The effect of L-thyroxine on oxidation and phosphorylation in a subcellular particulate fraction of baking yeast

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Content THE EFFECT OF L-THYROXINE ON OXIDATION AND
PHOSPHORYLATION IN A SUBCELLULAR
PARTICULATE FRACTION
OF BAKING YEAST
by
James Milton Hodges
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Nutrition)
August 1959
UMI Number: EP41342
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L O S A N G E L E S 7 . C A L IF O R N IA
This thesis, written by
JAMS..MJLTQJ5LRQD.G.E5.................
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and approved by all its members, has been pre
sented to and accepted by the Graduate School,
in partial fulfillm ent of requirements for the
degree of
MASTER OF SCIENCE
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Date August , . . . . 1 9 5 9 .
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U s e / . . . .
Chairman
TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION AND STATEMENT OF PROBLEM . .
II. HISTORICAL REVIEW.....................
III. MATERIALS ...........................
| Reagents.......... ................
I
! Solutions ........................
IV. EXPERIMENTAL PROCEDURES................
| Preparation of the particulate
i
j
fraction ......................
1
The determination of protein
nitrogen content ..............
The determination of oxygen consump-
i
j
I tion . . . ..................
The determination of inorganic
phosphate uptake ..............
i
i
The calculation of P/0 ratios . . .
V. RESULTS................ ..............
Respiration of the Particulate
Fraction ........................
PAGE
1
3
9
9
10
12
12
14
14
15
16
17
17
CHAPTER
j
I
--•
1X1
PAGE
The effect of disintegration
time ........................ 17
The effect of pH on respiration . . 17
Oxidation of succinate in the
presence of L-thyroxine .... 20
Oxidative Phosphorylation in the
Particulate Fraction ............ 20
The effect of crystalline bovine
plasma albumin .............. 20
The effect of magnesium on
oxidative phosphorylation ... 22
The effect of pH on oxidative
phosphorylation .............. 25
The Influence of L-Thyroxine on the
Particulate Fraction ............ 27
Uncoupling of phosphorylation from
oxidation by L-thyroxine ... 27
The effect of hypotonic pretreat
ment of the particulate fraction
on the uncoupling action of
L-thyroxine .......... 27
The effect of varying osmolarity
of the final reaction medium
on the uncoupling action of
L-thyroxine..........
The effect of pH on the uncoupling
action of L-thyroxine ....
The effect of magnesium on the
action of L-thyroxine . .
The effect of crystalline bovine
plasma albumin on the action
of L-thyroxine .......
Effects of L-Thyroxine on Processes
Involved in Determining P/0
Ratios ............ ........
The effect of L-thyroxine on Phos
phate liberation from glucose-
6-phosphate by the particulate
fraction .............
The effect of L-thyroxine on an
adenosine triphosphatase asso
ciated with the particulate
fraction .................. ,
V
CHAPTER PAGE
The effect of L-thyroxine on the
glucose-hexokinase trap .... 38
✓ . . .
VI. DISCUSSION . . . . . ............ 41
VII. SUMMARY ................... . . . 47
BIBLIOGRAPHY .............. .......... 49
LIST OF TABLES
TABLE PAGE
I. Effects of Bovine Plasma Albumin on
Oxidative Phosphorylation in a
Particulate Fraction of
Baking Yeast .......... 23
II. Effects of Magnesium Concentration on
Oxidative Phosphorylation in a
Particulate Fraction of
Baking Yeast............. .......... 24
III. Uncoupling of Phosphorylation by L-
Thyroxine in a Subcellular
Yeast Fraction........... .... 28
IV. Effect of Hypotonic Pretreatment of the
Particulate Fraction on the
Uncoupling Action of L-Thyroxine ... 29
V. Effect of Varying Osmolarity of Final
Reaction Medium on the Uncoupling
Action of L-Thyroxine ........ 31
TABLE PAGE
VI. The Effect of Magnesium on the Action
of L-Thyroxine................. 34
VII. The Effect of Crystalline Bovine Plasma
Albumin on the Action of L-Thyroxine . 35
VIII. The Effect of L-Thyroxine on Phosphate
Liberation from Glucose-6-Phosphate
by the Particulate Fraction ........ 37
IX. The Effect of L-Thyroxine on an Adenosine
Triphosphatase Associated with the
Particulate Fraction . ............ 39
X. The Effect of L-Thyroxine on the
Glucose-Hexokinase Trap ........... 40
LIST OF FIGURES
FIGURE PAGE
1. The Effect of Disintegration Time on
Oxidation in a Particulate Fraction
of Baking Yeast . .................. 18
2. The Effect of pH on Respiration in a
Particulate Fraction of Baking Yeast . 19
3. The Respiration of Non Phosphorylating
Particles in the Presence of
L-Thyroxine ........... 21
4. The Effect of pH on Oxidative
Phosphorylation .................. 26
5. The Effect of pH on the Uncoupling
Action of L-Thyroxine .............. 32
CHAPTER I
I
INTRODUCTION AND STATEMENT OF PROBLEM
The effect of the thyroid hormone in increasing
over-all metabolism rates of mammals is well known (1).
The biochemical mechanism whereby thyroxine exerts this j
effect on the rate of oxidation has been the target of
considerable speculation and research.
Activity of this hormone is not restricted to
o
*
mammalian organisms. The stimulation of metamorphosis of
tadpoles has been used to test for activity of thyroxine
analogues (2). More recently stimulatory effects on the
respiration of a bacterium, Aerobacter aerogenes (3), and
a fungus, Saccharomyces cerevisiae (4), have been j
demonstrated. j
The yeast was found to be remarkably sensitive to a
low concentration of L-thyroxine. This finding combined
with certain practical considerations such as ease of
obtaining material and working with a large number of
organisms confers certain advantages on this microorganism
as an experimental tool. Accordingly it was determined to
2
expand the investigation of the action of L-thyroxine on
I
Saccharomyces cerevisiae in an attempt to gain further
knowledge about the mechanism of action of the thyroid
hormone.
The present study is concerned with the effect of
L-thyroxine on oxidation and phosphorylation in a subcel-
lular particulate fraction of baking yeast (5,6,7).
CHAPTER II
HISTORICAL REVIEW
Hoch and Lipmann have hypothesized that the
thyroid hormone exerts its physiological effects by sup-
i
pressing oxidative phosphorylation in mitochondria thereby j
reducing the efficiency of catabolism (8). Early efforts
i
to demonstrate a direct action of the hormone on this
energy conserving process were not successful (9).
i
It was not until 1951 that evidence for the direct I
, j
action of thyroxine in reducing the efficiency of phosphate|
i
esterification was announced by Martius and Hess (10). ;
" i
These investigators isolated liver mitochondria from rats j
I
made hyperthyroid with 4 to 12 milligrams of thyroxine j
injected over a period of 24 to 72 hours. The uptake of j
qo
PJ labelled inorganic phosphate from the reaction medium j
was greatly depressed in the vessels containing mito
chondria from the thyroxine treated rats, as compared to
the control. Thyroxine added in vitro at concentrations
of 2 X TO'5 M and 5 X 10"^ M to normal mitochondria again
caused a marked suppression of inorganic phosphate j
4
disappearance. It was necessary to preincubate the hormone
with mitochondria in the absence of substrate to obtain an
effect with thyroxine added in vitro. These authors did
not mention oxygen uptake so P/0 ratios could not be
calculated.
It was soon confirmed by Maley and Lardy (11) that
i
-5 1
thyroxine at 1 X 10 M decreased P/0 ratios using rat ,
■ .. . |
kidney mitochondria and glutamate blocked with malonate as
substrate. An inhibition of the oxidation of glutamate
was observed in these experiments. No effect on the metabo
lism of other Krebs cycle intermediates could be demon
strated. i
i
|
In an effort to avoid permeability difficulties j
Hoch and Lippmann (8) used hamster liver mitochondria with j
glutamate as substrate. No preincubation of mitochondria j
i
t
and hormone was necessary to demonstrate a progressive !
I
lowering of the P/0 ratio, from a control value of 2.72 to
essentially zero, as the thyroxine concentration was
increased from 5 X 10 ^ M to 1 X 10' ^ M. Little effect
was noted on respiration. The presence of glucose and
i
hexokinase caused a marked increase in the respiration of j
I
[
normal rat liver mitochondria. This was considered a
mass action stimulation caused by removal of adenosine
5
triphosphate by the acceptor system. The P/0 ratios as
well as the respiration rate of mitochondria from rats
made hyperthyroid by the regime of Martius and Hess was
equal to that of normal mitochondria; however the stimu
latory effect by the acceptor system was abolished. The
authors concluded this to be an even more sensitive indi-
cation of thyroxine action than a reduction in P/0 ratios.
This is in accord with findings by Niemeyer et_ al. (9).
Differences in the affinity of hamster and rat
^ . ' i
I
liver mitochondria for thyroxine were confirmed by incu
bating 1 X 10“^ M 1^31 labelled thyroxine with each (8).
Only 45 per cent of the radioactivity was recovered with .
i
the rat mitochondria. The hamster mitochondria contained
84 per cent of the activity. It was emphasized that this j
i
procedure can not be expected to give the concentration 1
i
of the hormone within the mitochondria or at the active i
sites of phosphorylation.
Klemperer (12) showed thyroxine to be rapidly taken
up by rat liver mitochondria at G°C concomitantly with some
swelling. No increase in rate or magnitude of uptake was !
noted at 20°C. j
i
Since this early work a number of reports of
uncoupling by thyroxine have appeared (13,14,15) including
one by Brodie and Gray (16) using a bacterial extract and
by Tapley, Cooper and Lehninger (17) who obtained uncoupling
in rat liver mitochondria by pretreating with hypotonic
sucrose rather than a preincubation with the hormone.
It has been established that the substrate phos
phorylation occurring when alpha-keto-glutarate is
oxidized is not influenced by concentrations of thyroxine \
which uncouple respiratory chain phosphorylation (8).
Attempts to locate the exact site of action and determine ;
the mechanism whereby thyroxine inhibits oxidative phos
phorylation have met with little success. This is largely
I
due to the rather stringent requirements of an organized
j I
I structure to maintain the enzymes of the respiratory chain
in a configuration necessary for activity.
I
Recently Lehninger's group have fragmented rat ;
1
liver mitochondria with digitonin and obtained submito- I
condrial particles capable of phosphate esterification
coupled to the oxidation of beta-hydroxy-butyrate (18). |
Thyroxine had no action on these mitochondrial fragments. !
I
j
After observing that thyroxine accelerates the swelling |
of resting mitochondria suspended in isotonic and hypotonic ,
i
sucrose at concentrations of the hormone lower than that
affecting phosphorylation (19) it was proposed the primary
7
action of thyroxine is on the structure of mitochondria.
iDepression of P/0 ratios would be secondary to structural
damage. It has been noted that mitochondria from hyper-
thyroid tissues appear swollen (20).
However, Bronk (21) and Park, Meriwether, and Park
(22) have fragmented mitochondria in sonic disintegrators
and obtained preparations which are capable of oxidative
phosphorylation. In both of these systems added thyroxine
exerts an uncoupling action comparable to that seen in
intact mitochondria.
Bain (23) has demonstrated added magnesium to
reverse the uncoupling action of thyroxine when the Mjg to
thyroxine ratio is approximately 50 to one. Mudd et al.
(24) have confirmed this and Vitale et al. (25,26) have
shown diets high in magnesium to reverse the effect of
ingested thyroxine.
Recently Fairhurst et al,. (27) have demonstrated
uncoupling in rat liver mitochondria by physiological
concentrations of thyroxine. Thyroidectomized rats were
injected with 3 to 100 micrograms of L-thyroxine daily
for 14 days. With alpha-keto-glutarate as substrate,
control P/0 ratios of approximately 2.5 were reduced to
1 in an unwashed particulate preparation from the livers
■ ' ' v '8 ■
of rats given as little as 30 micrograms thyroxine daily.
No reduction in p/0 ratios was observed when the particulate
fraction was washed with sucrose.
i
CHAPTER III
MATERIALS
Reagents
The following materials were generously supplied
by the company indicated:
1. L-Thyroxine: Baxter Laboratories, Inc., and
r ' * •
Hoffman La Roche and Company, Inc.
i
2. Glass Beads, average diameter 0.02 mm.: The
Minnesota Mining and Manufacturing Company.
The following materials were purchased from
the source indicated:
1. Ethylenediaminetetracetic acid: Alrose
t
Chemical Company.
2. Adenosine 51 monophosphate: Schwartz
Laboratories.
%
3. Adenosine 5* diphosphate: Sigma Chemical
Company and Pabst Laboratories.
4. Adenosine 5‘ triphosphate: Schwartz
Laboratories and Pabst Laboratories.
10
5. Diphosphopyridine nucleotide: Pabst Labora
tories and California Corporation for
Biochemical Research.
6. Cytochrome c (horse heart): Sigma Chemical
Company and Nutritional Biochemicals
Corporation.
7. Coenzyme A: Sigma Chemical Company.
\:
8. Hexokinase, type III: Sigma Chemical Company.
9. Crystalline bovine plasma albumin: Armour
and Company.
10. Glucose-6-phosphate: Sigma Chemical Company.
11. Tris (hydroxymethyl) aminomethane, grade Sigma
7-9: Sigma Chemical Company.
12. Baking Yeast was a commercial preparation
(Fleischmann1s Active Dry Yeast) purchased in
local grocery stores.
13. All other chemicals used were of reagent grade.
Solutions
1. Solution A contained 0.5 M sucrose, 0.005 M
ethylenediaminetetracetic acid, and 0.1 M
potassium phosphate, pH 7.4.
2. Solution B contained 0.5 M sucrose, 0.002
. . . . . . . . . . . _ _ 1;L
ethylenediaminetetracetic acid, and 0.05 M
potassium phosphate, pH 7.4.
3. Solution G contained 0.5 M sucrose, and 0.02 M
potassium phosphate, pH 7.4.
CHAPTER IV
EXPERIMENTAL PROCEDURES
Preparation of the Particulate
Fraction
A particulate fraction having many of the proper
ties of mitochondria was prepared from yeast essentially
by the method of Linnane and Still (5).
Baking yeast (Fleischmann's Active Dry Yeast) was
suspended in approximately 100 ml. of 0.9 per cent KC1 at
room temperature and washed three times by centrifugation
in fresh 100 ml. portions of the same solution. One
package of yeast provided a convenient amount of cells
for each experiment. Upon decanting the wash solution
after the third centrifugation the yeast was weighed and
suspended in solution A so that each ten ml. contained
four grams wet weight cells. Ten ml. of this suspension
I
was placed in a stainless steel capsule and an equal
volume of glass beads, average diameter 0.02 mm., added
leaying an air space of two or three ml. The capsule
and its contents were brought to 0°C in an ice bath and
then securely locked in the shaking arm of a Nossal shaker
(7) which had been previously cooled with dry ice. The
shaker was ordinarily operated for thirty seconds, which
Iwas sufficient to disintegrate approximately one third of
\
the cells. Heating, which inactivated the preparation,
occurred with shaking periods appreciably longer than
thirty seconds. The capsule was removed from the shaker
and placed in an ice bath for a few minutes while the
beads were allowed to settle. The yeast suspension was
then decanted to a polyethylene centrifuge tube. The beads
and capsule were washed twice with ten ml. of cold solution
B. These washings were combined with the disintegrate and
kept in ah ice bath until sufficient yeast for the experi
ment had been processed. The suspension was placed in a
refrigerated centrifuge maintained at 0°C and sedimented
at 3,000 X G for twenty minutes to remove unbroken cells
and larger fragments. The supernatant was carefully
decanted and centrifuged at 25,000 X G for twenty minutes.
i
A pellet of light brown material was obtained which was
<
washed by suspending in approximately twenty ml. of cold
solution B, and re-qentrifuging at 25,000 X G for twenty
minutes. The packed particles were kept at 0°C under the
14
supernatant until just before use, when the latter was
decanted and a volume of suspension medium was added suf
ficient to provide 0.4 ml. for each flask.
The particles were usually used within two to three
hours after disintegration of the cells; however their
respiration remained essentially unchanged after storage
at 0°C for twenty-four hours in solution B.
The Determination of Protein
Nitrogen Content
Protein was precipitated in an aliquot of each
particle preparation with trichloroacetic acid. The
protein was digested in a sulfuric-phosphoric acid mixture
containing copper as a catalyst and the nitrogen content
was measured after nesslerization, in a Coleman Spectro
photometer Model 6A at 5,00 nju (28).
The Determination of Oxygen Consumption
Oxygen uptake was measured by conventional Warburg
technics using double side arm flasks of approximately
twenty ml. capacity with 0.1 ml. 30 per cent KOH and a
fluted filter paper in the center well to absorb CO2.
Unless stated otherwise the chilled particles were added
15
directly to the complete medium at room temperature. After
;equilibration for ten minutes the manometer stopcocks were
closed and oxygen consumption measured for twenty minutes
i
i
at 31°C. Total oxygen uptake for thirty minutes was
determined by extrapolation. The reaction was terminated
and protein precipitated by tipping in 0.2 ml. of 50 per j
i
cent trichloroacetic acid from the side arm. Unless stated
otherwise endogenous respiration was measured by including
duplicate flasks except that substrate was omitted. Oxygen,
j
uptake was corrected for this activity. j
I
The Determination of Inorganic !
i '
Phosphate Uptake I
Adenosine triphosphate formed during oxidative j
phosphorylation from adenosine diphosphate and inorganic
Sphosphate was trapped with glucose and hexokinase. This
reaction transfers the labile terminal phosphate of
adenosine triphosphate to glucose, forming stable glucose- |
i
i
6-phosphate and regenerating adenosine diphosphate. \
i
i
Phosphorylation was computed from the decrease of • ;
inorganic phosphate in the substrate containing flask as '
i
I
compared to the control flask used to measure endogenous
respiration. This eliminated the small correction neces- j
“ “ 16
sary for endogenous phosphorylation when the decrease was
measured from a "0 time1 1 flask.
After tipping in trichloroacetic acid to stop the
reaction and precipitate protein the contents of each
flask were diluted to ten ml. with distilled water, mixed
well and centrifuged. Inorganic phosphate was determined
on a suitable aliquot of the protein free supernatant by
the method of Taussky and Shorr (29).
!
The Calculation of P/0 Ratios
P/0 ratios were calculated by dividing microatoms
of oxygen consumed into micromoles of inorganic phosphate
iesterified in thirty minutes. This corresponds to the
I
i
P/2e ratio used by some workers.
,
j
I
I
CHAPTER V
RESULTS
I. RESPIRATION OF THE PARTICULATE FRACTION
i
The Effect of Disintegration Time
j
The subcellular particulate fraction of disinte
grated yeast which sedimented between 3,000 X G and 25,000 j
X G oxidized succinate rapidly and did not require the
addition of cofactors. There was an almost linear relation
ship between disintegration time, up to 25 seconds, and
. . . • , i
oxidizing capacity, with no appreciable diminution of
!
specific activity, as shown by the increase in slope when I
total oxygen consumption of the particles recovered was
i
i plotted against disintegration time in Figure 1. Although '
an acceptor system was present no phosphate esterification
I
was observed during this experiment, in which no bovine
I
plasma albumin was included. j
The Effect of pH on Respiration ]
Figure 2 shows a relatively sharp optimum around !
j
pH 6.6 for the oxidation of succinate. No attempt was }
18
w
20
TO 15 20
DISINTEGRATION TIME (SECONDS)
FIGURE 1
The Effect of Disintegration Time on Oxidation in
A Particulate Fraction of Baking Yeast
Experimental conditions: Cells were disintegrated for times
indicated and the particles suspended in equal volumes of
solution C. Each flask contained particles derived from 2
grams yeast, 5 mM "Tris" and 16 mM potassium phosphate buf
fer, 350'mM sucrose, 12.5 mM succinate, 6.25 mM M&C12, 5 mM
ATP, 1.5 mM AMP, 0.2 mM DPN, 0.01 mM cytochrome c, 25 mM
glucose and 140 K. M. units hexokinase. Total volume 2.0
ml. pH 6.6. The glucose and hexokinase were tipped in from
the side arm after 10 minutes equilibration and oxygen
uptake measured for 60 minutes at 31°C. No phosphorylation
was observed. Oxygen consumption was not corrected for
endogenous respiration.
19
o r f
W
I
8
0 £
H
S
M
W
H
O
-B
e
3
£D
SZ
W
P
►J
400
300
200
100
pH
FIGURE 2
The Effect of pH on Respiration in a Particulate
Fraction of Baking Yeast
Experimental conditions: Each flask contained particles
corresponding to 0.16 mg protein nitrogen suspended in solu
tion C, 17.3 mM "Tris" and 50 mM potassium phosphate buffer.
275 mM sucrose, 15 mM succinate, 0.3 mM AMP and 0.3 mM DPN.
Total volume 2.0. pH as indicated. The succinate was
tipped in from the side arm after 15 minutes equilibration
and oxygen uptake measured for 60 minutes at 31°C. Oxygen
uptake was not corrected for endogenous activity.
20
made in this experiment to demonstrate phosphorylation.
i
I
1
Oxidation of Succinate in the
Presence of L-Thyroxine
Many experiments were performed under a variety of
conditions in attempts to determine if L-thyroxine altered
the metabolism of succinate by the particles. Figure 3
shows an average of the results of three typical experi- !
ments conducted under identical conditions. It is apparent
i
there was no significant change in the rate of succinate j
oxidation or in endogenous respiration; furthermore, in j
i
|
the absence of phosphorylation, no significant effect on
i ,
respiration was observed under any of the conditions
studied. |
i
II. OXIDATIVE PHOSPHORYLATION IN THE !
PARTICULATE FRACTION i
i
The Effect of Crystalline Bovine j
Plasma Albumin i
t
i
Attempts to demonstrate an uptake of inorganic |
i
i
phosphate simultaneously with the oxidation of succinate j
1
were not successful when the particles were equilibrated
at 31°C without the acceptor system. When the particles
21
o 40
20 30 40
RESPIRATION TIME (MINUTES)
FIGURE 3
The Respiration of Non Fhosphorylating Particles
in the Presence of L-Thyroxine
Experimental conditions: As described in Figure 2 except
particles corresponding to 0.5 mg protein nitrogen, and pH
7.4 were used. Average of 3 experiments.
T
A
*
O
Succinate, Thyroxine 1 X 1 0 M
Succinate
Endogenous, Thyroxine 1 X 10
Endogenous
-4
M
22
were added to the complete system oxidative phosphorylation
i
was observed. Including a small amount of crystalline
bovine plasma albumin in the medium greatly enhanced phos
phorylation, as shown in Table I. The albumin became an
absolute requirement for phosphorylation when the particles
were exposed to hypotonic solution prior to use (Table
|
VII). In both instances the respiration rate was decreased
by the lowest concentration of albumin. As more albumin
was added to the particles treated with hypotonic solution !
a rate was attained approximately 50 per cent in excess
of that observed in the absence of albumin. The specific j
mechanism whereby bovine plasma albumin exerted this effect,
on oxidative phosphorylation is unknown.
i
I
The Effect of Magnesium on
Oxidative Phosphorylation
Magnesium was an essential cofactor for both the ■
i
glucose-hexokinase trap and oxidative phosphorylation. The
effects of magnesium are illustrated in Table II and VI. i
The only essential difference in experimental conditions
i
i
was exposure of the particles to a hypotonic medium prior
to use during the experiments shown in Table VI. This
hypotonic pretreatment apparently made added magnesium
TABLE I
EFFECTS OF BOVINE PLASMA ALBUMIN ON OXIDATIVE
PHOSPHORYLATION IN A PARTICULATE
FRACTION OF BAKING YEAST
Albumin
cone.
mg per flask
Inorganic
phosphate
uptake
Oxygen uptake P/0 Ratio
0.00
ju moles
5.4
M atoms
7.7 0.70
0.54 4.8 5.8 0.83
1.24 6.0 6.4 0.94
2.25 6.3 6.9 0.91
3.25 7.7 7.4 1.04
5.30 7.2 7.2 1.00 .
Experimental conditions: Each flask contained particles
corresponding to 0.4 mg protein nitrogen suspended in
solution C, 6.24 mM uTrisH and 16 mM potassium phosphate
buffer, 100 mM sucrose, 12.5 mM succinate, 6.25 mM MgCl2,
12.5 mMKF, 25 mM glucose, 0.25 mM DPN, 1.5 mM ADP* 140 K.
M. units hexokinase and crystalline bovine plasma albumin
as indicated. Total volume 2.0 ml. pH 7.2. Reaction
time 30 minutes.
V
TABLE II
EFFECTS OF MAGNESIUM CONCENTRATION ON OXIDATIVE
PHOSPHORYLATION IN A PARTICULATE
FRACTION OF BAKING YEAST
MgCl2
conc.
mM
Inorganic
phosphate
uptake
Oxygen uptake P/0 Ratio
0.0
ju moles
1.1
M atoms
3.9 0.28
1.0
2.7 4.3 0.63
2.0 2.6 4*0 0.65
4.0 2.9 3.9 0.74
12.5 ‘ 2.9 3.5 0.83
Experimental conditions: , As described for Table I,
except 3 mg albumin, MgCl2 as indicated, and particles
corresponding to 0.25 mg protein nitrogen were used.
25
more essential for phosphorylation, probably by allowing
bound magnesium to be liberated from the particles.
No significant effect by magnesium was noted on
oxygen uptake when the particles were not given the hypo
tonic pretreatment; however in the experiment using pre
treated particles a decrease in respiration was noted at
the lower concentrations. As more magnesium was added the
rate approached that attained in the absence of magnesium.
Very low P/0 ratios were obtained in the absence of mag
nesium. The decrease in respiration rate by small amounts
of magnesium may be due to its ability to stimulate phos
phorylation which, in turn, would cause a decrease in
respiration.
The Effect of pH on Oxidative
j Phosphorylation
Figure 4 indicates oxidative phosphorylation to
be most effective at a neutral pH with a relatively broad
optimum.
26
0.9
o
M
§
o
* 0.5
0.3
578 7.0 7.4
FIGURE 4
The Effect of pH on Oxidative Phosphorylation
Experimental conditions: 0.4 ml. of particle suspension,
corresponding to approximately 0.2 mg. protein nitrogen, in
0.05 M sucrose and 0.02 M potassium phosphate pH 7.4, was
added to the reaction medium. Each flask contained (final
composition): 6.5 mM "Tris” and 10 mM potassium phosphate
buffers, 65 mM sucrose, 12.5 mM succinate, 2 mM MgCl2, 1.5
mM ADP, 0.01 mM cytochrome c, 25 mM glucose, 3 mg. crystal
line bovine plasma albumin, and 130 k. M. units hexokinase.
Total volume 2.0 ml. Final pH as indicated. Bath tempera
ture 31°C. Reaction time 30 minutes.
(
27
III. THE INFLUENCE OF L-THYROXINE ON THE
PARTICULATE FRACTION
Uncoupling of Phosphorylation from
Oxidation by L-Thyroxine
Table III shows the results of experiments in
]
which the efficiency of phosphorylation coupled to the 1
oxidation of succinate was observed both in the presence
of and in the absence of L-thyroxine. Under the conditionsj
of these experiments L-thyroxine significantly depressed i
t
phosphate uptake with no appreciable effect on the respira-
j
tion rate. I
I
t
The Effect of Hypotonic Pretreatment of j
I
the Particulate Fraction on the '
i
Uncoupling Action of L-Thyroxine
i
Exposure to hypotonic sucrose media buffered with ;•
potassium phosphate was found to greatly enhance the uncou-
\
pling action of the hormone. The result of experiments
to determine the sucrose concentration giving the greatest i
i
effect by the hormone is presented in Table IV. An uncou
pling value of 80 per cent was obtained with 0.05 M
sucrose. It was further observed that as the sucrose
28
TABLE III
UNCOUPLING OF PHOSPHORYLATION BY L-THYROXINE IN
A SUBCELLULAR YEAST FRACTION
Thyrox
ine
cone.
No. of Ex
periments
Mean
oxygen
uptake
Mean
phosphate
uptake
Mean P/O
ratio and
range or
std. error
Uncou
pling
M X 10" 6 j a atoms ju moles Per cent
0 (3) 3.9 2.5 0.64
(0.56-0.71)
i
5 (3) 4.1 2.3 0.56
(0.46-0.63)
13
10
(3)
4.0 2.1 0.52
(0.46-0.58)
19
t
50 (3) 4.5 1.7 0.38
(0.31-0.41)
41 :
i
0 (12) 5.1 3.6 0.71-0.031*
I
i
1
100 (12) 4.9 1.0 i0.20*0.023* 72 j
*p for difference: ^ 0.001.
Experimental conditions: As described in Figure 4 except
4 mM potassium phosphate and final pH 7.4 L-Thyroxine,
where used, was dissolved in 0.008 M KOH and an equivalent
amount of KOH added to the control flask.
29
TABLE IV
EFFECT OF HYPOTONIC PRETREATMENT OF THE PARTICULATE
FRACTION ON THE UNCOUPLING ACTION OF L-THYROXINE
Sucrose cone.
during pre-
treatment
mM
P/0 Ratios
No I X 10’* M
thyroxine thyroxine
Per cent
uncoupling
25 0.52 0.34 35
50 0.50 0.10 80
75 0.58 0.35 40
100 0.61 0.33 46
150 0.74 0.34 54
200 0.78 0.41 47
275 0.79 0.58 27
Experimental conditions: As described in Table III
except 10 mM potassium phosphate and the particles were
suspended in sucrose concentration as indicated.
30
concentration was decreased the control P/0 ratios also
j diminished somewhat.
I
The Effect of Varying Osmolarity of the
Final Reaction Medium on the Uncoupling
Action of L-Thyroxine
Experiments conducted to determine the effect of
sucrose concentrations in the final reaction medium are
I
j shown in Table V. As the sucrose concentration was
i ■ • .
decreased below 0.09 M there was a slight drop in the j
control P/O ratio with no increase in the uncoupling actionj
of the hormone. !
| ‘ !
!The Effect of pH on the Uncoupling i
• ' ■
Action of L-Thyroxine j
I
The relationship of pH to the uncoupling action of |
the hormone is presented in Figure 5. There was a sharp
optimum for uncoupling between pH 7.4 and 7.6 with a
precipitous drop on each side. The decrease in effect
on the acid side may possibly be influenced by the solu
bility of thyroxine. After addition of the hormone to the
i
reaction medium turbidity was observed at lower pH values, i
This was considered to be a part of the hormone precipi
tating.
TABLE V
EFFECT OF VARYING OSMOLARITY OF FINAL REACTION
MEDIUM ON THE UNCOUPLING ACTION
OF L-THYROXINE
Sucrose cone.
in reaction
medium
mM
P/O Ratios
No IX 10 M
Thyroxine Thyroxine
Per cent
uncoupling
15 0.75 0.26 65
53 0.80 0.32 60
90 0.89 0.32 64
140 0.96 0.49 49
265 0.97 0.64 34
Experimental conditions: As described in Table III except j
0.075 M sucrose in pretreatment solution,) and sucrose j
concentration in final reaction medium as indicated.
32
80
60
40
20
6.7 6.9 7.3 7.9 7.1 7.5 7.7
pH
FIGURE 5
The Effect of pH on the Uncoupling Action of
L-Thyroxine
Experimental conditions: As described in Figure 4.
Thyroxine concentration 1 X 1 0 M.
The Effect of Magnesium on the
Action of L-Thyroxine
Magnesium was a necessary cofactor for both oxida
tive phosphorylation and the glucose-hexokinase trap.
Magnesium has also been demonstrated to interfere with the
uncoupling action as well as with the thyroxine induced
stimulation of respiration observed in experiments having
i
a relatively low control P/0 ratio. The results of these
experiments are presented in Table VI.
i
The Effect of Crystalline Bovine Plasma
i
j Albumin on the Action of L-Thyroxine !
i
Albumin enhanced oxidative phosphorylation and ;
became an absolute requirement when the particles were
I
exposed to hypotonic medium. As in the case of magnesium, '
ithe experiments given in Table VII show thyroxine to
I stimulate respiration, when a low control P/0 ratio was
obtained by limiting a required cofactor. This effect ,
was abolished by adding sufficient albumin to obtain a
' ‘ i
i
P/0 ratio great enough so that an appreciable rate of j
\
phosphorylation existed in the thyroxine treated particles. j
Larger amounts of albumin reduced the uncoupling action, !
possibly by binding of the hormone.
34
TABLE VI
THE EFFECT OF MAGNESIUM ON THE
ACTION OF L-THYROXINE
MgCl2
cone:
mM A* B**
*~X100
A
Q i c k r k Q****
Per cent
uncoupling
0.0 7.8 12.0
Experiment 1
55 0.061 0.032 48
2.0 4.5 6.8 50 0.55 0.07 87
6.3 5.9 5.4 -8 0.64 0.55 14
12.5 6.7 5.7 -15 0.83 0.86 +3
Experiment 2
0.0 6.2 11.4 83 0.048 0.026 46
1.0 4.5 6.8 51 0.48 0.14 70
2.0 5.9 6.1 5 0.56 0.15 73
4.0 5.8 3.7 -35 0.82 0.63 23
A: ju atoms oxygen consumed (Succinate)
**B: j j l atoms oxygen consumed (Succinate, Hormone)
***C: P/O Ratio (Succinate)
P/0 Ratio (Succinate, Hormone)
Experimental conditions: As described in Table III except,
10 mM potassium phosphate and MgCl2 concentration as
indicated.
35
TABLE VII
THE EFFECT OF CRYSTALLINE BOVINE PLASMA ALBUMIN
ON THE ACTION OF L-THYROXINE
Albumin
cone.
mg/flask A* B**
^XIOO
A Per cent
uncoupling
Experiment 1
i
i
0.0 4.1 9.0 118
c
0.085 0.017 80 j
j
3.0 5.1 5.0 -3 0.77 0.29
1
62
6.0 6.0 5.7 -5 0.87 0.64 26
i
12.0 J 6.7 6.9 +3 0.87 0.78
I
10
»
1
Experiment 2
1
0.0 4.9 8.8 82 0.052 0.017 67
i
0.14 3.5 9.4 172 0.116 0.016 86
0.25 2.8 9.0 218 0.159 0.044
72
i
0.60 2.9 8.5 193 0.225 0.035
- 84 1
*A: ja atoms oxygen consumed (Succinate)
**B: ju atoms oxygen consumed.(Succinate, Hormone) j
***C: P/0 Ratio (Succinate) i
P/0 Ratio (Succinate, Hormone) !
• |
i
Experimental conditions: As described in Table III except
10 mM potassium phosphate and crystalline.bovine plasma !
albumin concentration as indicated. j
36
IV. EFFECTS OF L-THYROXINE ON PROCESSES INVOLVED
IN DETERMINING P/O RATIOS
When demonstrating an uncoupling action of an agent
by measuring P/0 ratios calculated)from disappearance of
inorganic phosphate it is necessary to demonstrate that
the agent does not inhibit or accelerate a reaction which
may mimic uncoupling by causing an increase of inorganic
phosphate in the test flask. Accordingly experiments
were conducted to determine if the uncoupling action of
L-thyroxine could be attributed to such a process.
The Effect of L-Thvroxine on Phosphate
Liberation from Glucose-6-Phosphate by
the Particulate Fraction
As shown by the experiment given in Table VIII
there was a small amount of inorganic phosphate liberated
from glucose-6-phosphate. L-thyroxine did not significantly
affect this reaction.
i
TABLE VIII
THE EFFECT OF L-THYROXINE ON PHOSPHATE LIBERATION
FROM GLUCOSE-6-PHOSPHATE BY THE
PARTICULATE FRACTION
Phosphate Phosphate Phosphate liber Change due
zero time after incuba ated from glu- to thyrox
control tion cose-6-phosphate ine
A* B** A* B**
ju moles ju moles m moles M moles ;u moles M moles
8.9 9.3 9.4 0.4 0.5 +0.1
*A: No thyroxine
**B: 1 X 10"4 M Thyroxine
jExperimental conditions: As described in Table III except
115 mM "Tris" and the incubation was carried out in test
tubes with 10 mM potassium phosphate. Succinate, glucose,
hexokinase and ADP were omitted and 5 mM glucose-6-phos- •
phate was included. Particles were added to the zero time
control after adding TCA.
38
The Effect of L-Thyroxine or> an Adenosine
Triphosphatase Associated with the
Particulate Fraction
There was a potent adenosine triphosphatase asso
ciated with the particles. As shown by the experiment
given in Table IX, L-thyroxine had no significant effect
on the adenosine triphosphatase activity.
The Effect of L-Thyroxine on the
Glucose-Hexokinase Trap
Under the conditions of the experiment conducted
as described in Table X there was no significant effect
by L-thyroxine on the glucose-hexokinase reaction used
to trap the high energy phosphate generated by oxidative
phosphorylation.
39
TABLE IX
THE EFFECT OF L-THYROXINE ON AN ADENOSINE TRI
PHOSPHATASE ASSOCIATED WITH THE
PARTICULATE FRACTION
Phosphate
zero time
control
Phosphate
after incubation
A* B**
ATP
hydrolyzed
A* B**
Change due
to thyrox
ine
f j moles ju moles j a moles ju moles jj moles M moles
10.4 16.4 16.3 6.0 5.9 -0.1
10.6 16.6 16.5 6.0 5.9
i
o
•
11.0 14.6 14.0 3.6 3.0 -0.6
*A: No thyroxine
**B: 1 X 10"4 M thyroxine
Experimental conditions: As described in Table VII except
glucose-6-phosphate was omitted and 4 -mM ATP was included.
Increase in inorganic phosphate is expressed as ATP
hydrolyzed.
~4'0
TABLE X
THE EFFECT OF L-THYROXINE ON THE GLUCOSE-
HEXOKINASE TRAP
Phosphate Phosphate Glucose-6- Change due
zero time after incubation phosphate formed to thyroxine'
control A* B** A* B**
ju moles ju moles ju moles ju moles ju moles ju moles
16.4 12.7 12.5 3.7 3.9 +0.2
15.8 12.5 12.3 3.3 3.5 +0.2
16.0 12.8 12.4 3.2 3.6 +0.4
*A: No thyroxine .
**B: 1 X 1 0 M Thyroxine j
Experimental conditions: 6.5 K. M. units of hexokinase was!
incubated at 31°C, pH 7.4, for 30 minutes with 2 ml. medium
containing 11.3 mM "Tris" buffer, 65 mM sucrose, 2 mM j
MgCl2 4 tnM ATP, 12.5 mM succinate, 25 mM glucose, 0.01
i mM cytochrome c, 3 mg crystalline bovine plasma albumin.
0.1 mM L-Thyroxine, where used, was dissolved in 0.008
M KOH and an equivalent amount of KOH added to the control ;
tube. The zero time control was made 1 N with HCL before
adding hexokinase. The reaction was stopped by making 1 i
N with HCL and phosphate was determined on a suitable I
aliquot after hydrolysis at 100°C for 7 minutes. Glucose- |
6-phosphate formed is expressed as the decrease in non
labile phosphate. i
CHAPTER VI
DISCUSSION
The subcellular particles isolated from yeast
oxidize succinate and esterify inorganic phosphate with
maximal efficiency at a pH of approximately 6.9. In
accord with Utter et al. P/0 ratios up to 1 were observed
(6). This is in contrast to the theoretical maximum of
2 expected for the oxidation of succinate to fumarate.
It was suggested that this suboptimal activity may be due
to damage to the particles during isolation. Another pos
sibility is a loss of high energy phosphate due to
adenosine triphosphatase. This activity could be suppres
sed by fluoride but use of this inhibitor resulted in an
over-all reduction of P/0 ratios.
Thyroxine is known to be a potent uncoupler of
oxidative phosphorylation in animal mitochondria whether
added in vitro or in vivo. Early difficulties in demon
strating this effect have been attributed to the relative
impermeability to the hormone of mitochondria from the
organs of certain species. This problem has been resolved
42
by preincubating mitochondria and hormone or by exposing
the particles to hypotonic solution for a short time.
In the present study the greatest P/0 ratios were
obtained when the reaction was allowed to proceed in a
hypertonic sucrose medium. The maximal uncoupling action
of thyroxine, on the other hand, was manifest when the j
i
i
particles were exposed to hypotonic solution prior to use,
and when the final reaction medium was hypotonic. Reducing
the concentration of sucrose in the final reaction medium j
i
from 265 mM to 65 mM had little effect on phosphorylation.
Control P/0 ratios were lowered from 0.8 to 0.5 by reducing
i
the sucrose concentration of the pretreatment solution j
I
from 275 mM to 50 mM.
Oxidation of succinate was optimal at pH 6.6 but I
the highest P/0 ratios were obtained between pH 6.6 and
i
7.2. A pH of 7.5 was found to be optimal for the uncou- j
pling action of thyroxine, with a rapid decrease of
I
activity in more acid or alkaline medium. J
Magnesium was required both for the oxidative J
phosphorylation and the glucose-hexokinase trap. The
highest concentration of MjgCl£ used, 12.5 mM, gave the j
greatest P/0 ratios; however there was relatively little
increase in activity as the concentration was increased
43
above 1 mM. Two mM MgCl^ allowed the greatest reduction
in P/0 ratios by thyroxine. In accord with findings in
animal mitochondria the effect of the hormone was completely
abolished when larger amounts of magnesium were used.
Crystalline bovine plasma albumin was found to
be necessary for phosphorylation (30), but at all con
centrations it exerted an inhibitory effect on the uncou- i
pling action of thyroxine. This is probably due to binding
of the hormone by albumin. Albumin is known to bind a great
many substances and has been shown to combine with thyrox
ine in serum, when the hormone concentration exceeds the
j
capacity of the thyroxine-binding globulin (31). ;
j
L-thyroxine was shown to suppress phosphorylation i
' ' ' ' !
markedly, with little effect on respiration, at the highest|
1
-4 1
concentration used, 1 X 10 M. An average of twelve j
i
experiments with this amount gave a value of 72 per cent j
uncoupling. The effect diminished with decreasing con
centrations of the hormone. The lowest effective con
centration, in the presence of 0.15 per cent albumin, was
5 X 10 M. Greater effects were obtained when lower ! '
1
amounts of albumin were used. j
It is interesting that conditions most favorable j
for phosphorylation were not the best conditions for_____ |
44
demonstrating the uncoupling action of the hormone in the
system employed here. It is difficult to reproduce in
vitro the exact physiological conditions within the cells.
These conditions may be important in hormonal control in
higher organisms.
No stimulation of succinate oxidation by the
hormone was observed when sufficient magnesium and albumin i
j
were included to give optimal phosphate esterification.
When low control P/0 ratios of the order of 0.2 were j
obtained, by limiting either of these components so that !
|
P/0 ratios of below 0.05 were obtained in the presence of j
!
thyroxine, a marked stimulation of respiration was noted.
I
This is comparable with findings by Niemeyer e£ al. (9).
|
The addition of glucose and hexokinase to animal mito
chondria caused a marked increase in respiration rate. An
equal increase in oxidation was observed when thyroxine
was added to mitochondria with no acceptor system present; !
I
however when respiration was already stimulated by thyrox
ine, no further increase in respiration rate was produced
!
by addition of the hexokinase trap. The stimulation of |
j
respiration by the acceptor system was considered to be j
a mass action effect due to the removal of high energy j
I
intermediates. !
45
In the present study an increase in respiration
! rate was caused by thyroxine only when phosphorylation
was uncoupled to a very low rate. The possibility is
considered that respiration rate is controlled by the
concentrations of intermediates leading to the synthesis
of adenosine triphosphate. These concentrations are
influenced by the degree of uncoupling. Therefore, if
different systems have different critical threshold
concentrations of these intermediates, different degrees
of uncoupling might be required for the stimulation of
respiration by thyroxine. This may provide an explanation
for the variations in the action of thyroxine on mito
chondrial respiration observed by different investigators.
The possibility that the observed reduction in
P/0 ratios is a non-specific effect due to stimulation of
adenosine triphosphatase, inhibition of the glucose-
hexokinase trapping system, or increased liberation of
inorganic phosphorus from glucose-6-phosphate was inves
tigated. No significant effect by the hormone on any of
these processes was observed under the conditions used to
demonstrate uncoupling.
Several mechanisms, such as uncoupling of oxidative
phosphorylation, inhibition of the transhydrogenase
46
reaction, and structural damage to mitochondria, resulting
in lowered P/0 ratios, have been suggested as representing
the physiological activity of the thyroid hormone. None
of these proposals have met with complete acceptance.
Indeed the actual active form of the hormone is not known
with certainty.
I
Because of the unphysiologically high concentration!
of thyroxine necessary to demonstrate uncoupling of oxida
tive phosphorylation in the present study no additional ;
i
information on the mechanism of action of physiological
levels of the hormone may be presented. ,The experimental
i
data obtained support the contention that uncoupling may j
be responsible for many of the effects produced in animals ;
by large amounts of thyroid hormone. !
CHAPTER VII
SUMMARY
A subcellular particulate fraction corresponding
to mitochondria has been isolated from baking yeast. This
fraction oxidized succinate simultaneously with the esteri-
fication of inorganic phosphate. In addition to the usual
cofactors necessary for oxidative phosphorylation bovine
plasma albumin was required. P/0 ratios of 1 were obtained
under optimal conditions for phosphate uptake.
L-thyroxine was shown to depress P/0 ratios at the j
i
concentrations used to obtain suppression of phosphoryla- |
i
tion in animal mitochondria; however, conditions most
favorable for phosphorylation were not the best for j
j
demonstrating the maximal uncoupling effect of the hormone.
t
When low control P/0 ratios were produced, by I
f
limiting a required cofactor such as magnesium or albumin, I
I
a marked stimulation of succinate oxidation was observed. j
j
The relationship between uncoupling action and I
stimulation of respiration was discussed.
!
I
I
I
BIBLIOGRAPHY
• I
I
BIBLIOGRAPHY
1. DuBois, E. F. Basal Metabolism in Health and Disease.
Philadelphia: Lea, 1936, 3rd ed.
2. Wooley, D. W., J. Biol. Chem., 164, 11 (1946).
3. Wainfan, E., and Marx, W., J. Biol. Chem., 214,
441 (1955).
4. Gutenstein, M., and Marx, W., J. Biol. Chem., 599
(1957).
5. Linnane, A. W., and Still, J. L., Arch. Biochem. and i
Biophvs., 59, 383 (1955). j
6. Utter, M. F., Keech, D. B., and Nossal, P. M., j
Biochem. J., 6jJ, 431 (1958) . 1
^ 7. Nossal, P. M., Australian J. Exper. Biol, and Med.
Science, 31, 583 (1953). !
8. Hoch, F. L., and Lipmann, F., Proc. Nat. Acad. Sc., !
40, 909 (1954). i
r j
9. Niemeyer, H., Crane, R. K., Kennedy, E. P., and
Lipmann, F., Federation Proc., 10, 229 (1951). j
10. Martius, C., and Hess, B., Arch. Biochem. and
Biophys., 33, 486 (1951). j
I
11. Maley, G. F., and Lardy, H. A., J. Biol. Chem., 204. I
435 (1953). !
i
12. Klemperer, H. G., Biochem. J., 60, 128 (1955). .
I
13. Maley, G. F., and Lardy, H. A., J. Biol. Chem. 215, j
377 (1955). !
|
14. Dickens, F., and Salnony, D., Biochem. J., 64,
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50
15. Maley, G. F., J. Biol. Chem.,224, 1029 (1957).
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19. Lehninger, A. L., and Ray, B. L., Science, 125,
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!
20. Aebi, H., and Abelin, I., Biochem Z, 324, 364 (1953).
21. Bronk, J. R., Biochem. Biophys. Acta, 27, 667 (1958). !
22. Park, J. H., Meriwether, B. P., and Park, C. K.,
Biochem. Biophys. Acta, 28, 662 (1958).
23. Bain, J. A., J. Pharmacol. Exptl. Therap., 110, !
2 (1954). \
j
24. Mudd, S. H., Park, J. H., and Lipmann, F., Proc.
Nat. Acad. Sc., 41, 571 (1955). ;
25. Vitale, J. J., Hegsted, D. M., Nakamura, M., and
Connors, P., J. Biol. Chem., 226, 597 (1957).
26. Vitale, J. J*, Nakamura, M., and Hegsted, D. M.,
J. Biol. Chem., 228, 573 (1957). !
27. Fairhurst, A. S., Maher, J. M., and Smith R. E.,
Biochem. Biophys. Acta, 31, 296 (1959).
28. Simmons, J. S., and Gentzkow, C. J., Laboratory
Methods of the United States Army. Philadelphia:
Lea, 1944, 5th ed.
t
\
29. Taussky, H. H., and Shorr, E., J. Biol. Chem.. 202, !
675 (1953). ;
51
30. Polis, B. D., and Shmukler, H. W., J. Biol. Chem.,
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I
University of Southern California Library

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Creator Hodges, James Milton (author)

Core Title The effect of L-thyroxine on oxidation and phosphorylation in a subcellular particulate fraction of baking yeast

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Degree Master of Science

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