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The stability of the adaptive enzyme, tryptophan peroxidase
VOL. 31 (Z959)
SHORT COMMUNICATIONS
54z
The authors are grateful to Dr. G. C. BUTLER and to Dr. OLIVER SMITHIES for their interest and helpful advice.
Department o/Biochemistry, University el Toronto, Toronto, Ont. (Canada)
J. M. N E E L I N * G. E. CONNELL
1 p. F. DAVISON, Biochem. J., 66 (I957) 708. J. A. V. BUTLER AND P. F. DAVISON, Advances in Engymol.i i8 (I957) z7o. 8 L. B. SMXLLIE, G. C. BUTLER AND D. B. SMITH, Can. J. Biochom. and Physiol., 36 (1958) I. 4 C. F. CRAMPTON, W. H. STEIN AND S. MOORE, J. Biol. Chem., 225 (1957) 363 • 5 j . M. NEELIN AND G. C. BUTLER, in preparation. e j . M. NEELIN AND G. C. BUTLER, in preparation. ? O. SMITHIES, Biochem. J., 61 (I955) 629.
Received July 24th, 1958
The stability of the adaptive enzyme, tryptophan peroxidase The injection or feeding of tryptophan markedly increases the tryptophan peroxidase of rat liver 1. Cortisone is equally effective2. The present study was undertaken to determine the relative stability of tryptophan peroxidase induced by tryptophan and by cortisone and the possible role of the substrate as a stabilizer of the enzyme under both conditions of induction. It was found that the enzyme was unstable even at o ° and that tryptophan protected the enzyme from inactivation (Table I). It seemed likely that the somewhat greater stability of the tryptophan-induced enzyme was a reflection of the level of the substrate in the tissue. This view was supported by an independent experiment in which tryptophan was injected interperitoneaUy. 4 h later the liver still remained coated with tryptophan suggesting that the tryptophan level within the liver was high at the time of homogenization. Homogenates from such livers showed no loss of tryptophan peroxidase activity during an I8-h period at 2 °. Since induction by cortisone does not raise the basal level of tryptophan 3, this TABLE I THE STABILITY OF TRYPTOPHAN
PEROXIDASE
IN RAT-LIVeR
HOMOGENATE
DURING
STORAGE AT 2 °
The r a t s were anesthetized w i t h n e m b u t a l 4 h after injection or feeding. The liver was removed, chilled a n d homogenized at a 1:8 dilution. H o m o g e n a t e s were stored at 2 ° w i t h and w i t h o u t 0.0003 M DL-tryptophan. E n z y m e activity was d e t e r m i n e d b y incubation w i t h 0.0025 M t r y p t o p h a n for i h at 38 ° as d e s c r i b e d b y KNOX AND AUERBACH4. Activity is t h e net k y n u r e n i n e (#moles) formed in z h / g wet wt. tissue. Zero-time control values h a v e been subtracted.
Aaivity wi~.o~ ~tor~ w/tA ~ptophan trrptop~n
Method o] inclusion
Time at zo
2 g t r y p t o p h a n fed by stomach tube
o 18
41.8 IO.O
40.5
2.5 m g cortisone injected interperitoneally
o 18
38.2 4.8
22. 5
(h)
stored
* H o l d e r of a S t u d e n t s h i p of t h e Fisheries Research Board of Canada.
542
SHORT COMMUNICATIONS
VOL. 3 1
(1959)
offered the best conditions for a study of substrate stabilization. With this method of induction it was found that the half-life of the enzyme was only IO mid at 38 ° (Table II). The addition of tryptophan lowered the rate of inactivation. TABLE II STABILITY OF CORTISONE-INDUCED TRYPTOPHAN PEROXIDASE IN RAT-LIVER HOMOGENATE AT 38 ° All samples i n c l u d i n g c o n t r o l s for e a c h t i m e i n t e r v a l were placed in t h e 380 w a t e r b a t h a t zero t i m e . A t t h e t i m e s g i v e n in t h e t a b l e c o n t r o l s were r e m o v e d a n d a n a l y z e d for k y n u r e n i n e a l r e a d y formed. T r y p t o p h a n w a s a d d e d to t h e c o r r e s p o n d i n g e x p e r i m e n t a l v e s s e l s to give a final concn. of o:0025 M a n d t h e i n c u b a t i o n c o n t i n u e d for i h . V a l u e s are n e t k y n u r e n i n e (/,moles) s y n t h e s i z e d / g liver d u r i n g a i - h period b e g i n n i n g a t t h e t i m e indicated. T h e r a t e of k y n u r e n i n e s y n t h e s i s in t h e presence of t r y p t o p h a n r e m a i n e d c o n s t a n t for a 2-h period in t h i s e x p e r i m e n t .
Time rain o io 20 30 60
o.ooo3MuL-tvyptophan during homogeaiaatien absent prese~ 23.5 14. 3 4.8 2.5 0.6
29.3 28. 5 27.6 25.2 i8.o
The changes in level of this basically unstable enzyme are consistent with the view that the enzyme protein is being continually synthesized and broken down. When the cellular level of tryptophan rises more of the newly synthesized enzyme is stabilized. It is not necessary to assume in the case of tryptophan peroxidase that the substrate initiates the synthesis of new enzyme protein. Whatever the mode of action of cortisone the increase ill enzyme concentration is not a result of increased stability. The fact that the tryptophan level does not rise after cortisone administration 4 supports the view that the substrate is not essential for the increase in enzyme concentration 5. This investigation was supported in part b y Research Grants No. A498 and HI613(CIo) from the National Institute of Health and by a financial grant from Mr. EDWARD H . H E L L E R , Atherton, California. The authors wish to express their thanks to Dr. S. LEPKOVSKYfor his advice and encouragement and the use of the facilities of the Department of Poultry Husbandry at the University of California at Berkeley and to Dr. A. MEHLER for helpful discussions of this problem. JACOB W. DUBNOFF Division o[ Biology, Pasadena, Calf]. (U.S.A.) MILDRED DIMICK
Cali]ornia Institute o/Technology, and the Department o[ Poultry Husbandry, University o/Cali/ornia, Berkeley, Cal~/. (U.S.A.) 1 W. E. KNOX AND A. W. IV~EHLER,Science, I13 (i951) 237. J W. E. KNOX, J. Expa. Pathol., 32 (1951) 462. 3 M. CIV~N AND W. E. KNOX, Federation Proc., 16 (1957) I65. 4 W . E. KNOX AND V. H. AUER.BACH, J. Biol. Chem., 214 (1955) 3o7 • 6 j . W. DUBNOFF. Amino Acid Metabolism. T h e J o h n s H o p k i n s U n i v . P r e s s (1955) P. 198.
Received July 2Ist, 1958
SHORT COMMUNICATIONS
54z
The authors are grateful to Dr. G. C. BUTLER and to Dr. OLIVER SMITHIES for their interest and helpful advice.
Department o/Biochemistry, University el Toronto, Toronto, Ont. (Canada)
J. M. N E E L I N * G. E. CONNELL
1 p. F. DAVISON, Biochem. J., 66 (I957) 708. J. A. V. BUTLER AND P. F. DAVISON, Advances in Engymol.i i8 (I957) z7o. 8 L. B. SMXLLIE, G. C. BUTLER AND D. B. SMITH, Can. J. Biochom. and Physiol., 36 (1958) I. 4 C. F. CRAMPTON, W. H. STEIN AND S. MOORE, J. Biol. Chem., 225 (1957) 363 • 5 j . M. NEELIN AND G. C. BUTLER, in preparation. e j . M. NEELIN AND G. C. BUTLER, in preparation. ? O. SMITHIES, Biochem. J., 61 (I955) 629.
Received July 24th, 1958
The stability of the adaptive enzyme, tryptophan peroxidase The injection or feeding of tryptophan markedly increases the tryptophan peroxidase of rat liver 1. Cortisone is equally effective2. The present study was undertaken to determine the relative stability of tryptophan peroxidase induced by tryptophan and by cortisone and the possible role of the substrate as a stabilizer of the enzyme under both conditions of induction. It was found that the enzyme was unstable even at o ° and that tryptophan protected the enzyme from inactivation (Table I). It seemed likely that the somewhat greater stability of the tryptophan-induced enzyme was a reflection of the level of the substrate in the tissue. This view was supported by an independent experiment in which tryptophan was injected interperitoneaUy. 4 h later the liver still remained coated with tryptophan suggesting that the tryptophan level within the liver was high at the time of homogenization. Homogenates from such livers showed no loss of tryptophan peroxidase activity during an I8-h period at 2 °. Since induction by cortisone does not raise the basal level of tryptophan 3, this TABLE I THE STABILITY OF TRYPTOPHAN
PEROXIDASE
IN RAT-LIVeR
HOMOGENATE
DURING
STORAGE AT 2 °
The r a t s were anesthetized w i t h n e m b u t a l 4 h after injection or feeding. The liver was removed, chilled a n d homogenized at a 1:8 dilution. H o m o g e n a t e s were stored at 2 ° w i t h and w i t h o u t 0.0003 M DL-tryptophan. E n z y m e activity was d e t e r m i n e d b y incubation w i t h 0.0025 M t r y p t o p h a n for i h at 38 ° as d e s c r i b e d b y KNOX AND AUERBACH4. Activity is t h e net k y n u r e n i n e (#moles) formed in z h / g wet wt. tissue. Zero-time control values h a v e been subtracted.
Aaivity wi~.o~ ~tor~ w/tA ~ptophan trrptop~n
Method o] inclusion
Time at zo
2 g t r y p t o p h a n fed by stomach tube
o 18
41.8 IO.O
40.5
2.5 m g cortisone injected interperitoneally
o 18
38.2 4.8
22. 5
(h)
stored
* H o l d e r of a S t u d e n t s h i p of t h e Fisheries Research Board of Canada.
542
SHORT COMMUNICATIONS
VOL. 3 1
(1959)
offered the best conditions for a study of substrate stabilization. With this method of induction it was found that the half-life of the enzyme was only IO mid at 38 ° (Table II). The addition of tryptophan lowered the rate of inactivation. TABLE II STABILITY OF CORTISONE-INDUCED TRYPTOPHAN PEROXIDASE IN RAT-LIVER HOMOGENATE AT 38 ° All samples i n c l u d i n g c o n t r o l s for e a c h t i m e i n t e r v a l were placed in t h e 380 w a t e r b a t h a t zero t i m e . A t t h e t i m e s g i v e n in t h e t a b l e c o n t r o l s were r e m o v e d a n d a n a l y z e d for k y n u r e n i n e a l r e a d y formed. T r y p t o p h a n w a s a d d e d to t h e c o r r e s p o n d i n g e x p e r i m e n t a l v e s s e l s to give a final concn. of o:0025 M a n d t h e i n c u b a t i o n c o n t i n u e d for i h . V a l u e s are n e t k y n u r e n i n e (/,moles) s y n t h e s i z e d / g liver d u r i n g a i - h period b e g i n n i n g a t t h e t i m e indicated. T h e r a t e of k y n u r e n i n e s y n t h e s i s in t h e presence of t r y p t o p h a n r e m a i n e d c o n s t a n t for a 2-h period in t h i s e x p e r i m e n t .
Time rain o io 20 30 60
o.ooo3MuL-tvyptophan during homogeaiaatien absent prese~ 23.5 14. 3 4.8 2.5 0.6
29.3 28. 5 27.6 25.2 i8.o
The changes in level of this basically unstable enzyme are consistent with the view that the enzyme protein is being continually synthesized and broken down. When the cellular level of tryptophan rises more of the newly synthesized enzyme is stabilized. It is not necessary to assume in the case of tryptophan peroxidase that the substrate initiates the synthesis of new enzyme protein. Whatever the mode of action of cortisone the increase ill enzyme concentration is not a result of increased stability. The fact that the tryptophan level does not rise after cortisone administration 4 supports the view that the substrate is not essential for the increase in enzyme concentration 5. This investigation was supported in part b y Research Grants No. A498 and HI613(CIo) from the National Institute of Health and by a financial grant from Mr. EDWARD H . H E L L E R , Atherton, California. The authors wish to express their thanks to Dr. S. LEPKOVSKYfor his advice and encouragement and the use of the facilities of the Department of Poultry Husbandry at the University of California at Berkeley and to Dr. A. MEHLER for helpful discussions of this problem. JACOB W. DUBNOFF Division o[ Biology, Pasadena, Calf]. (U.S.A.) MILDRED DIMICK
Cali]ornia Institute o/Technology, and the Department o[ Poultry Husbandry, University o/Cali/ornia, Berkeley, Cal~/. (U.S.A.) 1 W. E. KNOX AND A. W. IV~EHLER,Science, I13 (i951) 237. J W. E. KNOX, J. Expa. Pathol., 32 (1951) 462. 3 M. CIV~N AND W. E. KNOX, Federation Proc., 16 (1957) I65. 4 W . E. KNOX AND V. H. AUER.BACH, J. Biol. Chem., 214 (1955) 3o7 • 6 j . W. DUBNOFF. Amino Acid Metabolism. T h e J o h n s H o p k i n s U n i v . P r e s s (1955) P. 198.
Received July 2Ist, 1958