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Studies on activities of gluconeogenic enzymes in sheep liver
190
SHORT COMMUNICATIONS
BBA 23 23Z
Studies on activities of gluconeogenic enzymes in sheep liver In ruminating animals such as the sheep and the cow no glucose is absorbed from the rumen and it must be therefore synthesized exclusively from simple precursors. Cellulose and other carbohydrates are metabolized into acetate, butyrate, propionate and other metabolites in the rumen of the sheep and cow and these metabolites serve as the major source of energy and as precursors for glucose synthesis. Metabolism of acetate and butyrate has been extensively studied in these animals l a, and it has been shown that in sheep and cow liver acetate and butyrate can be rapidly utilized in vitro and that carbon from butyrate is incorporated into glucose 1. in the past few years gluconeogenesis has been a very active area of investigation under various experimental conditions and it is well documented now that pyruvate carboxylase (EC 6.4.i.I ) (refs. 4, 5) ~-ketoglutarate carboxylase 6, phosphoenolpyruvate carboxykinase (EC 4.I.1.32) (refs. 7, 8) glucose-6-phosphatase (EC 3.1.3.9) (ref. 9) and fructose-x,6-diphosphatase (EC 3.I.3.xi) (ref. IO) play a key role in gluconeogenesis and it was, therefore, of interest to measure these enzymes in sheep liver. In the work reported here these various key gluconeogenie enzymes have been assayed in normal sheep livers. Normal rat and sheep liver was obtained by killing the animals by decapitation and the livers were excised and homogenized (x g of wet liver/3.o ml of o.i54 M KC1 containing nicotinamide) and the homogenate was centrifuged at 6oo "7 g for IO min. The supernatant obtained after the removal of nuclei was centrifuged at I2 ooo , ,v for I5 min, and the mitochondrial fraction was resuspended in KCI and sonicated in Raytheon sonic oscillator and recentrifuged at 2o ooo ), g for 15 min. The supernatant obtained was assayed for pyruvate carboxylase activity by incubating an aliquot of supernatant fraction (o. 7 mg protein) with 2o/~moles of potassium pyruvate, 5o t~moles of NaH~4COa (5.o I~C) 3.3/zmoles of MgClz, 1.25/,moles ATP, o.38/,moles acetyl-CoA and 5o ~moles of Tris-HC1 buffer (pH 7.6) At the end of IO rain the / incubation mixture was deproteinized by precipitation with o.5 ml of IO o,o trichloroacetic acid and gassed with CO 2 for 3 rain. All steps were carried out at 2 ~'. Fixed 14CO2 was counted from an aliquot of filtrate in an anthracene-packed cuvette in a Packard Tricarb scintillation counter, c~-Ketoglutarate carboxylase activity was measured by incubating the extract obtained from the mitochondria by sonication (o.7 mg protein) with 4 °/zmoles of c~-ketoglutarate, 5o ~moles of NaH14COa (5.o ILC), 3.o/,moles MgC12, 1.25/xmoles ATP, 2.o/,moles T P N H in phosphate buffer (pH 7.~)). fixed CO,, was then assayed as described earlier. Phosphoenolpyruvate carboxykinase was assayed on the Io5 ooo :," g supernatant as described earlierL The results of these studies are given in Table I. Glucose-6-phosphatase activity was measured in the whole homogenate incubated at 3 °0 in citrate buffer (pH 6.4) and determined as P~ reteased from glucose-6-phosphate per g wet liver per 3o min incubation. Fructose1,6-diphosphatase activity was measured in the whole homogenate in glycine buffer (pH 9-4) with fructose diphosphate and Mg 2+, and the results are expressed as/~moles PI released from fructose-i,6-diphosphate per g wet liver per 3o min. These results are included in Table I. It m a y be seen from Table I that pyruvate carboxylase, phosphoenolpyruvate carboxykinase and ~-ketoglutarate carboxylase are present in the sheep-liver prepaBiochim. Biophys. Acla, i2I (i966) 1 9 o - i 9 I
SHORT COMMUNICATIONS
191
TABLE I ACTIVITIES OF PHOSPHOENOLPYRUVATE CARBOXYKINAS1~, PYRUVATE CARBOXYLASE, ~-KETOGLUTARATE CARBOXYLASE, GLUCOSE-6-PHOSPHATASE AND FRUCTOSE-I,6-DIPHOSPHATASE IN NORMAL SHEEP AND R A T L I V E R
Enzymes
P h o s p h o e n o l p y r u v a t e carboxykinase* P y r u v a t e carboxylase* a - K e t o g l u t a r a t e carboxylase* Glucose-6-phosphatase * * Fructose-I,6-diphosphatase~*
Activity in sheep liver
rat liver
12.6 2o.2 24. 5 74 ° 463
5.2 lO.5 12.8 36o 256
~~ ~: 4±
1. 4 2. 3 1.8 55 56
-~ ± ~ ~ -L
4.6 0.9 1.2 48 25
* Activity of these enzymes is expressed a s / z m o l e s CO 2 fixed per g w e t liver per h. ** Activity of these enzymes is expressed a s / , m o l e s Pt released per g per 3o min.
rations and that their activity per gram liver is twice as great as that for rat-liver preparations. Both glucose-6-phosphatase and fructose-I,6-diphosphatase show a similar pattern. CO s fixation may play a key role in the formation of oxaloacetate, and phosphoenolpyruvate n from various precursors such as propionate, pyruvate and a-ketoglutarate for the conversion to glucose. The increased activity of various gluconeogenic enzymes in sheep liver is thus explained as the sheep is called upon to synthesize its entire blood glucose, no glucose being absorbed from the rumen. The various keto-acid carbons must play an active role in this process in the sense that they provide the influx of carbon into the cycle which is necessary for the biosynthesis of glucose and amino acids. This investigation was supported by a Public Health Service Research Grant No. AM-o479 o and AM-o8242 for the National Institute of Arthritic and Metabolic Diseases and Training grant No. GM-953. One of us (P. N.) is a Predoctoral Fellow supported by Training Grant No. GM-953.
Department of Pharmacology, Indiana University Medical School, Indianapolis, Ind. (U.S.A.)
S. R. WAGLE P, NELSON
I A. L. BLACK,M. t~LEIBER AND A. M. BROWN, j . Biol. Chem., 236 (1961) 2399. 2 E. S. HOLDSWORTH, E. NEVILLE, C. NADER, I. G. JARRETT AND O. H. FILSELL, Biochim. Biophys. Acta, 86 (1964) 240. 3 H. W. ESSIG, H. W. NORTON AND B. C. JOHNSON, Proc. Soc. Exptl. Biol. Med., lO8 (1961) 194. 4 S. R. WAGLE, Biochim. Biophys. Res. Commun., 14 (1964) 533. 5 H . V . HENNING, I. SEIFFERT AND W. SEUBERT,Biochim. Biophys. Acta, 77 (1963) 345. 6 S. R. WAGLE, Biochim. Biophys. Aeta, 97 (1965) 142. 7 S. R. WAGLE AND J. ASHMORE, Bioehim. Biophys. Acta, 74 (1963) 564 • 8 ~xT.SHARAGO, H. A. LARDY, R. E. NORDIC AND D. FOSTER, j . Biol. Chem., 238 (1963) 36o3 . 9 J- ASHMORE, A. B. HASTINGS AND F. B. NESBETT, Proc. Natl. Acad. Sci. U.S., 4 ° (1954) 673. IO L. C. 1VIOKRASCH, W. D. DAVIDSON AND R. W. MCGILVERY, J. Biol. Chem., 222 (1956) 179. n S. R. WAGLE, Diabetes, 15 (1966) 19 .
Received January 24th, 1966 Biochim. Biophys. Acta, 121 (1966) i 9 o - i 9 i
SHORT COMMUNICATIONS
BBA 23 23Z
Studies on activities of gluconeogenic enzymes in sheep liver In ruminating animals such as the sheep and the cow no glucose is absorbed from the rumen and it must be therefore synthesized exclusively from simple precursors. Cellulose and other carbohydrates are metabolized into acetate, butyrate, propionate and other metabolites in the rumen of the sheep and cow and these metabolites serve as the major source of energy and as precursors for glucose synthesis. Metabolism of acetate and butyrate has been extensively studied in these animals l a, and it has been shown that in sheep and cow liver acetate and butyrate can be rapidly utilized in vitro and that carbon from butyrate is incorporated into glucose 1. in the past few years gluconeogenesis has been a very active area of investigation under various experimental conditions and it is well documented now that pyruvate carboxylase (EC 6.4.i.I ) (refs. 4, 5) ~-ketoglutarate carboxylase 6, phosphoenolpyruvate carboxykinase (EC 4.I.1.32) (refs. 7, 8) glucose-6-phosphatase (EC 3.1.3.9) (ref. 9) and fructose-x,6-diphosphatase (EC 3.I.3.xi) (ref. IO) play a key role in gluconeogenesis and it was, therefore, of interest to measure these enzymes in sheep liver. In the work reported here these various key gluconeogenie enzymes have been assayed in normal sheep livers. Normal rat and sheep liver was obtained by killing the animals by decapitation and the livers were excised and homogenized (x g of wet liver/3.o ml of o.i54 M KC1 containing nicotinamide) and the homogenate was centrifuged at 6oo "7 g for IO min. The supernatant obtained after the removal of nuclei was centrifuged at I2 ooo , ,v for I5 min, and the mitochondrial fraction was resuspended in KCI and sonicated in Raytheon sonic oscillator and recentrifuged at 2o ooo ), g for 15 min. The supernatant obtained was assayed for pyruvate carboxylase activity by incubating an aliquot of supernatant fraction (o. 7 mg protein) with 2o/~moles of potassium pyruvate, 5o t~moles of NaH~4COa (5.o I~C) 3.3/zmoles of MgClz, 1.25/,moles ATP, o.38/,moles acetyl-CoA and 5o ~moles of Tris-HC1 buffer (pH 7.6) At the end of IO rain the / incubation mixture was deproteinized by precipitation with o.5 ml of IO o,o trichloroacetic acid and gassed with CO 2 for 3 rain. All steps were carried out at 2 ~'. Fixed 14CO2 was counted from an aliquot of filtrate in an anthracene-packed cuvette in a Packard Tricarb scintillation counter, c~-Ketoglutarate carboxylase activity was measured by incubating the extract obtained from the mitochondria by sonication (o.7 mg protein) with 4 °/zmoles of c~-ketoglutarate, 5o ~moles of NaH14COa (5.o ILC), 3.o/,moles MgC12, 1.25/xmoles ATP, 2.o/,moles T P N H in phosphate buffer (pH 7.~)). fixed CO,, was then assayed as described earlier. Phosphoenolpyruvate carboxykinase was assayed on the Io5 ooo :," g supernatant as described earlierL The results of these studies are given in Table I. Glucose-6-phosphatase activity was measured in the whole homogenate incubated at 3 °0 in citrate buffer (pH 6.4) and determined as P~ reteased from glucose-6-phosphate per g wet liver per 3o min incubation. Fructose1,6-diphosphatase activity was measured in the whole homogenate in glycine buffer (pH 9-4) with fructose diphosphate and Mg 2+, and the results are expressed as/~moles PI released from fructose-i,6-diphosphate per g wet liver per 3o min. These results are included in Table I. It m a y be seen from Table I that pyruvate carboxylase, phosphoenolpyruvate carboxykinase and ~-ketoglutarate carboxylase are present in the sheep-liver prepaBiochim. Biophys. Acla, i2I (i966) 1 9 o - i 9 I
SHORT COMMUNICATIONS
191
TABLE I ACTIVITIES OF PHOSPHOENOLPYRUVATE CARBOXYKINAS1~, PYRUVATE CARBOXYLASE, ~-KETOGLUTARATE CARBOXYLASE, GLUCOSE-6-PHOSPHATASE AND FRUCTOSE-I,6-DIPHOSPHATASE IN NORMAL SHEEP AND R A T L I V E R
Enzymes
P h o s p h o e n o l p y r u v a t e carboxykinase* P y r u v a t e carboxylase* a - K e t o g l u t a r a t e carboxylase* Glucose-6-phosphatase * * Fructose-I,6-diphosphatase~*
Activity in sheep liver
rat liver
12.6 2o.2 24. 5 74 ° 463
5.2 lO.5 12.8 36o 256
~~ ~: 4±
1. 4 2. 3 1.8 55 56
-~ ± ~ ~ -L
4.6 0.9 1.2 48 25
* Activity of these enzymes is expressed a s / z m o l e s CO 2 fixed per g w e t liver per h. ** Activity of these enzymes is expressed a s / , m o l e s Pt released per g per 3o min.
rations and that their activity per gram liver is twice as great as that for rat-liver preparations. Both glucose-6-phosphatase and fructose-I,6-diphosphatase show a similar pattern. CO s fixation may play a key role in the formation of oxaloacetate, and phosphoenolpyruvate n from various precursors such as propionate, pyruvate and a-ketoglutarate for the conversion to glucose. The increased activity of various gluconeogenic enzymes in sheep liver is thus explained as the sheep is called upon to synthesize its entire blood glucose, no glucose being absorbed from the rumen. The various keto-acid carbons must play an active role in this process in the sense that they provide the influx of carbon into the cycle which is necessary for the biosynthesis of glucose and amino acids. This investigation was supported by a Public Health Service Research Grant No. AM-o479 o and AM-o8242 for the National Institute of Arthritic and Metabolic Diseases and Training grant No. GM-953. One of us (P. N.) is a Predoctoral Fellow supported by Training Grant No. GM-953.
Department of Pharmacology, Indiana University Medical School, Indianapolis, Ind. (U.S.A.)
S. R. WAGLE P, NELSON
I A. L. BLACK,M. t~LEIBER AND A. M. BROWN, j . Biol. Chem., 236 (1961) 2399. 2 E. S. HOLDSWORTH, E. NEVILLE, C. NADER, I. G. JARRETT AND O. H. FILSELL, Biochim. Biophys. Acta, 86 (1964) 240. 3 H. W. ESSIG, H. W. NORTON AND B. C. JOHNSON, Proc. Soc. Exptl. Biol. Med., lO8 (1961) 194. 4 S. R. WAGLE, Biochim. Biophys. Res. Commun., 14 (1964) 533. 5 H . V . HENNING, I. SEIFFERT AND W. SEUBERT,Biochim. Biophys. Acta, 77 (1963) 345. 6 S. R. WAGLE, Biochim. Biophys. Aeta, 97 (1965) 142. 7 S. R. WAGLE AND J. ASHMORE, Bioehim. Biophys. Acta, 74 (1963) 564 • 8 ~xT.SHARAGO, H. A. LARDY, R. E. NORDIC AND D. FOSTER, j . Biol. Chem., 238 (1963) 36o3 . 9 J- ASHMORE, A. B. HASTINGS AND F. B. NESBETT, Proc. Natl. Acad. Sci. U.S., 4 ° (1954) 673. IO L. C. 1VIOKRASCH, W. D. DAVIDSON AND R. W. MCGILVERY, J. Biol. Chem., 222 (1956) 179. n S. R. WAGLE, Diabetes, 15 (1966) 19 .
Received January 24th, 1966 Biochim. Biophys. Acta, 121 (1966) i 9 o - i 9 i