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Effect of hypophysectomy on dehydrogenase activity of rat tissues
382
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL. 19
(1956)
T A B L E i1 HORMONAL EFFECTS ON TI)N-DIAPHORASE ACTIVITY*
Condition o/rats
Number o! rats
Normal Thyroidectonlized Hypophysectomized Thyroidectomizedt h y r o x i n treated * * *
Lo 3 4 4
TPN diaphorase Specificactivity'* 37.0 34-3 31.9 33.5
( _= io.o) ( - , 7 -°) ( ~ 6-3) ( k t.8)
" Conditions of a s s a y w e r e essentially as described in footnote toTable I, except t h a t 3.6" IO-SM 2,6-dichlorophenol-indophenol was s u b s t i t u t e d for cytochrome c as an electron acceptor. #moles 2,6-dichlorophenol-indophenol reduced ** Specific activity -g protein × minute. *** 1 mg DL-thyroxin injected intraperitoneally daily for 1- 4 days.
dition, in no previous case has evidence been obtained to indicate t h a t variation in enzymic activity was not due to variation in the concentration of the protein m o i e t y of the e n z y m e being studied. I n s u m m a r y , the activity of hepatic T P N - c y t o c h r o m e c reductase a p p e a r s to be under thyroid control, and it is suggested t h a t control of the activity of this enzyme m a y represent a p r i m a r y site of action of the t h y r o i d hormone. The a u t h o r s wish to express their appreciation to Dr. SAMUEL P. ASPEN for generously providing the thyroidectomized r a t s used in these experiments. This investigation was s u p p o r t e d b y a research g r a n t (H-173 i) from the National I n s t i t u t e s of Health, U.S. Public H e a l t h Service.
Department o/Physiological Chemistry, The Johns Hopkins University School of 2V[edicine, Baltimore, Md. (U.S.A.)
ALVAH H . PI-IILLIPS ROBERT G. LANGDON*
DOADY, F. D. \V. LUKENS AND S. GURIN, J. Biol. Chem., 193 (1951) 459. LANGDON, J. Am. Chem. Soc., 77 (I955) 5z9 o. HOGEBOOM AND W. C. SCHNEIDER, J. Biol. Chem., 186 (195o) 417 . MAHLER, N. K. SARKAR, L. P. VERNON AND R. A. ALBERTY, J. Biol. Chem., 199 (1952) 5 8 5 . GLOCK AND P. MCLEAN, Biochem. J., 55 (1953) 4 oo. 6 N . O. I~APLAN, S. P . COLOWICK AND E . F. NEUFELD, J. Biol. Chem., 205 (1953) 1. 7 H. R. MAHLER, A. S. FAIRHURST AND ]3. MACKLEN, J. Am. Chem. Soc., 77 (1955) 1514. 8 S. B. BARKER, Physiol. Revs., 31 (1951) 205. Received N o v e m b e r i4th, 1955 1 R. R. 3 G. 4 H. 5 G.
O. G. H. R. E.
* This work was done during the t e n u r e of a Lederle Medical F a c u l t y Award.
Effect of hypophysectomy on dehydrogenase activity of rat tissues High levels of activity of glucose-6-phosphate (G6P) dehydrogenase have been reported for the adrenal cortex and lactating m a m m a r y glandsl, 2. M e a s u r e m e n t s of t h e activities h a v e also been made in a v a r i e t y of m a m m a l i a n tissues. I t was suggested t h a t the direct oxidative p a t h w a y m i g h t play a significant role in adrenal m e t a b o l i s m since the cortex showed high activity 2. Selected tissues from control and h y p o p h y s e c t o m i z e d (postoperative 7 days) rats* were assayed for G6P dehydrogenase and 6-phosphogluconate (6PG) dehydrogenase activities. The t w o - s u b s t r a t e m e t h o d of GLOCK AND MCLEAN4 was used. E a c h cell contained 0.24 m M tris ( h y d r o x y methyl) amino m e t h a n e buffer p H 7.45, o . 4 / z M t r i p h o s p h o p y r i d i n e nucleotide and suitable aliquots of the s u p e r n a t a n t from tissue h o m o g e n a t e s to a total volume of 3.o ml. The * Control and h y p o p h y s e c t o m i z e d r a t s were p u r c h a s e d from H o r m o n e Assay Laboratories, Chicago, Ill.
voI.. 19 (1956)
SHOI~TCOlVrMUNICATIONS, PRELIMINARY NOTES
383
first cell contained 5 /*zll of 6 PG ~, the second cell 5 t t M of G6P and 5 IbM of 6PG, while the third cell contained no s u b s t r a t e and served as a blank. The data (Table I) s h o w t h a t the G6P dehydrogenase activities per g r a m of tissue of the secondary sex glands were reduced seven days after h y p o p h y s e c t o m y , while the gonads and adrenals showed little change. The G6P dehydrogenase activity of the kidney was increased in the operated g r o u p from 122 to 155 units for females and 198 to 248 units for males. The enzyme activities of the liver, brain, and h e a r t were not changed by the r e m o v a l of the p i t u i t a r y ; the average levels were 83, 154, and 29 units respectively. Activities of 6PG dehydrogenase of the h y p o p h y s e c t o m i z e d groups were lower in the adrenals, w i t h little change observed for the gonads and secondary sex glands. The activity in kidney was increased from 82 to 12o units for females and 13o to 15o units for males following h y p o p h y s e c t o m y . Liver, brain, and h e a r t showed little or no change in levels of enzyme activity, average values being 125, 39, and 33 units respectively. TABLE I G6P-AND 6PG-DEHYDROGENASE ACTIVITIES IN UNITS PER GRAM TISSUE FROM CONTROL AND HYPOPHYSECTOMIZED RATS (One u n i t is the a m o u n t of enzyme which will reduce O.Ol # M of T P N per m i n u t e at 25 ° C). The values are expressed as means with s t a n d a r d deviations. The n u m b e r s in parentheses are the n u m b e r of animals.
Female Control
MaN Hypox
Control
Hypox
Glucose-6-phosphate dehydrogenase Uterus Prostate Sem. Vesicles Adrenal Gonads
217±
80 (3) ---
794 ± 77 (4) 493-L 55 (3)
15o~
15 (6)
-842 ~2~145 (6) 5 8 4 ± 3 4 8 (6)
. I75 ~= 109 ~ 791 ~ 55±
. . . . . 52 (4) 89 ~: 18 45 (4) 62 ~ 13 86 (4) 769 4 271 4(4) 4 5 ± 12
(4)
(5) (5) (5)
6-phosphogluconate dehydrogenase Uterus Prostate Sem. Vesicles Adrenal Gonads
80 ~
20 (3)
62 ~
i i (6)
--
356-~ II3~
54 (4) 25(3)
267± lO2~
79 (6) 28(6)
-59 ± 14 (4) 69 -~- 21 (4) 425~1o4(4) 6o-k 15(4)
-51 + 45 -E 256~ 79q:
21 (4) 16 (5) 78 (5) 18(5)
The levels in the various tissues f r o m n o r m a l r a t s are in general agreement with those found b y others 1, 2. H y p o p h y s e c t o m y did not change the G6P dehydrogenase activity per unit weight in the p r i m a r y t a r g e t organs (gonads and adrenals) even t h o u g h these tissues were atrophied; the secondary sex glands also decreased in weight while the activity of the enzyme decreased. The activity of 6 P C dehydrogenase of the adrenals decreased while t h a t of t h e gonads and secondary sex glands showed no appreciable change. This m a y indicate t h a t the oxidative p a t h w a y for glucose m e t a b o l i s m m a y be i m p o r t a n t in the accessory sex glands and u n d e r the control of estrogens and androgens. GUILFORD G. RUDOLPH Radioisotope and Research Laboratories, NORMAN S. OLSEN
Veterans ,4 dministration Hospital and Department o[ Biochemistry, Vanderbilt University School o] Medicine, Nashville, Tenn. (U.S.A.) 1 G. 2 T. s G. 4 j.
E. GLOCK AND P. McLEAN, Biochem. J., 56 (1954) 171. L. KELLY, E. D. NIELSON, R. B. JOHNSON AND C. S. VESTLING, J. Biol. Chem., 212 (1955) 545. E. GLOCK AND P. MCLEAN, Bloc\era. J., 55 (1953) 4 °°E. SEEGMILLER AND B. L. HORECKER, J. Biol. Chem., 192 (1951) 175. Received N o v e m b e r 8th, 1955
SHORT COMMUNICATIONS, PRELIMINARY NOTES
VOL. 19
(1956)
T A B L E i1 HORMONAL EFFECTS ON TI)N-DIAPHORASE ACTIVITY*
Condition o/rats
Number o! rats
Normal Thyroidectonlized Hypophysectomized Thyroidectomizedt h y r o x i n treated * * *
Lo 3 4 4
TPN diaphorase Specificactivity'* 37.0 34-3 31.9 33.5
( _= io.o) ( - , 7 -°) ( ~ 6-3) ( k t.8)
" Conditions of a s s a y w e r e essentially as described in footnote toTable I, except t h a t 3.6" IO-SM 2,6-dichlorophenol-indophenol was s u b s t i t u t e d for cytochrome c as an electron acceptor. #moles 2,6-dichlorophenol-indophenol reduced ** Specific activity -g protein × minute. *** 1 mg DL-thyroxin injected intraperitoneally daily for 1- 4 days.
dition, in no previous case has evidence been obtained to indicate t h a t variation in enzymic activity was not due to variation in the concentration of the protein m o i e t y of the e n z y m e being studied. I n s u m m a r y , the activity of hepatic T P N - c y t o c h r o m e c reductase a p p e a r s to be under thyroid control, and it is suggested t h a t control of the activity of this enzyme m a y represent a p r i m a r y site of action of the t h y r o i d hormone. The a u t h o r s wish to express their appreciation to Dr. SAMUEL P. ASPEN for generously providing the thyroidectomized r a t s used in these experiments. This investigation was s u p p o r t e d b y a research g r a n t (H-173 i) from the National I n s t i t u t e s of Health, U.S. Public H e a l t h Service.
Department o/Physiological Chemistry, The Johns Hopkins University School of 2V[edicine, Baltimore, Md. (U.S.A.)
ALVAH H . PI-IILLIPS ROBERT G. LANGDON*
DOADY, F. D. \V. LUKENS AND S. GURIN, J. Biol. Chem., 193 (1951) 459. LANGDON, J. Am. Chem. Soc., 77 (I955) 5z9 o. HOGEBOOM AND W. C. SCHNEIDER, J. Biol. Chem., 186 (195o) 417 . MAHLER, N. K. SARKAR, L. P. VERNON AND R. A. ALBERTY, J. Biol. Chem., 199 (1952) 5 8 5 . GLOCK AND P. MCLEAN, Biochem. J., 55 (1953) 4 oo. 6 N . O. I~APLAN, S. P . COLOWICK AND E . F. NEUFELD, J. Biol. Chem., 205 (1953) 1. 7 H. R. MAHLER, A. S. FAIRHURST AND ]3. MACKLEN, J. Am. Chem. Soc., 77 (1955) 1514. 8 S. B. BARKER, Physiol. Revs., 31 (1951) 205. Received N o v e m b e r i4th, 1955 1 R. R. 3 G. 4 H. 5 G.
O. G. H. R. E.
* This work was done during the t e n u r e of a Lederle Medical F a c u l t y Award.
Effect of hypophysectomy on dehydrogenase activity of rat tissues High levels of activity of glucose-6-phosphate (G6P) dehydrogenase have been reported for the adrenal cortex and lactating m a m m a r y glandsl, 2. M e a s u r e m e n t s of t h e activities h a v e also been made in a v a r i e t y of m a m m a l i a n tissues. I t was suggested t h a t the direct oxidative p a t h w a y m i g h t play a significant role in adrenal m e t a b o l i s m since the cortex showed high activity 2. Selected tissues from control and h y p o p h y s e c t o m i z e d (postoperative 7 days) rats* were assayed for G6P dehydrogenase and 6-phosphogluconate (6PG) dehydrogenase activities. The t w o - s u b s t r a t e m e t h o d of GLOCK AND MCLEAN4 was used. E a c h cell contained 0.24 m M tris ( h y d r o x y methyl) amino m e t h a n e buffer p H 7.45, o . 4 / z M t r i p h o s p h o p y r i d i n e nucleotide and suitable aliquots of the s u p e r n a t a n t from tissue h o m o g e n a t e s to a total volume of 3.o ml. The * Control and h y p o p h y s e c t o m i z e d r a t s were p u r c h a s e d from H o r m o n e Assay Laboratories, Chicago, Ill.
voI.. 19 (1956)
SHOI~TCOlVrMUNICATIONS, PRELIMINARY NOTES
383
first cell contained 5 /*zll of 6 PG ~, the second cell 5 t t M of G6P and 5 IbM of 6PG, while the third cell contained no s u b s t r a t e and served as a blank. The data (Table I) s h o w t h a t the G6P dehydrogenase activities per g r a m of tissue of the secondary sex glands were reduced seven days after h y p o p h y s e c t o m y , while the gonads and adrenals showed little change. The G6P dehydrogenase activity of the kidney was increased in the operated g r o u p from 122 to 155 units for females and 198 to 248 units for males. The enzyme activities of the liver, brain, and h e a r t were not changed by the r e m o v a l of the p i t u i t a r y ; the average levels were 83, 154, and 29 units respectively. Activities of 6PG dehydrogenase of the h y p o p h y s e c t o m i z e d groups were lower in the adrenals, w i t h little change observed for the gonads and secondary sex glands. The activity in kidney was increased from 82 to 12o units for females and 13o to 15o units for males following h y p o p h y s e c t o m y . Liver, brain, and h e a r t showed little or no change in levels of enzyme activity, average values being 125, 39, and 33 units respectively. TABLE I G6P-AND 6PG-DEHYDROGENASE ACTIVITIES IN UNITS PER GRAM TISSUE FROM CONTROL AND HYPOPHYSECTOMIZED RATS (One u n i t is the a m o u n t of enzyme which will reduce O.Ol # M of T P N per m i n u t e at 25 ° C). The values are expressed as means with s t a n d a r d deviations. The n u m b e r s in parentheses are the n u m b e r of animals.
Female Control
MaN Hypox
Control
Hypox
Glucose-6-phosphate dehydrogenase Uterus Prostate Sem. Vesicles Adrenal Gonads
217±
80 (3) ---
794 ± 77 (4) 493-L 55 (3)
15o~
15 (6)
-842 ~2~145 (6) 5 8 4 ± 3 4 8 (6)
. I75 ~= 109 ~ 791 ~ 55±
. . . . . 52 (4) 89 ~: 18 45 (4) 62 ~ 13 86 (4) 769 4 271 4(4) 4 5 ± 12
(4)
(5) (5) (5)
6-phosphogluconate dehydrogenase Uterus Prostate Sem. Vesicles Adrenal Gonads
80 ~
20 (3)
62 ~
i i (6)
--
356-~ II3~
54 (4) 25(3)
267± lO2~
79 (6) 28(6)
-59 ± 14 (4) 69 -~- 21 (4) 425~1o4(4) 6o-k 15(4)
-51 + 45 -E 256~ 79q:
21 (4) 16 (5) 78 (5) 18(5)
The levels in the various tissues f r o m n o r m a l r a t s are in general agreement with those found b y others 1, 2. H y p o p h y s e c t o m y did not change the G6P dehydrogenase activity per unit weight in the p r i m a r y t a r g e t organs (gonads and adrenals) even t h o u g h these tissues were atrophied; the secondary sex glands also decreased in weight while the activity of the enzyme decreased. The activity of 6 P C dehydrogenase of the adrenals decreased while t h a t of t h e gonads and secondary sex glands showed no appreciable change. This m a y indicate t h a t the oxidative p a t h w a y for glucose m e t a b o l i s m m a y be i m p o r t a n t in the accessory sex glands and u n d e r the control of estrogens and androgens. GUILFORD G. RUDOLPH Radioisotope and Research Laboratories, NORMAN S. OLSEN
Veterans ,4 dministration Hospital and Department o[ Biochemistry, Vanderbilt University School o] Medicine, Nashville, Tenn. (U.S.A.) 1 G. 2 T. s G. 4 j.
E. GLOCK AND P. McLEAN, Biochem. J., 56 (1954) 171. L. KELLY, E. D. NIELSON, R. B. JOHNSON AND C. S. VESTLING, J. Biol. Chem., 212 (1955) 545. E. GLOCK AND P. MCLEAN, Bloc\era. J., 55 (1953) 4 °°E. SEEGMILLER AND B. L. HORECKER, J. Biol. Chem., 192 (1951) 175. Received N o v e m b e r 8th, 1955