration for maximal reaction could be due to a secondary e ffe c t of s a lt on the high spin concentration which accelerates the electron tran sfer step. Therefore, the ra tio of i n i t i a l rate of malate- induced spin state change ys. the P II induced by the K3 PO1, changes at d iffe re n t K3 PO1/ concentration was examined. The KsPOtj e ffe c t on this r a tio , shown in Fig. 19, is also a bell-shaped curve which is sim ilar to the K3 PCU e ffe c t on the i n it ia l rate shown in Fig. 17. This ra tio decreases as mitochondria becomes more aged, although the to ta l spin state change induced by malate in these aged mitochondria is larger. This increase of to tal spin state change is due to the high cholesterol- bound P450<-££ levels in these aged mitochondria, as shown in Fig. 21. The e ffe c t of Na3 P04 , . K3 PO4 , Na2 S0 4 , K2 SO4 , MgCl2 , NaCl and KC1 on the ra tio of the in it ia l rate of the malate induced low spin spectrum to the P II is shown in Fig. 20. Both Na3P0i, and K3 PO4 give sim ilar bell shaped curves. The bell shaped curve of MgCU again shows its maximal a c tiv ity at a lower s a lt concentration (15 m M ) than fo r Na3 P0 4 (20 m M) or K3 PO4 (40 mM), The rest of the salts do not give d is tin c t bell shaped curves. A much smaller high spin to low spin change in P450<~££ is induced by is o c itra te in ra t mitochondria, as compared to that in bovine mito chondria, Because of this small change in spin s ta te , the high spin to 6$. pregnenolone formation ra te , the malate-induced spectrum also shows a biphasic change in the time course, as seen in Fig. 15. Fig. 16 shows that the malate-induced spin state changes and P II values correspond to P450 concentration. As shown in Fig. 17, the in it ia l rate of the malate-induced spectrum increases as mitochondria are aged, e ith e r at 4° C or at room temperature. Prolonged aging eventually in h ib its this rate of spin state change. To maintain the enzymatic reaction, a certain degree of membrane in te g rity is required. Leakage of the cofactor, NADP, or the soluble protein, adrenodoxin, may become lim itin g factors in the prolonged aged mitochondria. On the other hand, aging of mitochondria irre v e rs ib ly increases P II, Salt effects on reconstituted mitochondrial P450 side chain cleavage and 118 hydroxylase system have been demonstrated by Lambeth,. Leybert and Kamin. (1979) recently. The e ffe c t of KsPOi, on the in it ia l rate of the malate-induced reverse type I spectra in beef mitochondria aged at 4° C or at room temperature fo r d iffe re n t periods of time is shown in Fig. 17, Increasing K3PCU in the buffer at f i r s t enhances the rate of the malate-induced spin state tra n s itio n , which reaches a maximal rate and subsequently fa lls as K3PO4 is further increased, resulting in a bell-shaped curve, The i n it ia l rate of the malate induced reverse type I spectrum in the presence of varying con centrations of several other salts including Na3 P0 4 , K2 SO4 , Na2 S0i,, K2 S(U was studied. These data are shown in Fig. 18. NasPOi* and MgCl2 also show the bell shaped curves produced by K3 P O 4 . The maximal in it ia l rate of the malate induced reverse type I spectral change fo r MgCl2 6 ,1 ' • FIGURE 15 DUAL WAVELENGTH MEASUREMENTS OF THE REVERSE TYPE I (AA 390-420 rim ) DIFFERENCE SPECTRA INDUCED BY MALATE O R PREGNENOLONE AT DIFFERENT CONCENTRATIONS OF BOVINE ADRENOCORTICAL MITOCHONDRIA P450 Bovine adrenal cortical mitochondria equivalent to 0.09 yM (A), or 0.18 yM (BL or 0,27 yM (C) of P450 were suspended in buffer B containing 25 m M K3P0tt and incubated at room temperature fo r 10 to 15 min to exhaust endogenous reducing equivalents. 62 M a l a t e Pregnenolone FIGURE 16 RATE OF FORMATION OF THE REVERSE TYPE I DIFFERENCE SPECTRUM INDUCED BY MALATE Bovine mitochondria equivalent to 0,18 pM of P450 were suspended in buffer B containing 25 m M K3P0i, and incubated at room temperature fo r 10 to 15 min to exhaust endogenous electron donors. The rate of spin state change induced by malate was calculated from the 10 sec (x) 1 min (•) and 2 min (A) AA changes. The maximal malate-induced spin state change and pregnenolone measurements are also recorded. 200 * 10 sec * 1 min * 2 min o -J x 150 T 3 50 - 0. 0 0.09 0 . 1 8 0.27 p 450 FIGURE 17 THE EFFECT OF K3P0* O N INITIAL RATE OF MALATE-INDUCED REVERSE TYPE I SPECTRUM IN BOVINE ADRENOCORTICAL MITOCHONDRIA AGED FOR DIFFERENT PERIODS OF TIME Bovine adrenal cortical mitochondria equivalent to 0.18 yM P450 were used to measure the in it ia l rate of AA change 10 sec a fte r malate in itia te d the reaction. Mitochondria aged at 4° C for less than 1 hr (A ), 2-3 hr (B ), or 5-6 hr (C), or room temperature for 1 hr (D) were - used. Mitochondria was suspended in buffer B containing various concentrations of K3P0i* and incubated at room temperature fo r 10 to 15 min to exhaust endogenous electron donors. I A J U J •' O d ' O I Initial Rate of Malate Induced Low Spin ( A A 4 2 0 - 3 9 0 n m / m i n x 1 0 - 4 ) FIGURE 18 THE EFFECT OF SALTS O N INITIAL RATE OF THE MALATE-INDUCED REVERSE TYPE I DIFFERENCE SPECTRUM IN BOVINE ADRENOCORTICAL MITOCHONDRIA Mitochondria equivalent to 0.2 yM P450 were used to measure the in it ia l rate as described in Fig. 17. Initial Rate of Malate Induced Low Spin (AA420 3 g Q nm/min x10"4) 3 S US \ d 240| occurs at a lower s a lt concentration (15 m M) than fo r Na3P0it and K3P0i» (40 mM). The rest of the salts do not produce bell shaped curves. Increasing s a lt has been reported to increase the dissociation constant of the FLP*ADX complex (Lambeth and Seybert e t al., 1979). I t has been demonstrated by Lambeth et a l . (1979) and subsequently sup ported by Hanukoglu et aV. (1980) that ADX serves as an electron c a rrie r by shuttling between FLP and P450 to drive the P450 redox cycle. In the system where the formation of substrate-bound P450 high spin complex is not lim ite d , the redox state of ADX or reduced ADX concentration w ill therefore determine the hydroxylation fa te . This bell-shaped curve observed in isolated bovine mitochondria indicates that a change in the ra te -lim itin g step has occurred as K3PCh increases in the buffer. The rising arms occur at low K3PCU where oxidized and reduced ADX remain tig h tly associated with the FLP. Transfer of reducing equivalents from reduced ADX to cytochrome P450 w ill be slow. Therefore, the dissociation of reduced ADX from tig h tly bound FLP*ADX complex is the ra te -lim itin g step. As K3PCh increases, the reaction rate reaches its maximum and starts to f a l l . At very high K3P0i* concentration, formation o f the in it ia l Fl-Pre(j»ADXox complex becomes ra te -lim itin g . A high dissociation rate of oxidized ADX from the in it ia l complex w ill prevent the ra te -lim itin g electron transfer from FLP to ADX and thus in h ib it a c tiv ity . Maximal reaction rate should occur at the K .3P0i* concentfation where oxidized ADX is tig h tly associated with FLP, thereby allowing e ffic ie n t FLP to ADX electron tran sfer and where reduced ADX can dissociate read ily from the complex, Fig. 17, at the same time, shows that the bell-shaped area increases as 70 FIGURE 19 THE EFFECT OF K3P0* O N THE RATIO OF THE INITIAL RATE OF MALATE-INDUCED REVERSE TYPE I SPECTRUM TO THE PREGNENOLONE-INDUCED SPECTRUM (P II) IN BOVINE .TOENOCO'RTrCA'L 4 MITOCHONDRIA AGED FOR DIFFERENT PERIODS OF TIME The i n it ia l rates of the malate-induced spectrum were taken from Fig. 17. The P II measurements were s im ilar mitochondrial preparations (see Fig. 21). The conditions fo r curves A, B and C correspond to those in Fig. 17. Initial Rate of Malate Induced Low Spin/ p 3 1 ro to -t* b b ‘ o b ► o • > 00 . IO • ► X -j N > FIGURE 20 THE EFFECT OF SALTS O N THE RATIO OF THE INITIAL RATE OF MALATE-INDUCED REVERSE TYPE I SPECTRUM TO THE PREGNENOLONE-INDUCED SPECTRUM (P II) IN BOVINE ADRENAL CORTICAL MITOCHONDRIA The in it ia l rates were taken from Fig. 18. The P II measurements were carried out iii a same mitochondria preparation. Initial Rate of Malate Induced Low Spiij/^PIT 3 2 OQ ■ v i - t - FIGURE 21 THE EFFECT OF AGING O N THE KaPO^-DEPENDENT PREGNENOLONE-INDUCED SPECTRUM IN BOVINE ADRENAL CORTICAL MITOCHONDRIA P II measurements of 0.18 yM P450 were made on the same bovine adrenal cortical mitochondrial preparation used in Fig, 17. 120 - 'o Y — X £ % 8 0 - 0) C O I o C M < < 3 w M 0. 40 120 80 40 K3P04 mM low spin changes are completed rap id ly. Consequently, measurement of the in it ia l rate is not p ra c tic a l. Since the spin state change at the end of one minute or two minutes, up to completion, goes along with the i n it ia l rate as shown in Fig. 16, changes of spin state obtained in rat mitochondria at the end of one minute were used to examine the X3P0i, e ffe c t on the isocitrate-induced high spin to low spin change. Sim ilar to fresh bovine mitochondria, ACTH-treated ra t mitochondria also show a f l a t bell-shaped curve in response to increasing K3PCU concentration, as shown in Fig. 22. The figure also Shows that aging of the ra t mitochondria w ill fu rth er reduce this ra te , regardless of the increasing P II produced by the aging process. This spectral study is not sophis ticated enough to allow an examination of the possible cycloheximide e ffe c t on this s a lt-s en sitiv e process in ACTH-treated ra t mitochondria. Correlation of P II and is o c itra te supported i n it ia l rate of cholesterol SCC a c tiv ity as measured by pregnenolone formed in one minute a fte r reaction is in itia te d by is o c itra te was examined. Mito chondria isolated from aerobic and anaerobic glands from ACTH or ACTH plus cycloheximide-treated rats in various concentrations of K3P0i* were used to study the e ffe c t of high spin concentration on the in it ia l rate of pregnenolone formation, The reaction was started a fte r 10 minutes of preincubation at 27° C to exhaust the endogenous reducing equivalents. The pregnenolone formed in ACTH-treated adrenals during the preincubation, as shown in Fig, 7, is subtracted from the to tal pregnenolone formed one minute a fte r is o c itra te in itia te s the choles terol SCC a c tiv ity . Under a s im ilar condition (s im ila r buffer and s im ila r time la g ), PIT was measured, PIT levels from variously treated 77 FIGURE 22 THE EFFECT OF K3P O « * O N THE RATE OF ISOCITRATE-INDUCED REVERSE TYPE I SPECTRUM IN ACTH-TREATED RAT MITOCHONDRIA The rate of formation of low spin P450$cc 'induced by is o c itra te in mitochondria equivalent to 0,5 yM P450 from ACTH-treated ra t adrenals was measured at the end of one minute. Freshly isolated mitochondria suspension exposed to room temperature fo r 10 min ( • ) or exposed to room temperature for more than 1 hr (o) were used. 78 GL isocitrate induced Low Spin P 4 5 Q ( A A42o - 390 nm x 10“4) 0 0 o O '- adrenals, at d iffe re n t K3 P0i* concentrations, are shown in Fig. 23.~ The correlation of P II and pregnenolone formation, as shown in Fig. 24, is not a 'rlinear relationsh ip, but increases exponentially as P II is increased. Because the P II induced by KaPO^ correlated lin e a rly with the high spin P450 measured as difference type I spectrum induced by K3 PO 4 , as shown in Fig. 25, apparently increasing concentration of cholesterol-P450cjQC high spin hemoprotein accelerates the in it ia l rate of cholesterol SCC a c tiv ity . At the low P II range, ACTH-treated mito chondria show higher SCC a c tiv ity than that of ACTH plus cycloheximi de treated mitochondria. As the P II value increases, the in it ia l rates become s im ilar between the two groups. C rivello and Jefcoate (1978), in a related study, found a lin e a r correlation between mitochondrial P II and plasma corticosterone lev els . There are several differences between the two studies. Changes in plasma corticosterone levels do not necessarily r e fle c t the i n i t i a l rate of cholesterol SCC a c tiv ity . More im portantly, the P II levels measured at room temperature depend greatly on the conditions under which the:.mitochondria were examined. Depending on the condition used, the P II value may bear l i t t l e re la tio n ship to the steady-state level of the c hol est er olc om pl ex in vivo. Because of the s e n s itiv ity of the P450scc system to salts described by Lambeth et a l, (1979), the e ffe c t o f NasPCU and MgCll as well as K3 P 0 i» on the re la tiv e rate constant of cholesterol *P450SCC ------ ^ pregnenolone + P450<.££ was studied, The effects of Na3 P0it and MgCl2 on the re la tiv e rate constant expressed as the ra tio of the in it ia l rate of pregnenolone formation to the P II in the mitochondria of 80' FIGURE 23 THE K3 P0^-INDUCED PREGNENOLONE SPECTRUM IN MITOCHONDRIA FROM ACTH O R ACTH PLUS CYCLOHEXIMIDE-TREATED AEROBIC O R ANAEROBIC ADRENALS AFTER EXHAUSTION OF ENDOGENOUS ELECTRON DO NO RS Mitochondria equivalent to 0.2 yM P450 from ACTH-treated aerobic adrenals (A1) , or anaerobic adrenals (A) and ACTH plus cycloheximide treated aerobic adrenals ( B ') s or anaerobic adrenals (B) were suspended in incubation buffer B (pH = 7,0) with 6 yM rotenone and 0.2 m M NADP. K3 PO 4 was added to mitochondria and diluted with buffer B to obtain the fin a l concentration. The mitochondrial suspension was exposed to room temperature fo r 10 to 15 min before P II was measured. 120 - a B o 8 0 - X E c 0 o> C O 1 o <? < 0 A 40 W Q _ o • ACTH Mitochondria A A ACTH-CHI Mitochondria 40 80 120 K3 PO 4 mM .0 0 N 5 FIGURE 24 THE EFFECT OF VARIATION IN THE P II SPECTRUM INDUCED BY K3 PO 4 O N THE RATE OF PREGNENOLONE FORMATION IN MITOCHONDRIA FROM ACTH O R ACTH PLUS CYCLOHEXIMIDE-TREATED AEROBIC OR ANAEROBIC ADRENALS Pregnenplone formation 1 min a fte r the hydroxylation was in itia te d by is o c itra te was measured in 0.2 ml mitochondria equivalent to 0.2 yM P450 from ACTH-treated aerobic adrenals or anaerobic adrenals and ACTH plus cycloheximide-treated aerobic adrenals or anaerobic adrenals. Buffer B w ith 6 yM rotenone, 0,2 -m M NADP f 10 yM Win and d iffe re n t K3 P0i* concentrations (from 0 to 120 mM) were used to obtain d iffe re n t levels of P II. Pregnenolone formed in the same buffer during 10 min at 27° C preincubation was substrated as the blank value. 8.3 8 - v 2- o ACTH,aerobic mitochondria • ACTH,anaerobic mitochondria a ACTH “ CHI,aerobic mitochondria 4 ACTH- CHI,anaerobic mitochondria 0.004 0.008 0.0120 Pregnenolone Induced Spectrum a A 420 - 390nm j 0.2 fxM P 450 84 FIGURE 25 CORRELATION OF THE PREGNENOLONE-INDUCED REVERSE TYPE I SPECTRUM AND TYPE I DIFFERENCE SPECTRUM INDUCED BY K3P0^ The Type I difference spectrum was obtained as mentioned in Fig. 1. Mitochondria equivalent to 0 . 25 piM P450 from ACTH-treated adrenals were suspended in buffer B, D ifferen t K3P0., concentrations were obtained in the sample cuvette to obtain the Type I difference spectrum. Corre sponding PIT difference spectrum (PII<.-PIIn) was obtained from the same mitochondrial suspension. 85- Pregnenolone Induced Difference Spectrum (A A 420-390 nm xi<r4) £ X 3 ® ro . H o g m4 % *H h 0 ( D D O 0 C O "O 0 CO c K ACTH-treated anaerobic adrenals are shown in Fig. 26. Both Na3P0i* and MgCl2 in hib ited cholesterol SCC a c tiv ity at low s a lt concentrations. The re la tiv e rate constants are enhanced with fu rth e r increase of NaaPOu and MgCl2 and reach th e ir maxima above 70 mM. The MgCl2 require ment fo r optimal mitochondrial cholesterol SCC a c tiv ity is almost double the concentration required in the reconstituted system studied by Lambeth e t a l. (1979). In addition, the i n it ia l in h ib itio n observed in in tac t mitochondria is not seen in the reconstituted system. The K3P0i* e ffe c t, as shown in Fig. 27, the mitochondria from ACTH (A) or ACTH plus cycloheximide (B )-treated anaerobic adrenals show an increased re la tiv e rate constant fo r the cholesterol SCC a c tiv ity which reaches a maximum a t 40 mM. This constant is maintained as the K^PO* concentra tion increases to 120 mM. For mitochondria from ACTH plus cyclohexi mide-treated aerobic adrenals (Bl ) , the apparent rate constant does not reach a maximum u n til the K3PCU stim ulation of the apparent rate con stant was also observed in the KsPOi* e ffe c t on PIT (Fig. 23). The cause of this difference between aerobic and anaerobic adrenals is not clear, The K3P04 e ffe c t on the re la tiv e rate constant of is o c itra te or malate-induced i n i t i a l spin change/PII ra tio in ra t mitochondria is d iffe re n t from that in bovine mitochondria (Fig, 18), Therefore, the difference possibly is due to differences in in trin s ic properties of these mitochondria. They are also d iffe re n t in th e ir degree of aging and possibly in th e ir perm eability to K3P04, Examination of the re la tiv e rate constants at d iffe re n t K3P(K concentrations of cycloheximide-treated mitochondria shows th a t, in 8 7- FIGURE 26 THE EFFECT OF NaaPO* AND MgCl2 O N THE RELATIVE RATE CONSTANT IN THE MITOCHONDRIA FROM ACTH-TREATED ANAEROBIC ADRENALS Pregnenolone and PIT measurements are carried out in a p a ra llel mito chondria suspension as described in f ig , 23 and Fig, 24, nmoles of Pregnenolone/nmole R/s o / pIT o » O c n to O o o 3 2 00 o — A o > o -GO A Q FIGURE 27 THE EFFECT OF KgPOi, O N THE RELATIVE RATE CONSTANT IN THE MITOCHONDRIA FRO M ACTH AND ACTH PLUS CYCLOHEXIMIDE-TREATED ADRENALS Pregnenolone measurements are shown in Fig. 24; P II values are shown in Fig. 23. N a. c I 0) c o o c o e o o h . a. !/} © o E c 12 • 8u 4 - • ACTH Mitochondria a a a CTH-CHI Mitochondria 40 0 80 120 K3P04 mM 91 addition to a decrease in cholesterol binding to P450 (Figs. 11, 12, 13, 14), the c a ta ly tic rate of cholesterol SCC a c tiv ity is also decreased. Due to these effects on the cholesterol SCC system, CHI in h ib its the ACTH-stimulated steroidogenesis. V, DISCUSSION AND SUM M ARY Cycloheximide could in h ib it the stim ulation of cholesterol side chain cleavage a c tiv ity by ACTH by reducing substrate binding or reducing c a ta ly tic a c tiv ity of p450^C (,, or both. A change in choles terol-bound P450S£C formation may resu lt from decreased a f f in it y of the enzyme fo r cholesterol, or from a reduced cholesterol pool for enzyme binding. The c a ta ly tic a c tiv ity of P450scc could be inhibited by changes in the in trin s ic properties of the P450 enzyme or by changes in interaction of the oxidase with its electron transport system, or both. The overall rate: of-cholesterol SCC a c tiv ity is determined by the rate^l.im iting step which is believed to be the second electron tran sfer (S lig a r and Gunsalus, 1976^ Pederson et a l , , 1977) from ADX red to the cholesterol bound high spin P450<jC(, fo r 22 hydroxycholesterol formation (Burstein and Gut, 1976), I f the P II measured represents the steady-state level of cholesterol *P450< -q£*ADX high spin complex, the i n it ia l rate of pregnenolone formation measured at a time when P II remains constant should be proportional to P II in any given experimental condition. This relationship also applies: to the measurement of the malate or is o c itra te induced i n it ia l rate of low spin P450 or reverse type I spectrum formation in which cholesterol is depleted from the P450$cc bound high spin state by hydroxylation conditions. The ra tio of i n it ia l rate of pregnenolone formation to P II or in it ia l rate of to low spin P450 conversion to P II is equivalent to the re la tiv e 93 rate constant fo r the cholesterol SCC a c tiv ity . The re la tiv e rate constant of a second order reaction can be expressed as [ADX ^gOr = [ i n i t i a l rate o f pregnenolone formation] — [cholesterol•P450scc»ADXqx] or = [ i n i t i a l rate of malate induced low spin] [cholesterol *P450c .qq*ADXox] Experimentally, the i n it ia l rate of pregnenolone formation measured one minute (27° C) a fte r is o c itra te or malate in itia te s hydroxylation/ while the i n it ia l rate of malate induced low spin P450 tran s itio n is estimated from the formation of a AA 420-390 nm difference spectrum 10 seconds a fte r addition of substrate. The P IT measurement estimates the cholesterol*P450Sq£»ADXox complex in the mitochondria. P II levels depend on the a v a ila b ility of cholesterol and also free ADXqx (Lambeth and Seybert et a l , , 1980), Since i t has been suggested that the levels of both oxidized and reduced forms of free ADX are regulated by s a lt concentration, at least in certain reconstituted side chain cleavage systems (’Lambeth and Seybert et a l , , 1979b), the s a lt e ffe c t on SCC a c tiv ity in in tac t mitochondria was tested and the e ffe c t of s a lt on the re la tiv e rate constant expressed as k.*-lADXrecj] was examined. The effects of several salts such as Na2S0i», K2S0i,, MgCl2 , NaCl and KC 1 on P II, the i n it ia l rate of malate induced reverse type I spectrum and the re la tiv e rate constant were studied in order to test the s p e c ific ity of the K^PCi* e ffe c t which is a major ion component (K+ , .113 mM~; POi,- , 157 mM) in c e ll flu id . As shown in Fig. 6, except in the case of MgCl2 , increasing s a lt concentration increases PIT values, Among the tested 94 s a lts , K3 PO 4 produced the most marked e ffe c t on P II. In order to determine i f cycloheximide affects this re la tiv e rate constant, the P II measured has to re fle c t the steady-state level of the mitochondria cholesterol-bound P450<.£C induced by the p a rtic u la r experimental circumstances and not a rtifa c tu a l changes resulting from the iso latio n procedure. A number of investigators have previously reported that P II levels in the isolated mitochondria depend on the treatment of the animal, state of anoxia, pH and temperature. I t has been found in this study that other factors such as aging, time and temperature of preincubation, and buffer components also a ffe c t the mitochondrial P II level (Fig. 4,5,13,18,21 and Table I I I ) . Since the P II values reported in the lite r a tu re following various treatments of the animals have been made.us'iingv mitochondria isolated and stored under a va riety of conditions, and using d iffe re n t procedures fo r the pregnenolone t it r a t io n , i t is d if f ic u lt to correlate the cholesterol- bound P450<-q£ levels and th e ir relationship to cholesterol SCC a c tiv ity in mitochondria derived from animals with sim ilar treatment schedules, The cholesterol-bound P450 measured by the pregnenolone induced spectrum CPU) may not often determine accurately the steady-state level of high spin P 4 5 0 ^ Endogenous electron donors in the fresh mitochondria can drive the cholesterol SCC a c tiv ity in the diluted mitochondrial suspension (Fig. 7, 8 and Table I , I I ) , when i t is warmed to room temperature, to perform the pregnenolone binding study. Con sequently, the P II measured under these conditions represents the remaini ng cholesterol-bound P450<.££ complex a fte r exhaustion of endogenous electron donors, 95 As can be seen in Fig. 9, the P II measurement of the mitochondria from ACTH-treated adrenals in buffer B, which has no added K3 P0h, and an active endogenous electron donor-supported SCC, is low. Therefore, the is o c itra te in itia te d i n it ia l rate of pregnenolone formation from endogenous cholesterol is also low. When 10 m M or 100 m M of K3 P0i* is added to buffer B, the endogenous electron donor-supported SCC a c tiv ity is in h ib ited . High P II levels of 0.0041 and 0.0085/0.2 pM P450 are obtained, respectively, as compared to .0026/0.2 pM P450 in the mito chondria in buffer B. Consequently, the is o c itra te in itia te d in it ia l rate of pregnenolone formation is increased corresponding to the increased P II le v e l. The e ffe c t of K3 P0i, in h ib itio n of electron trans fe r from FLP to cytochrome P450 oxidase on the cholesterol SCC a c tiv ity is shown in F ig .'7. The corresponding P II levels from a s is te r mito chondrial preparation which is measured a fte r completion of endogenous electron donor-supported SCC a c tiv ity are shown in Fig. 23 (A and A 1). These data indicate that the P II induced by K^PCK is at least p a r tia lly due to an in h ib itio n of the mitochondrial endogenous electron donor- supported cholesterol SCC a c tiv ity , Fig. 10 shows that K3 P(H fu rth e r increases the P II in mitochondria which are suspended in a low pH buffer where cholesterol SCC a c tiv ity is negligible (Table I ) , K3 P 0 4 also increases the PIT of the aged or frozen and thawed mitochondria in which endogenous electron donor-supported cholesterol SCC a c tiv ity has been exhausted (see Figs, 2 9 13 and 21), At a fixed K3PCU concentra-ii tio n , PIT! induced by K 3 PQ 4 is a time-dependent process (.see pig, 11), Mitochondria from ACTH-treated adrenals reach th e ir maximal P II faste r than those from ACTH plus cycloheximide-treated adrenals. Apparently 96' the CHI treatment in terferes in some way with the K3 PO 4 -induced increase in cholesterol binding to P450. In these various studies of the K3 PO 1* e ffe c t on P II value, other factors which may change the P II values are held constant. S alt is reported to be able to increase the dissociation constant of the FLP*ADX complex, resulting in a high level of free ADX (Lambeth and Seybert et a l ., 1979b), The free ADX may be e ith er oxidized ADX or reduced ADX. The redox state of free ADX depends on the s a lt con centration which affects the association of oxidized ADX with reduced FLP, electron tran sfer from FLP to ADX, and the dissociation of reduced ADX from the complex. That oxidized ADX does not bind as well as reduced ADX to P450 is suggested in the ADX involved shuttle mechanism. At high levels of oxidized ADX, i t can compete with the reduced ADX fo r P45Q oxidase binding. Th.i.s w ill resu lt in in h ib itio n of hydroxylation a c tiv ity (Hanukoglu and Jefcoate, 1980}, Binding of ADX to P450gC (, enhances cholesterol binding and con sequently increases the level o f high spin cholesterol*P450^^C*ADX complex in the isolated mitochondria. I t is hypothesized that the increase in this complex induced by K3PCU in fresh mitochondria results from an increase in the dissociation constant of the ADX*FLP complex which in h ib its endogenous electron donor-supported cholesterol SCC a c tiv ity , and in an enhanced binding of cholesterol to P450scc? the cholesterol pool size is not subject to the K3 P(H e ffe c t. This is supported by the fact that the to ta l pregnenolone formation from endo genous and exogenous reducing equivalent-supported SCC a c tiv ity observed in the absence of KaPOu (buffer B )? or in the presence of 10 m M or 100 97; m M K 3P0i, b u ffer, is no d iffe re n t, even though the P I I reading is much higher in high ^PO* buffer (Fig. 9 ). The e ffe c t o f mitochondrial aging on this membrane-associated P450* cholesterol complex is undoubtedly very complicated. The observed P II increase in the aged mitochondria may be p a r tia lly due to a loss of endogenous energy sources or to a loss of essential ions or cofactors necessary fo r the dehydrogenase to generate reducing equivalents. Such conditions would lead to a conservation o f the P450s^(,-bound choles- * t e r o l. In addition, aging could also a lte r the dissociation constant o f the ADX*FLP complex and increase the a v a ila b ility of free ADX for P450<.££ binding. Consequently, formation of high spin cholesterol* P450$cc*ADX complex would be favored, resulting in an increase of P II (Figs. 13 and 21). The e ffe c t of various cations in reconstituted and P450jj^ enzyme systems on pregnenolone and corticosterone formation, respec tiv e ly , has been reported to give bell-shaped curves (Lambeth and Seybert et a l . , 1979b). The K3P0i* e ffe c t on the SCC a c tiv ity in bovine and ra t mitochondria was examined by determining the in it ia l rate of the high spin to low spin state changes induced by Krebs cycle in te r mediates, as shown in Figs. 17 and 22. In the bovine adrenal cortical mitochondria, a larger s h ift of high spin to low spin is in itia te d by malate. This rate fu rth er increases as the mitochondria are aged, e ith e r at 4° C or room temperature. In bovine mitochondria, the in it ia l rate o f the $pi.n state change was recorded in the f i r s t 10 seconds a fte r addition of electron donor. In the case o f ra t mitochondria, due to the small spin state change induced by is o c itra te , the spin state changes at 98 the end of one minute were used to estimate cholesterol SCC a c tiv ity . This may contribute to the difference in the bell-shaped curves of the two mitochondria, along with possible species differences. Several salts including NaCl, K C 1 and MgCl2 used in the reconstituted system were also studied for th e ir e ffe c t on the i n it ia l rate of the malate- induced low spin spectrum as shown in Fig. 18. Na3 P0i* and MgCl2 also show the bell-shaped curves produced by K3 P0i*. The s a lt effects on the re la tiv e rate constant were obtained by calculating the ra tio of e ith e r the i n it ia l rate of malate-induced low spin s ta te /P II (Fig. 19, 20) or the ra tio of i n i t i a l rate of pregneno lone form ation/PII (Fig. 26, 27) of bovine or ra t mitochondria. Again, in the bovine mitochondria, MgCl2 , Na3 P0it, and K3 P0i, f i r s t increase th e ir rate constants, then reduce that as these s a lt concentrations are increased, resulting in a bell-shaped curve. Aging decreases this re la tiv e rate constant by substantially increasing its P II levels re la tiv e to the i n it ia l rate (Fig. 21). In ra t mitochondria, the rate constant increases and reaches its maximum at 40 m M K3 PO 4 , A fter th is , the rate constant is maintained. The s e n s itiv ity of the rate constant toward K 3 P 0 if in ra t mitochondria is d iffe re n t from that of bovine mitochondria, In the mitochondria of ACTH plus cycloheximide treated adrenals, K 3 PO* is alsp found to in h ib it the endogenous energy-supported cholesterol SCC a c tiv ity . Table I shows that this endogenous electron- supported cholesterol SCC a c tiv ity is less active in this type of mitochondria as compared to the ACTH-treated control mitochondria, Despite this fa c t, the portion of P II which can be induced by high 9.9 K3P O t,. is much higher in cycloheximide-treated mitochondria, as shown in Figs. 11, 13 and 14. A ctu ally, in certain mitochondrial preparations, high K3P04 releases the cycloheximide in h ib itio n of the P II level which is observed at low K3P0i*. A decrease in the cholesterol-bound P450<-££ at low K3P0i* concentrations and a blunted response to increase in K3P0if at the low range were observed in the mitochondria o f cyclo heximide-treated adrenals. Buffer containing about 10 m M K3P0i* has generally been used to study the cycloheximide e ffe c t on P450 binding to cholesterol. Gycloheximide-inhibited cholesterol SCC a c tiv ity is also observed at the low K3P0i, range, where the high spin cholesterol- bound P450S££ complex formation or P II is also suppressed (Simpson et a l . , 1972; Alfano et a l . , 1973; Jefcoate e t a l . , 1974). When the re la tiv e rate constant fo r pregnenolone formation in mitochondria from ACTH or ACTH plus cycloheximide is compared, as shown in Fig. 27, i t is clear that cycloheximide treatment suppresses this rate constant in the low K3PCk range. This rate constant increases rapidly as the K3P04 concentration is increased in the bu ffer. At high K3P0i* le v e ls , the rate constant becomes very s im ilar fo r both ACTH and ACTH plus cyclo- hextmi.de treatments, In other words, high K3P0it does away with the cycloheximide e ffe c t on the ACTH-stimulated formation of cholesterol- bound P450<.C£, in the i n i t i a l rate of pregnenolone formation, and consequently in the apparent rate constant, The s ite of cycloheximide e ffe c t on ACTH stimulated cholesterol SCC a c tiv ity is demonstrated to be on P450<j££ binding of cholesterol to form the high spin complex and also on the c a ta ly tic a c tiv ity o f the P450scc enzyme. Both substrate binding and cholesterol SCC a c tiv ity 1 00 are subject to the K3PCU e ffe c t by regulating the a v a ila b ility of free ADX. By comparing the K 3 P(V e ffe c t on these two parameters, i t was found that cycloheximide in h ib itio n was overcome as the ^POi, concentra tion increas_ed‘to high le v e ls r it^if|nofcibelieved that this K3 P(K e ffe c t results from disruption of the mitochondrial membrane leading to an increase in the active substrate pool (Fig. 9 ), since the mitochondria are normally exposed to these levels o f K3 PO 4 in vivo . In addition, this high s a lt concentration in the buffer does not increase the a b ility of the mitochondrial SCC system to u t iliz e NADPH as an electron donor as would be expected in disrupted mitochondria. A possible explanation fo r this phenomenon is that the increasing s a lt concentration increases the dissociation constant of ADX*FLP, which in turn affects the redo* state of ADX and also the concentration of free ADX. Since the redox state of free ADX determines the choles tero l SCC a c tiv ity and the concentration of free ADX regulates the binding of cholesterol to P45Q<.£C? both of these conditions could over come an e ffe c t of CHI, The scheme on the next page shows the formation of high spin SH«P450*-ADX (SH = cholesterol) and a number of p artial reactions which are believed to occur in the process of oxygen activa tion ('SH*P450Fe3+‘ 02“ ) and cholesterol hydroxyTation (SOH). Under these assumptions and the observation of K3 P0i, release of the cycloheximide in h ib itio n in isolated mitochondria, i t is inferred that free ADX levels in the cycloheximide treated mitochondria are reduced. The ADX levels are restored, reaching that of control mito chondria, i f dissociation of ADX from FLP is promoted by high. K3 P0it, Subsequently, the suppression of the high spin cholesterol*P450SCC*ADX idl NADPH v NADP fLP(red]-ADX(ox) } O X ) ‘ ADX(red) ADXfox; + FL P [red) A D X fred;+ FlP(o x) ADX SH-P450Fe+ 3 -ADXro x ; (HIGH SPIN) SH-P150Fe*3 -ADXfre tW (HIGH SPIN) H2O+SOH + P 450Fe+ 3 1 02 formation by cycloheximide treatment is released and the electron c a rrie r, ADX, can shuttle between FLP and P450< >£ q oxidase e ffic ie n tly . Since mitochondria in vivo are normally exposed to high concentra tions of potassium and phosphate, i t might be asked why cycloheximide treatment is not blocked by these ions in vivo. Two p o s s ib ilitie s can be suggested. In the in ta c t animal, the cycloheximide e ffe c t is con tinuously maintained, while in the isolated mitochondria this is not the case. This difference may allow the isolated mitochondria to recover from the cycloheximide e ffe c t. In addition, the permeability of ions in the mitochondria is highly regulated and the buffer con stituents used here probably do not represent the r n "yjvo environment of mitochondria. Therefore, although these ions in cell cytosol may be high, the tig h tly regulated perm eability of the mitochondria membrane may prevent these ionst f rom-enteringtthe 'imi toehohdri alcc'empartmenttto the same degree as may occur in v it r o , The s p e c ific ity of ACTH and cycloheximide on cholesterol SCC a c tiv ity , as compared to other hydroxylation systems, may r e fle c t the dependence of cholesterol binding to P450<-£q on ADX binding to this oxidase. The other mitochondrial P450 oxidase, 11B hydroxylase, which presumably u tiliz e s the same electron transport system, has not been shown to be dependent on ADX binding to enhance its binding of sub strates. The lack of e ffe c t of these agents on the remaining adrenal hydroxylase located in the smooth endoplasmic reticulum would, of course, be expected since electron transport to the P450 oxidase does not involve ADX or, as fa r as is known, any other activators of substrate binding. 1 l 0 > 3 ' . The dogma which states that ACTH stimulates synthesis of a la b ile protein(s) which accelerates P450<.£q binding of cholesterol may be incorporated into these foregoing hypotheses by assuming that this protein(s) control the association of ADX and FLP. In the absence of this la b ile protein , the dissociation constant of ADX*FLP might be low. 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Isolation and characterization of intestinal alkaline phosphatase

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Studies of carotenoid and vitamin A complexes with protein in plasma and tissues

Asset Metadata

Creator Ts'ao, Kathleen Sye (author)

Core Title The influence of K3O4 on the regulation of cholesterol side chain cleavage by ACTH and cycloheximide

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-607506

Unique identifier UC11353993

Identifier DP21609.pdf (filename),usctheses-c17-607506 (legacy record id)

Legacy Identifier DP21609.pdf

Dmrecord 607506

Document Type Dissertation

Rights Ts'ao, Kathleen Sye

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA