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Kinetic characterization of lactate dehydrogenase of normal and virus-infected chorioallantoic membrane of the chick embryo
177
SHORT COMMUNICATIONS
work was supported in part by the Antituberculosis League, Cleveland, Ohio and by a U.S. Public Grant E-I742.
Departments of Biochemistry and Medicine, Western Reserve University School of Medicine, Cleveland, Ohio (USA)
R U N E L . STJERNHOLM RUDOLF E. NOBLE DIETER KOCH-WESER
z H, P. KLEIN, J. Bacteriol., 73 (1957) 53 °. 2 S. J. WAKIL, J. Am, Chem. Soc., 80 (I958) 6465. 3 M. FLAVIN, P. J. ORTIZ AND S. OCHOA, Nature, 176 (1955) 823. ¢ W. S. BECK, M. FLAVIN AND S. OCHOA, J. Biol. Chem., 229 (1957) 997. 5 W. S. BECK AND S. OCHOA, J. Biol. Chem., 232 (1958) 931. 6 R, W. SWlCK AND H. G. WOOD, Proc. Natl. Acad. Sci. U.S., 46 (196o) 28. S. GURNANI, S. P. 3~ISTRY AND B. C. JOHNSON, Biochim. Biophys. Acta, 38 (I96O) 187. 8 R. STJERNHOLM AND H. G. WOOD, Proc. Natl. Acad. Sci. U.S., 47 (1961) 3o3. 9 p. LENGVEL, R. MAZUMDER AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 46 (I96O) 1312. t0 W. B. SCHAEFER, J. Bacteriol., 73 (1955) 52. u E. STADTMAN AND H. A. BARKER, J. Biol. Chem., 184 (195 o) 769. lz A. R. THOMPSON, Australian J. Sci. Research, B, 4 (195o) 18o.
Received March 24th, 1962 Biochim. Biophys. Acta, 64 (1962) 174-177
sc 11005
Kinetic characterization of lactate dehydrogenase of normal and virus.infected chorioallantoic membrane of the chick embryo We have repeatedly observed that the concentration of glycolytic enzymes is markedly increased in chorioallantoic membranes immediately preceding and during rapid virus multiplication 1-3. The characteristic increase in glycolytic activity, most probably caused by an increased rate of synthesis of glycolytic enzymes, is accompanied by the appearance of aerobic glycolysis, normally absent in this tissue, and an increased turnover of the hexose monophosphate pathway of carbohydrate oxidation, without the concomitant increase in the amount of enzymes of the hexose 6-phosphate-oxidizing metabolic cycles. The key to this increased rate of hexose monophosphate oxidation is the increased amount of lactate dehydrogenase (Llactate: DPN + oxidoreductase, EC 1.1.1.29), capable of oxidizing T P N H by pyruvate, thus activating hexose 6-phosphate oxidation by the removal of the normally ratelimiting T P N H *. We have previously proposed that the increased rate of enzyme formation which occurs after virus infection may be due to a direct effect of some component(s) of the infective virus on the enzyme-synthesizing system of the chorioallantoic cells. The new steady-state of metabolism of the infected cell, reached as a consequence of the increased quantity of certain enzyme proteins, is then sufficient "biochemical cause" to affect cell growth or the synthesis of viral material s . This working hypothesis requires a detailed testing at every level of its complexity. We were first interested to discover whether or not a newly formed enzyme is a catalytically different protein from the one present in the uninfected tissue. Since increase in lactate dehydrogenase plays a central role in the alteration of carbohydrate Biochim. Biophys..4cta, 64 (I962) 177-179
178
SHORT COMMUNICATIONS
m e t a b o l i s m o f t h e c h o r i o a l l a n t o i c t i s s u e , t h i s e n z y m e w a s c h o s e n for p r e l i m i n a r y studies. A n a l o g s o f p y r i d i n e n u c l e o t i d e s , i n t r o d u c e d b y KAPLAN a n d h i s associates4, 5 h a v e b e e n u s e d as k i n e t i c p a r a m e t e r s
for l a c t a t e d e h y d r o g e n a s e s . An a r b i t r a r y
a m o u n t o f t h e s e a n a l o g s w a s e m p l o y e d b y o t h e r w o r k e r s a n d tile r e l a t i v e r a t e s o f r e a c t i o n s w e r e c o n s i d e r e d t o b e s u f f i c i e n t f o r t h e c h a r a c t e r i z a t i o n o f d e h y d r o g e n a s e s ~. I t s e e m s t o u s t h a t t h i s u s e o f p y r i d i n e n u c l e o t i d e a n a l o g s s u f f e r s f r o m t h e disa d v a n t a g e t h a t t h e a r b i t r a r i l y c h o s e n c o n c e n t r a t i o n s m a y or m a y n o t s a t u r a t e t h e e n z y m e , o r e v e n c a u s e e n z y m e i n h i b i t i o n {in c a s e o f a n a b n o r m a l k i n e t i c b e h a v i o r as f o u n d w i t h m a n y c o e n z y m e s ) , so t h a t r e a c t i o n r a t e s e x p r e s s e d u n d e r s u c h c o n ditions h a v e a l i m i t e d m e a n i n g . In o r d e r to avoid these possibile difficulties we h a v e determined the apparent Michaelis constant of each coenzyme, coenzyme analog and substrate at various pH values. In addition chromatographic isolation of lactate dehydrogenase of normal and virus-infected tissues was attempted,
in o r d e r t o
a s c e r t a i n w h e t h e r or n o t several p r o t e i n c o m p o n e n t s w i t h e n z y m i c activities could b e o b t a i n e d . I f t h i s is n o t d o n e , o n e m a y b e f a c e d w i t h c o n f u s i n g k i n e t i c d a t a , s i n c e
TABLE I KINETIC
CONSTANTS
OF
LACTATE
DEHYDROGENASE
OF THE
CHICK
OF
CHORIOALLANTOIC
MEMBRANES
EMBRYO
All measurements were carried out with the Zeiss PMQn spectrophotometer, in I-ml cuvettes (light path I c m ) . Buffers were o.i M phosphate (pH 6.o and 7.4) and Tris (pH 9.0). Km values refer to the first compound shown in Column I ("Substrate"), while the component in brackets indicates conditions of the reaction./~m determinations were carried out under conditions yielding Vmax. with respect to the second component, which was kept at constant concentrations. All coenzymes and analogs were purchased from Pabst Laboratories (Milwaukee, Wise.). Vmax (in terms of/*moles coen~ymelmg #~otein/min)
K m (M) "Substrate"
pH
Normal Pyruvate (DPNH) Pyruvate (DPNH) Pyruvate (TPNH) Pyruvate (TPNH) D P N H (pyruvate) D P N H (pyruvate) T P N H (pyruvate) T P N H (pyruvate) Lactate (DPN+) DPN+ (lactate) A P D P N +** (lactate) Lactate (APDPN+) D es-NH2-DPN +*** (lactate) Lactate (des-NH2-DPN+) SH-DPN+§ (lactate) Lactate (SH-DPN+)
7-4 6.0 7-4 6.0 7.4 6.o 7-4 6.0 9.0 9.0 9-0 9.0 9.o 9.0 9.o 9 .o
5.55' lO-5 1.62- lO -5 3.7 '1o-a 4.o • lO-4 I.O - io -~ 1.54. lO -5 5.o • IO-5 2.5 " lO -5 2.5 "1o -3 1.58. lO -5 2-33" lO-5 1.34. lO-a 3.2 • IO-4 2.75" lO -3 2.7 "io -4 3.3 "IO-2
Virus-infected* 5.55" lO-5 1.62" lO -5 2.o - IO-3 4.0 - lO-4 I.O
" I O
- 6
Normal
Virusinfected*
o-525 o.295 o.o28 0.054
1.66 o.91o o.o55 o.195
- -
- -
1.54. lO -5 3.3 " I O - 5
__
__
--
--
2,O
--
--
" 10 -5
2.5 " IO--3 1.58. 10 -5 2,33. IO-5 1.34"
1°-3
3.6 " lO -4 2.75. lO -8 3.3 " lO-4 3.4
" 1°-2
O.128 __ 0.036 --
o.143 __ o,147 --
0-357 -O.133 --
0.660 __ o.50o --
* The tissues were harvested 48 h after inoculation with the virus. ** Acetylpyridine analog of DPN +. *** Deamino_DPN +. § Thionicotinamide-DPN+.
Biochim. Biophys. Acta, 64 (1962) 177-179
179
SHORT COMMUNICATIONS
a mixture of several lactate dehydrogenases with various kinetic constants may simulate "different species" of enzymes. The preparation and collection of normal, and avian pox-virus infected chorioallantoic membranes of chick embryos was carried out as described earlier 3 except on a large scale (14 dozen eggs were used for each experiment). The pooled, frozen tissues were homogenized in o.15 M KC1 in a blendor; particulate material was removed by high-speed centrifugation (2o ooo × g for 3 ° min at o°), then the soluble protein was precipitated by saturation with solid (NH4)2SO 4. The precipitate, dissolved in a minimum volume of deionized water, was freed from salts by passing through a Sephadex G-25 column. Enzyme assays were performed by the spectrophotometric measurements of either the reoxidation of reduced coenzymes by pyruvate or the reduction of oxidized coenzymes or coenzyme analogs by lactate. The protein solution was subjected to gradient-elution chromatography on DEAE-cellulose and the enzyme eluted with o.oo5 M potassium phosphate of pH 7.2. This procedure yielded a 5-fold purification of the lactate dehydrogenase, obtained from both normal and virus-infected tisues, without any sign of separation into enzymically active protein components. It is noteworthy that while the D P N H - p y r u v a t e reaction exhibits marked substrate inhibition with concentrations of pyruvate above IO-4 M, such inhibition by pyruvate does not exist in case of the T P N H - p y r u v a t e reaction. This kinetic behavior stresses the important role of this reaction (i.e., T P N H - p y r u v a t e ) in tissues where increased glycolysis yields high concentrations of pyruvate. From these results, summarized in Table I, it is concluded that the lactate dehydrogenase formed in the chorion tissue of chick embryo after infection with canary pox-virus is kinetically identical, with the enzyme, present in non-infected tissues. This work was supported by grants. C-468I and C-5884, C-32II of the U.S. Public Health Service and by a grant of the American Heart Association, Incorporated.
Departments of Pharmacology and Biochemistry, University of California, School of Medicine, San Francisco, Calif., and
ERNEST KUN D. W. FANSHIER JUNE E. AYLING
Department of Pathology, University of Oregon Medical School, Portland, Oreg. (U.S.A.)
B E N J A M I N V, SIEGEL
KoN AND M. H. D. SMITH, Proc. Soc. Exptl. Biol. Med., 73 (195 o) 628. H. D. SMITH AND E. I{UN, Brit. J. Exptl. Pathol., 34 (1954) I. KoI% J. E. AYLING AND B. V. SIEGEL, Proc. Natl. Acad. Sci. U.S., 46 (196o) 622. O. KAPLAN, in G. E. W. WOLSTENHOLME AND C. i . O'CONNOR, The steric mechanisms involved in the reaction of lactic acid, Ciba F o u n d a t i o n , S t u d y G r o u p No. 2. March 17, 1959. Little, B r o w n and Co., Boston. •. O. KAPLAN AND M. M. CIOTTI, Biochim. Biophys. Acta, 49 (I96I) 425 .
1 E. 2 M. 3 E. ¢ N.
Received March I2th, 1962. Biochim. Biophys. Acta, 64 (1962) I 7 7 - [ 7 9
SHORT COMMUNICATIONS
work was supported in part by the Antituberculosis League, Cleveland, Ohio and by a U.S. Public Grant E-I742.
Departments of Biochemistry and Medicine, Western Reserve University School of Medicine, Cleveland, Ohio (USA)
R U N E L . STJERNHOLM RUDOLF E. NOBLE DIETER KOCH-WESER
z H, P. KLEIN, J. Bacteriol., 73 (1957) 53 °. 2 S. J. WAKIL, J. Am, Chem. Soc., 80 (I958) 6465. 3 M. FLAVIN, P. J. ORTIZ AND S. OCHOA, Nature, 176 (1955) 823. ¢ W. S. BECK, M. FLAVIN AND S. OCHOA, J. Biol. Chem., 229 (1957) 997. 5 W. S. BECK AND S. OCHOA, J. Biol. Chem., 232 (1958) 931. 6 R, W. SWlCK AND H. G. WOOD, Proc. Natl. Acad. Sci. U.S., 46 (196o) 28. S. GURNANI, S. P. 3~ISTRY AND B. C. JOHNSON, Biochim. Biophys. Acta, 38 (I96O) 187. 8 R. STJERNHOLM AND H. G. WOOD, Proc. Natl. Acad. Sci. U.S., 47 (1961) 3o3. 9 p. LENGVEL, R. MAZUMDER AND S. OCHOA, Proc. Natl. Acad. Sci. U.S., 46 (I96O) 1312. t0 W. B. SCHAEFER, J. Bacteriol., 73 (1955) 52. u E. STADTMAN AND H. A. BARKER, J. Biol. Chem., 184 (195 o) 769. lz A. R. THOMPSON, Australian J. Sci. Research, B, 4 (195o) 18o.
Received March 24th, 1962 Biochim. Biophys. Acta, 64 (1962) 174-177
sc 11005
Kinetic characterization of lactate dehydrogenase of normal and virus.infected chorioallantoic membrane of the chick embryo We have repeatedly observed that the concentration of glycolytic enzymes is markedly increased in chorioallantoic membranes immediately preceding and during rapid virus multiplication 1-3. The characteristic increase in glycolytic activity, most probably caused by an increased rate of synthesis of glycolytic enzymes, is accompanied by the appearance of aerobic glycolysis, normally absent in this tissue, and an increased turnover of the hexose monophosphate pathway of carbohydrate oxidation, without the concomitant increase in the amount of enzymes of the hexose 6-phosphate-oxidizing metabolic cycles. The key to this increased rate of hexose monophosphate oxidation is the increased amount of lactate dehydrogenase (Llactate: DPN + oxidoreductase, EC 1.1.1.29), capable of oxidizing T P N H by pyruvate, thus activating hexose 6-phosphate oxidation by the removal of the normally ratelimiting T P N H *. We have previously proposed that the increased rate of enzyme formation which occurs after virus infection may be due to a direct effect of some component(s) of the infective virus on the enzyme-synthesizing system of the chorioallantoic cells. The new steady-state of metabolism of the infected cell, reached as a consequence of the increased quantity of certain enzyme proteins, is then sufficient "biochemical cause" to affect cell growth or the synthesis of viral material s . This working hypothesis requires a detailed testing at every level of its complexity. We were first interested to discover whether or not a newly formed enzyme is a catalytically different protein from the one present in the uninfected tissue. Since increase in lactate dehydrogenase plays a central role in the alteration of carbohydrate Biochim. Biophys..4cta, 64 (I962) 177-179
178
SHORT COMMUNICATIONS
m e t a b o l i s m o f t h e c h o r i o a l l a n t o i c t i s s u e , t h i s e n z y m e w a s c h o s e n for p r e l i m i n a r y studies. A n a l o g s o f p y r i d i n e n u c l e o t i d e s , i n t r o d u c e d b y KAPLAN a n d h i s associates4, 5 h a v e b e e n u s e d as k i n e t i c p a r a m e t e r s
for l a c t a t e d e h y d r o g e n a s e s . An a r b i t r a r y
a m o u n t o f t h e s e a n a l o g s w a s e m p l o y e d b y o t h e r w o r k e r s a n d tile r e l a t i v e r a t e s o f r e a c t i o n s w e r e c o n s i d e r e d t o b e s u f f i c i e n t f o r t h e c h a r a c t e r i z a t i o n o f d e h y d r o g e n a s e s ~. I t s e e m s t o u s t h a t t h i s u s e o f p y r i d i n e n u c l e o t i d e a n a l o g s s u f f e r s f r o m t h e disa d v a n t a g e t h a t t h e a r b i t r a r i l y c h o s e n c o n c e n t r a t i o n s m a y or m a y n o t s a t u r a t e t h e e n z y m e , o r e v e n c a u s e e n z y m e i n h i b i t i o n {in c a s e o f a n a b n o r m a l k i n e t i c b e h a v i o r as f o u n d w i t h m a n y c o e n z y m e s ) , so t h a t r e a c t i o n r a t e s e x p r e s s e d u n d e r s u c h c o n ditions h a v e a l i m i t e d m e a n i n g . In o r d e r to avoid these possibile difficulties we h a v e determined the apparent Michaelis constant of each coenzyme, coenzyme analog and substrate at various pH values. In addition chromatographic isolation of lactate dehydrogenase of normal and virus-infected tissues was attempted,
in o r d e r t o
a s c e r t a i n w h e t h e r or n o t several p r o t e i n c o m p o n e n t s w i t h e n z y m i c activities could b e o b t a i n e d . I f t h i s is n o t d o n e , o n e m a y b e f a c e d w i t h c o n f u s i n g k i n e t i c d a t a , s i n c e
TABLE I KINETIC
CONSTANTS
OF
LACTATE
DEHYDROGENASE
OF THE
CHICK
OF
CHORIOALLANTOIC
MEMBRANES
EMBRYO
All measurements were carried out with the Zeiss PMQn spectrophotometer, in I-ml cuvettes (light path I c m ) . Buffers were o.i M phosphate (pH 6.o and 7.4) and Tris (pH 9.0). Km values refer to the first compound shown in Column I ("Substrate"), while the component in brackets indicates conditions of the reaction./~m determinations were carried out under conditions yielding Vmax. with respect to the second component, which was kept at constant concentrations. All coenzymes and analogs were purchased from Pabst Laboratories (Milwaukee, Wise.). Vmax (in terms of/*moles coen~ymelmg #~otein/min)
K m (M) "Substrate"
pH
Normal Pyruvate (DPNH) Pyruvate (DPNH) Pyruvate (TPNH) Pyruvate (TPNH) D P N H (pyruvate) D P N H (pyruvate) T P N H (pyruvate) T P N H (pyruvate) Lactate (DPN+) DPN+ (lactate) A P D P N +** (lactate) Lactate (APDPN+) D es-NH2-DPN +*** (lactate) Lactate (des-NH2-DPN+) SH-DPN+§ (lactate) Lactate (SH-DPN+)
7-4 6.0 7-4 6.0 7.4 6.o 7-4 6.0 9.0 9.0 9-0 9.0 9.o 9.0 9.o 9 .o
5.55' lO-5 1.62- lO -5 3.7 '1o-a 4.o • lO-4 I.O - io -~ 1.54. lO -5 5.o • IO-5 2.5 " lO -5 2.5 "1o -3 1.58. lO -5 2-33" lO-5 1.34. lO-a 3.2 • IO-4 2.75" lO -3 2.7 "io -4 3.3 "IO-2
Virus-infected* 5.55" lO-5 1.62" lO -5 2.o - IO-3 4.0 - lO-4 I.O
" I O
- 6
Normal
Virusinfected*
o-525 o.295 o.o28 0.054
1.66 o.91o o.o55 o.195
- -
- -
1.54. lO -5 3.3 " I O - 5
__
__
--
--
2,O
--
--
" 10 -5
2.5 " IO--3 1.58. 10 -5 2,33. IO-5 1.34"
1°-3
3.6 " lO -4 2.75. lO -8 3.3 " lO-4 3.4
" 1°-2
O.128 __ 0.036 --
o.143 __ o,147 --
0-357 -O.133 --
0.660 __ o.50o --
* The tissues were harvested 48 h after inoculation with the virus. ** Acetylpyridine analog of DPN +. *** Deamino_DPN +. § Thionicotinamide-DPN+.
Biochim. Biophys. Acta, 64 (1962) 177-179
179
SHORT COMMUNICATIONS
a mixture of several lactate dehydrogenases with various kinetic constants may simulate "different species" of enzymes. The preparation and collection of normal, and avian pox-virus infected chorioallantoic membranes of chick embryos was carried out as described earlier 3 except on a large scale (14 dozen eggs were used for each experiment). The pooled, frozen tissues were homogenized in o.15 M KC1 in a blendor; particulate material was removed by high-speed centrifugation (2o ooo × g for 3 ° min at o°), then the soluble protein was precipitated by saturation with solid (NH4)2SO 4. The precipitate, dissolved in a minimum volume of deionized water, was freed from salts by passing through a Sephadex G-25 column. Enzyme assays were performed by the spectrophotometric measurements of either the reoxidation of reduced coenzymes by pyruvate or the reduction of oxidized coenzymes or coenzyme analogs by lactate. The protein solution was subjected to gradient-elution chromatography on DEAE-cellulose and the enzyme eluted with o.oo5 M potassium phosphate of pH 7.2. This procedure yielded a 5-fold purification of the lactate dehydrogenase, obtained from both normal and virus-infected tisues, without any sign of separation into enzymically active protein components. It is noteworthy that while the D P N H - p y r u v a t e reaction exhibits marked substrate inhibition with concentrations of pyruvate above IO-4 M, such inhibition by pyruvate does not exist in case of the T P N H - p y r u v a t e reaction. This kinetic behavior stresses the important role of this reaction (i.e., T P N H - p y r u v a t e ) in tissues where increased glycolysis yields high concentrations of pyruvate. From these results, summarized in Table I, it is concluded that the lactate dehydrogenase formed in the chorion tissue of chick embryo after infection with canary pox-virus is kinetically identical, with the enzyme, present in non-infected tissues. This work was supported by grants. C-468I and C-5884, C-32II of the U.S. Public Health Service and by a grant of the American Heart Association, Incorporated.
Departments of Pharmacology and Biochemistry, University of California, School of Medicine, San Francisco, Calif., and
ERNEST KUN D. W. FANSHIER JUNE E. AYLING
Department of Pathology, University of Oregon Medical School, Portland, Oreg. (U.S.A.)
B E N J A M I N V, SIEGEL
KoN AND M. H. D. SMITH, Proc. Soc. Exptl. Biol. Med., 73 (195 o) 628. H. D. SMITH AND E. I{UN, Brit. J. Exptl. Pathol., 34 (1954) I. KoI% J. E. AYLING AND B. V. SIEGEL, Proc. Natl. Acad. Sci. U.S., 46 (196o) 622. O. KAPLAN, in G. E. W. WOLSTENHOLME AND C. i . O'CONNOR, The steric mechanisms involved in the reaction of lactic acid, Ciba F o u n d a t i o n , S t u d y G r o u p No. 2. March 17, 1959. Little, B r o w n and Co., Boston. •. O. KAPLAN AND M. M. CIOTTI, Biochim. Biophys. Acta, 49 (I96I) 425 .
1 E. 2 M. 3 E. ¢ N.
Received March I2th, 1962. Biochim. Biophys. Acta, 64 (1962) I 7 7 - [ 7 9