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Hydrogen transfer from tritium-labeled pyridine nucleotides to fatty acids synthesized by homogenate fractions of lactating-rat mammary gland
424
PRELIMINARY NOTES
Hydrogen transfer from tritium-labeled pyridine nucleotides to fatty acids synthesized by homogenate fractions of lactating-rat mammary gland The roles of DPNH and TPNH as hydrogen donors in fatty acid synthesis have been widely discussed 1-3, but the actual introduction of hydrogen from these reduced pyridine nucleotides has thus far not been demonstrated. TPN + and DPN + containing tritium (T_) in the 4-position of the pyridine ring [TPN(T)+ and DPN(T)+l were prepared by a modification of the method outlined by SAN PIETRO4. By reducing these tritiated nucleotides with purified enzymes according to the methods of VENNESLAND and coworkers we obtained TPN-aT_ (glucose 6phosphate dehydrogenase~), TPN-flT (isocitric dehydrogenase6), DPN-aT (glutamie dehydrogenasee and DPN-fl_T (yeast alcohol dehydrogenaseT). The tritium-labeled reduced pyridine nucleotides were incubated under varying cofactor conditions with particle-free supernatant fractions prepared from lactatingrat mammary glands 8. In the presence of citrate (Table I), the tritium of TPN-aT was more readily incorporated into fatty acids than was the isotope of T P N - f f ~ TABLE INCORPORATION INTO
FATTY FROM
OF
ACIDS
BY
HYDROGEN
FROM
PARTICLE-FREE
HOMOGENATES
OF
I REDUCED
PYRIDINE
SUPERNATANT
LACTATING-RAT
NUCLEOTIDES
FRACTIONS MAMMARY
PREPARED
GLAND
0.75 m l of p a r t i c l e - f r e e s n p e r n a t a n t f r a c t i o n p r e p a r e d f r o m 3 : 1 h o m o g e n a t e s of l a c t a t i n g - r a t m a m m a r y g l a n d s in 0.25 M sucrose w a s i n c u b a t e d for i h a t 3 °0 in air w i t h 1 2 o / z m o l e s glycylg l y c i n e ( p H 7.2), 5 / z m o l e s K H C O z, 3 5 / z m o l e s MgC1 v I / , m o l e MnC1 v 3 o # m o l e s r e d u c e d g l u t a t h i o n e , o . i / z m o l e C o A , 5 / ~ m o l e s A T P , a n d 3 / z m o l e s a c e t a t e in a f i n a l v o l u m e of 1.75 m l w i t h f u r t h e r a d d i t i o n s as s h o w n b e l o w . T h e r a d i o a c t i v e p y r i d i n e n u c l e o t i d e s c o n t a i n e d 4" l ° s counts/rain per assay. Pyridine nucleotides added ( z ~mole) Ci#~.t ( ~5 ,umoles)
Tri.H.um-labeled DPNH
Counts/rain recovered as fatty acids
Unlabeled
TPNH
DPN+
R a t I*
Rat 2"
Rat 3*
TPN+
+
--
a
+
--
61,23 °
44,12o
33,44 °
+
--
a
--
--
60,600
35,33 °
28,360
+
__
fl
+
--
31,6oo
12,87o
12,OLO
+
--
fl
--
__
35,320
8,870
13,44o
--
--
~
--
--
47 °
235
19o
__
__
fl
--
--
21o
235
~
-
17o
14o
90
__
fl
__
--
--
240
250
lOO
+ +
a ~
---
---
+ --
11,73 ° I6,I6O
7,590 6,270
6,790 lO,47o
+ +
fl fl
---
-__
+ __
13,35o 20,900
5,450 13,85o
5,120 14,ooo
-
-
-
-
-
-
-
* E a c h a s s a y c o n t a i n e d lO.2 m g ( R a t I) o r 6.0 m g ( R a t s 2 a n d 3) of p r o t e i n . A b b r e v i a t i o n s : D P N +, D P N H , o x i d i z e d a n d r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e ; T P N +, T P N H , oxidized a n d r e d u c e d d i p h o s p h o p y r i d i n e nucleotide; CoA, c o e n z y m e A; A T P , a d e n o s i n e triphosphate. Biochim.
Biophys.
A c t a , 47 (1961) 4 2 4 - 4 2 6
PRELIMINARY NOTES
425
I n the absence of citrate, the i n c o r p o r a t i o n of t r i t i u m from T P N T or D P N T i n t o f a t t y acids was negligible, regardless of stereo-configuration of the isotope. I n the presence of citrate, the isotopes of D P N - ~ T a n d of D P N - f l T were i n c o r p o r a t e d i n t o f a t t y acids to a b o u t the same e x t e n t . U n d e r our o p t i m u m conditions, i . e . , i n the presence of citrate, the recovery of t r i t i u m i n f a t t y acids was three t i m e s as great in the e x p e r i m e n t s w i t h T P N T as i n those w i t h D P N T . The results o b t a i n e d with the oxidized, labeled p y r i d i n e nucleotides are shown i n Table I I . Significant conversion of the isotope from either nucleotide i n t o f a t t y acids d i d not occur i n the absence of citrate. I n the presence of citrate, glucose 6-phosphate i n h i b i t e d the t r i t i u m transfer from DPN(T)+ i n t o f a t t y acids b u t enhanced this transfer from TPN(T) +. This s t i m u l a t i o n can be explained b y the relative a c t i v i t i e s of glucose 6-phosphate dehydrogenase a n d isocitric dehydrogenase i n the m a m m a r y g l a n d s u p e r n a t a n t fractions 8. T P N +, when added to our m a m m a r y - g l a n d s u p e r n a t a n t fraction, was more readily reduced b y glucose 6-phosphate t h a n b y citrate. Citrate act s b y i n c r e a s i n g the i n c o r p o r a t i o n of acetate carbon i n t o f a t t y acids 8, whereas glucose TABLE INCORPORATION INTO
FATTY
OF HYDROGEN
II
FROM
ACIDS BY PARTICLE-FREE
FROM
HOMOGENATES
OXIDIZED
PYRIDINE
SUPERNATANT
OF LACTATING-RAT
NUCLEOTIDES
FRACTIONS
PREPARED
MAMMARY GLAND
For incubation conditions see Table I. 4" IOS counts/min of TPN(T) + or DPN(T) + were incubated in each assay. -
C~trate 25 Itmoles
Glucose 6-pkosphate (20 t, moles)
Pyridine nucleotides addc,d (x #mole) Tritium-labeled
Countslmin recovered as fatty acids
Unlabeled
DPN +
TPN+
DPN +
TPN +
---
+ +
---
---
---
-+
- -
- -
+
- -
- -
- -
- -
+
+
- -
- -
- -
--+ + + --
__ ---+ --
__ ----+
+ + + + + +
-+ -+ + +
+ + ---+
Rat z*
Rat 2*
Rat 3*
300 400 21o 80 13,44° 5,040 39,200 64,400 127,4oo 2,280
18o 200 7° 65 8,030 2,820 21,7oo 42,400 lOl,5OO I,IO5
14o 18o 7° 7° 8,040 2,400 13,3oo 26,500 57,8oo 840
* See Table I. 6-phosphate acts b y p o s i t i o n i n g the t r i t i u m in the proper configuration for its t r a n s f e r to f a t t y acids. Thus, the results given i n Table I agree closely with those recorded i n Table II. A n e v a l u a t i o n of the stereospecificity of DPN(T) + r e d u c t i o n i n the s u p e r n a t a n t fractions from l a c t a t i n g r a t - m a m m a r y g l a n d is not possible at this t i m e as t h e stereospecificity of the enzymes i n v o l v e d is n o t known. Comparable e x p e r i m e n t s with p r e p a r a t i o n s from r a t - l i v e r homogenates (supern a t a n t plus microsomes 9) yielded results s i m i l a r to those recorded above. I n this case, Biochim.
Biophys.
Acta,
47 (1961) 424-426
426
PRELIMINARY NOTES
however, the incorporation of tritium into f a t t y acids was tess than that observed with the mammary-gland preparations. This work was aided by a grant from the National Science Foundation.
Department of Physiology, University of California, Berkeley, Calif. (U.S.A.)
S. ABRAHAM K . J . MATTHES*
I. L . CHAIKOFF 1 p. HELE, Brit. Med. Bull., 14 (1958) 2Ol. 2 j . W . PORTER, S. J. WAKIL, A. TIETZ, M. I. JACOB AND D. M. GIBSON, Biochim. Biophys. Acta, 25 (1957) 35. s F. LYNEN, J. Cell. Comp. Physiol., 54 (1959) 33. 4 A. SAN PIETRO, J. Biol. Chem., 217 (1955) 579. 5 B. K. STERN AND B. VENNESLAND, J. Biol. Chem., 235 (196o) 2o 5. e T. NAKAMOTO A.ND ]3. VENNESLAND, J. Biol. Chem., 235 (196o) 202. ]3. K. STERN AND B. VENNESLAND, J. Biol. Chem., 235 (196o) 209. S. ABRAHAM, K. J. ]V[ATTHES AND I. L. CHAIKOFF, u n p u b l i s h e d o b s e r v a t i o n s . 9 S. ABRAHAM, K. J. MATTHES AND I. L. CHAIKOFF, J. Biol. Chem., 235 (196o) 2551.
Received November 2ist, 196o * P o s t d o c t o r a l Fellow of t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . P r e s e n t a d d r e s s : Medizinischen Universit~tsklinik, Munster (Germany).
Biochim. Biophys. Acta, 47 (1961) 424-426
The effect of parathyroid extract on the isocitric dehydrogenase activity of bone tissue Since parathyroid extract has been shown to increase the release and/or the production of citrate in bone 1, 2 it became of importance to know whether this phenomenon is accompanied b y changes in the enzyme activities of aconitase and/or isocitric dehydrogenase. In this connection it is of interest to note that DIXON AND PERKINSs found extremely low values for the isocitric dehydrogenase activity of bone tissue. However, VAI~ REEN AND LOSEE 4 and VAN REEN 5 found much higher values using another method for estimating the activity. A modification of this method has been used in this study. The P T E (Eli Lilly) was administered as a single dose b y subcutaneous injection (IOO USP units/kg) to a number of male rabbits, 8-12 months old, weighing (2900 ~ 250) g. They were killed b y stunning and bleeding 16 h later. The control animals received a placebo injection, composed of diluted human serum with added phenol, with the same protein and phenol concentration as in PTE. The long bones of the legs were carefully dissected and freed of periosteum. After removing the epiphyses, the remaining shafts were cut into pieces and freed of adhering bone marrow. W i t h the help of a h e a v y stainless-steel pounder the pieces of bone were then powdered as far as possible while keeping the temperature at 1-4 ° . The bone powder was extracted for 4 h at 4 ° with 2 volumes of a solution containing 0.o25 M Tris (pH 7.4) and 0.5" lO -3 M citrate. The addition of citrate was found to be essential for preventing a rapid decline especially of the aconitase activity. The mixture was then centrifuged for io min at 25,000 × g, and in the water-clear supernatant the A b b r e v i a t i o n s : P T E , p a r a t h y r o i d e x t r a c t ; Tris, t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e .
Biochim. Biophys. Acta, 47 (1961) 426-427
PRELIMINARY NOTES
Hydrogen transfer from tritium-labeled pyridine nucleotides to fatty acids synthesized by homogenate fractions of lactating-rat mammary gland The roles of DPNH and TPNH as hydrogen donors in fatty acid synthesis have been widely discussed 1-3, but the actual introduction of hydrogen from these reduced pyridine nucleotides has thus far not been demonstrated. TPN + and DPN + containing tritium (T_) in the 4-position of the pyridine ring [TPN(T)+ and DPN(T)+l were prepared by a modification of the method outlined by SAN PIETRO4. By reducing these tritiated nucleotides with purified enzymes according to the methods of VENNESLAND and coworkers we obtained TPN-aT_ (glucose 6phosphate dehydrogenase~), TPN-flT (isocitric dehydrogenase6), DPN-aT (glutamie dehydrogenasee and DPN-fl_T (yeast alcohol dehydrogenaseT). The tritium-labeled reduced pyridine nucleotides were incubated under varying cofactor conditions with particle-free supernatant fractions prepared from lactatingrat mammary glands 8. In the presence of citrate (Table I), the tritium of TPN-aT was more readily incorporated into fatty acids than was the isotope of T P N - f f ~ TABLE INCORPORATION INTO
FATTY FROM
OF
ACIDS
BY
HYDROGEN
FROM
PARTICLE-FREE
HOMOGENATES
OF
I REDUCED
PYRIDINE
SUPERNATANT
LACTATING-RAT
NUCLEOTIDES
FRACTIONS MAMMARY
PREPARED
GLAND
0.75 m l of p a r t i c l e - f r e e s n p e r n a t a n t f r a c t i o n p r e p a r e d f r o m 3 : 1 h o m o g e n a t e s of l a c t a t i n g - r a t m a m m a r y g l a n d s in 0.25 M sucrose w a s i n c u b a t e d for i h a t 3 °0 in air w i t h 1 2 o / z m o l e s glycylg l y c i n e ( p H 7.2), 5 / z m o l e s K H C O z, 3 5 / z m o l e s MgC1 v I / , m o l e MnC1 v 3 o # m o l e s r e d u c e d g l u t a t h i o n e , o . i / z m o l e C o A , 5 / ~ m o l e s A T P , a n d 3 / z m o l e s a c e t a t e in a f i n a l v o l u m e of 1.75 m l w i t h f u r t h e r a d d i t i o n s as s h o w n b e l o w . T h e r a d i o a c t i v e p y r i d i n e n u c l e o t i d e s c o n t a i n e d 4" l ° s counts/rain per assay. Pyridine nucleotides added ( z ~mole) Ci#~.t ( ~5 ,umoles)
Tri.H.um-labeled DPNH
Counts/rain recovered as fatty acids
Unlabeled
TPNH
DPN+
R a t I*
Rat 2"
Rat 3*
TPN+
+
--
a
+
--
61,23 °
44,12o
33,44 °
+
--
a
--
--
60,600
35,33 °
28,360
+
__
fl
+
--
31,6oo
12,87o
12,OLO
+
--
fl
--
__
35,320
8,870
13,44o
--
--
~
--
--
47 °
235
19o
__
__
fl
--
--
21o
235
~
-
17o
14o
90
__
fl
__
--
--
240
250
lOO
+ +
a ~
---
---
+ --
11,73 ° I6,I6O
7,590 6,270
6,790 lO,47o
+ +
fl fl
---
-__
+ __
13,35o 20,900
5,450 13,85o
5,120 14,ooo
-
-
-
-
-
-
-
* E a c h a s s a y c o n t a i n e d lO.2 m g ( R a t I) o r 6.0 m g ( R a t s 2 a n d 3) of p r o t e i n . A b b r e v i a t i o n s : D P N +, D P N H , o x i d i z e d a n d r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e ; T P N +, T P N H , oxidized a n d r e d u c e d d i p h o s p h o p y r i d i n e nucleotide; CoA, c o e n z y m e A; A T P , a d e n o s i n e triphosphate. Biochim.
Biophys.
A c t a , 47 (1961) 4 2 4 - 4 2 6
PRELIMINARY NOTES
425
I n the absence of citrate, the i n c o r p o r a t i o n of t r i t i u m from T P N T or D P N T i n t o f a t t y acids was negligible, regardless of stereo-configuration of the isotope. I n the presence of citrate, the isotopes of D P N - ~ T a n d of D P N - f l T were i n c o r p o r a t e d i n t o f a t t y acids to a b o u t the same e x t e n t . U n d e r our o p t i m u m conditions, i . e . , i n the presence of citrate, the recovery of t r i t i u m i n f a t t y acids was three t i m e s as great in the e x p e r i m e n t s w i t h T P N T as i n those w i t h D P N T . The results o b t a i n e d with the oxidized, labeled p y r i d i n e nucleotides are shown i n Table I I . Significant conversion of the isotope from either nucleotide i n t o f a t t y acids d i d not occur i n the absence of citrate. I n the presence of citrate, glucose 6-phosphate i n h i b i t e d the t r i t i u m transfer from DPN(T)+ i n t o f a t t y acids b u t enhanced this transfer from TPN(T) +. This s t i m u l a t i o n can be explained b y the relative a c t i v i t i e s of glucose 6-phosphate dehydrogenase a n d isocitric dehydrogenase i n the m a m m a r y g l a n d s u p e r n a t a n t fractions 8. T P N +, when added to our m a m m a r y - g l a n d s u p e r n a t a n t fraction, was more readily reduced b y glucose 6-phosphate t h a n b y citrate. Citrate act s b y i n c r e a s i n g the i n c o r p o r a t i o n of acetate carbon i n t o f a t t y acids 8, whereas glucose TABLE INCORPORATION INTO
FATTY
OF HYDROGEN
II
FROM
ACIDS BY PARTICLE-FREE
FROM
HOMOGENATES
OXIDIZED
PYRIDINE
SUPERNATANT
OF LACTATING-RAT
NUCLEOTIDES
FRACTIONS
PREPARED
MAMMARY GLAND
For incubation conditions see Table I. 4" IOS counts/min of TPN(T) + or DPN(T) + were incubated in each assay. -
C~trate 25 Itmoles
Glucose 6-pkosphate (20 t, moles)
Pyridine nucleotides addc,d (x #mole) Tritium-labeled
Countslmin recovered as fatty acids
Unlabeled
DPN +
TPN+
DPN +
TPN +
---
+ +
---
---
---
-+
- -
- -
+
- -
- -
- -
- -
+
+
- -
- -
- -
--+ + + --
__ ---+ --
__ ----+
+ + + + + +
-+ -+ + +
+ + ---+
Rat z*
Rat 2*
Rat 3*
300 400 21o 80 13,44° 5,040 39,200 64,400 127,4oo 2,280
18o 200 7° 65 8,030 2,820 21,7oo 42,400 lOl,5OO I,IO5
14o 18o 7° 7° 8,040 2,400 13,3oo 26,500 57,8oo 840
* See Table I. 6-phosphate acts b y p o s i t i o n i n g the t r i t i u m in the proper configuration for its t r a n s f e r to f a t t y acids. Thus, the results given i n Table I agree closely with those recorded i n Table II. A n e v a l u a t i o n of the stereospecificity of DPN(T) + r e d u c t i o n i n the s u p e r n a t a n t fractions from l a c t a t i n g r a t - m a m m a r y g l a n d is not possible at this t i m e as t h e stereospecificity of the enzymes i n v o l v e d is n o t known. Comparable e x p e r i m e n t s with p r e p a r a t i o n s from r a t - l i v e r homogenates (supern a t a n t plus microsomes 9) yielded results s i m i l a r to those recorded above. I n this case, Biochim.
Biophys.
Acta,
47 (1961) 424-426
426
PRELIMINARY NOTES
however, the incorporation of tritium into f a t t y acids was tess than that observed with the mammary-gland preparations. This work was aided by a grant from the National Science Foundation.
Department of Physiology, University of California, Berkeley, Calif. (U.S.A.)
S. ABRAHAM K . J . MATTHES*
I. L . CHAIKOFF 1 p. HELE, Brit. Med. Bull., 14 (1958) 2Ol. 2 j . W . PORTER, S. J. WAKIL, A. TIETZ, M. I. JACOB AND D. M. GIBSON, Biochim. Biophys. Acta, 25 (1957) 35. s F. LYNEN, J. Cell. Comp. Physiol., 54 (1959) 33. 4 A. SAN PIETRO, J. Biol. Chem., 217 (1955) 579. 5 B. K. STERN AND B. VENNESLAND, J. Biol. Chem., 235 (196o) 2o 5. e T. NAKAMOTO A.ND ]3. VENNESLAND, J. Biol. Chem., 235 (196o) 202. ]3. K. STERN AND B. VENNESLAND, J. Biol. Chem., 235 (196o) 209. S. ABRAHAM, K. J. ]V[ATTHES AND I. L. CHAIKOFF, u n p u b l i s h e d o b s e r v a t i o n s . 9 S. ABRAHAM, K. J. MATTHES AND I. L. CHAIKOFF, J. Biol. Chem., 235 (196o) 2551.
Received November 2ist, 196o * P o s t d o c t o r a l Fellow of t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . P r e s e n t a d d r e s s : Medizinischen Universit~tsklinik, Munster (Germany).
Biochim. Biophys. Acta, 47 (1961) 424-426
The effect of parathyroid extract on the isocitric dehydrogenase activity of bone tissue Since parathyroid extract has been shown to increase the release and/or the production of citrate in bone 1, 2 it became of importance to know whether this phenomenon is accompanied b y changes in the enzyme activities of aconitase and/or isocitric dehydrogenase. In this connection it is of interest to note that DIXON AND PERKINSs found extremely low values for the isocitric dehydrogenase activity of bone tissue. However, VAI~ REEN AND LOSEE 4 and VAN REEN 5 found much higher values using another method for estimating the activity. A modification of this method has been used in this study. The P T E (Eli Lilly) was administered as a single dose b y subcutaneous injection (IOO USP units/kg) to a number of male rabbits, 8-12 months old, weighing (2900 ~ 250) g. They were killed b y stunning and bleeding 16 h later. The control animals received a placebo injection, composed of diluted human serum with added phenol, with the same protein and phenol concentration as in PTE. The long bones of the legs were carefully dissected and freed of periosteum. After removing the epiphyses, the remaining shafts were cut into pieces and freed of adhering bone marrow. W i t h the help of a h e a v y stainless-steel pounder the pieces of bone were then powdered as far as possible while keeping the temperature at 1-4 ° . The bone powder was extracted for 4 h at 4 ° with 2 volumes of a solution containing 0.o25 M Tris (pH 7.4) and 0.5" lO -3 M citrate. The addition of citrate was found to be essential for preventing a rapid decline especially of the aconitase activity. The mixture was then centrifuged for io min at 25,000 × g, and in the water-clear supernatant the A b b r e v i a t i o n s : P T E , p a r a t h y r o i d e x t r a c t ; Tris, t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e .
Biochim. Biophys. Acta, 47 (1961) 426-427