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Distribution Studies of Glycine and Serine Enzymes in Chicks
Distribution Studies of Glycine and Serine Enzymes in Chicks C. N . C O O N * AND J. R. COUCH
Department of Poultry Science, Texas Agricultural Experiment Station, Texas A&M College Station, Texas 77843
University,
(Received for publication April 4, 1974)
POULTRY SCIENCE 54: 116-118, 1975
INTRODUCTION
T
HE capacity for glycine synthesis has been reported to be greater in mature male chickens than in adult rats (Sugahara and Ariyoshi, 1967). The requirement of glycine or serine in broiler rations have been consistently shown for optimum growth. In addition to the synthesis of serine from glycine in the chicken liver (Scrimgeour and Huennekens, 1960), serine can be formed from amino nitrogen and glucose via a "phosphorylated" or a "nonphosphorylated" pathway (Walsh and Sallach, 1966). In chicken liver both pathways exist but the enzymes of the phosphorylated pathway have a higher activity. In other vertebrates, the workers have found that the relative activities of the two pathways differed in various organs. The purpose of this investigation was to evaluate each target organ for glycine and serine metabolic enzymatic activity. MATERIALS AND METHODS Twenty crossbred day-old broiler chicks were placed in electrically heated chick batteries with raised screen floors and fed a *Presently with the Department of Poultry Nutrition, University of Maryland, College Park, Maryland, 20742.
chick starter (Table 1) and water ad libitum for three weeks. The chicks were then sacrificed by cervical dislocation and liver, kidney, heart, spleen and brain removed for enzyme determination. The extracted organs from ten chicks were placed in iced 0.1 M potassium phosphate buffer and the remaining chick organs were frozen in dry ice and reagent grade acetone at —60° C. The tissues in cold 0.1 M potassium phosphate were prepared and assayed for D-3phosphoglycerate dehydrogenase (E.C. 1.1.l.s), serine dehydratase (E.C. 4.2.1.13), and phosphoserine phosphatase (E.C. 3.1.3.3) by the method of Fallon et al. (1966). The supernatant from the phosphate buffer preparations was utilized for the determination of glycerate dehydrogenase by the method of Willis and Sallach (1964). Acetone powders of the organ tissues were prepared by freezing in dry ice and acetone by the procedure of Blakely (1954). Hydroxypyruvate-Prglutamate transaminase and serine transhydroxymethylase (E.C. 2.1.2.1) activities were determined from the powdered tissue by the method of Walsh and Sallach (1966) and Schirch and Mason (1962), respectively. Protein determinations were made according to the method of Lowry et al. (1951) using
116
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ABSTRACT Day-old broiler type chicks were fed a practical starter ration for three weeks, sacrificed and the D-3-phosphoglycerate dehydrogenase (E.C. 1.1.1 .s), phosphoserine phosphatase (E.C. 3.1.3.3), glycerate dehydrogenase (E.C. 1.1.1.29), hydroxypyruvate-P:glutamate transaminase, serine transhydroxymethylase (E.C. 2.1.2.1), and serine dehydratase (E.C. 4.2.1.13) enzyme activities were determined in liver, kidney, heart, spleen and brain tissue in evaluating glycine and serine metabolism. The distribution of the various glycine and serine biosynthetic enzymes in the chick tissues studied show that the major portion of glycine and serine synthesis occurs in hepatic tissue via the "phosphorylated pathway."
117
GLYCINE AND SERINE ENZYMES
TABLE 1.—Composition of chick starter diet Ingredient
g-/kg. 260.0 268.2 323.2 61.5 48.5 25.0 2.5 0.3 0.3 0.5 10.0
1 The vitamin mixture fortified each kg. of diet with the following: 7 mg. thiamine • HC1, 7 mg. riboflavin, 8 mg. pyridoxine. HC1, 60 mg. niacin, 30 mg. D-calcium pantothenate, 2 mg. folic acid, 2 mg. menadione sodium bisulfite, 220 (i.g. vitamin B 12 , 9000 I.U. vitamin A palmitate, 450 I.C.U. vitamin D 3 , 44 I.U. dl-a-tocopherol acetate, and 1.65 gm. choline chloride.
crystalline bovine serum albumin as a standard. RESULTS AND DISCUSSION D-3-phosphoglycerate dehydrogenase activity was found in all organs studied (Table
TABLE 2.—Organ distribution of glycerate dehydrogenase, hydroxypyruvate-P:glutamate transaminase, serine transhydroxymethylase, serine dehydratase, D-3-phosphoglycerate dehydrogenase and phosphoserine phosphatase activities in 3-week old chicks Enzymes Liver
Kidney
Specific activity' Brain Spleen
D-3-phosphoglycerate dehydrogenase mn.. Moles NAD/mg. pro9.7 ± .3 4.3 ± .3 tein/hour x 10 " 3 Phosphoserine phosphatase m(x. 62 ± 4 145 ± 11 Moles Pi/mg. protein/hour Glycerate dehydrogenase my.. Moles 259 ± 28 177 ± 16 NADH/mg. protein/hour Hydroxypyruvate-P:glutamate transaminase mpu Moles a-ketoglutarate/mg. protein/hour 120 ± 7 91 ± 5 Serine transhydroxymethylase m(ju Moles TPNH/mg. protein/hour x 205 ± 16 66 ± 5 10"' Serine dehydratase m|x. Moles 73 ± 5 242 ± 19 NAD/mg. protein /hour 'Means of two observations/tissue ± SEM. Each observation T h e dash represents activity too low to measure.
3.4 ± .2 34 ± 4
2.2 ± .1
*
100 ± 11
63 ± 6
86 ± 4
7±1 41 ± 3
consisted of five chicks.
Heart 1.7 ± .1 18 ± 2
*
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on November 8, 2014
Corn Milo Soybean meal (50% protein) Fish meal (65% protein) Soybean oil Defluorinated rock phosphate Salt Zinc oxide Manganese sulfate Sulfaquinoxoline Vitamin mixture'
2). The activity decreased progressively in liver, kidney, brain, spleen and heart tissues. The first enzyme in the ' 'nonphosphorylated'' pathway, glycerate dehydrogenase (E.C. 1.1.1.29), is found in the same relative proportions in the various tissues as is D-3phosphoglycerate dehydrogenase (Table 2). There was no measurable activity of this enzyme in the heart tissue (Table 2). A general observation in comparing the first enzyme of the "nonphosphorylated" and "phosphorylated" pathways is that glycerate dehydrogenase has 26.7 fold higher activity than D-3-phosphoglycerate dehydrogenase in chick liver. Hydroxypyruvate-P:glutamate transaminase is the second enzyme in the "phosphorylated" pathway for serine biosynthesis. The activity for this enzyme was highest in hepatic tissue; kidney and brain each had approximately 2 / 3 of the activity of the liver (Table 2). Hydroxypyruvateialanine transaminase was not determined because previous reports have shown avian hepatic tissue contained only minor amounts (Walsh and Sallach, 1966).
118
C. N. COON AND J. R. COUCH
Phosphoserine phosphatase and serine dehydratase activity in chick tissues was 2 to 3-fold higher in kidney as compared to activity in the liver (Table 2). The spleen was the only organ studied which contained no measurable phosphoserine phosphatase activity. Serine dehydratase activity was not observed in the brain, spleen or heart (Table 2). Serine transhydroxymethylase, the reversible enzyme for glycine and serine intercon-
found in kidney and spleen tissues (Table 2). The activity for this enzyme was not evident in brain and heart tissue. The distribution studies of the various glycine and serine biosynthetic enzymes in chicks demonstrate that the major portion of glycine and serine synthesis occurs in the hepatic tissue
via the
"phosphorylated"
pathway. The catabolism of serine to pyruvate occurs primarily in the kidney. REFERENCES Blakely, R. L., 1954. The interconversion of serine
NEWS AND NOTES (Continued from page 115)
necessary expertise to local people. ' 'The Shaver Poultry Breeding Farms make an award annually to a graduate student in the Department of Animal and Poultry Science, Ontario Agricultural College, to assist in research and training. A similar fellowship is awarded to a graduate student in a department of the Ontario Veterinary College who is pursuing studies related to poultry diseases. In addition, generous contributions of stock for experimental purposes have been made. "It is fitting that Donald McQueen Shaver be awarded an O.A.C. Centennial Medal for his contributions to the poultry industry throughout the World, and to this campus."
D. C. WARREN HONORED Dr. D. C. Warren, Fremont, California, received two honours in 1972, a D.Sc. degree at Indiana University, and an Award for Distinguished Service in the Field of Agriculture at Kansas State University. The citation for the Doctor of Science degree read, in part: "An eminent geneticist who was named to the Agricultural Hall of Fame in 1971 for his contributions in the field of poultry breeding, Dr. Don C. Warren grew up on a farm in eastern Indiana near Saratoga, where his chores included helping tend the family's flock of chickens. After graduation from high school he spent a summer at Indiana State Normal College and, thus equipped, became a teacher of a
(Continued on page 154)
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on November 8, 2014
version, activity was determined to be the highest in liver tissue, with moderate activity
and glycine:Role of pteroylglutamic acid and other cofactors. Biochem. J. 58: 448-462. Fallon, H. J., E. J. Hackney and W. L. Byrne, 1966. Serine biosynthesis in rat liver. J. Biol. Chem. 241: 4157-4166. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265. Schirch, L., and M. Mason, 1962. Serine transhydroxymethylase: Spectral properties of the enzymebound pyridoxal-5-phosphate. J. Biol. Chem. 237: 2578-2581. Scrimgeour, K. G., and F. M. Huennekens, 1960. Serine hydroxymethylase. Methods in Enzymology, Volume V, (editors S. P. Colawick and N. O. Kaplan) Academic Press, Inc., New York, pp. 838-843. Sugahara, M., and S. Ariyoshi, 1967. Metabolic amount of glycine in the rat. Agr. Biol. Chem. 31: 106-114. Walsh, D. A., and H. J. Sallach, 1966. Comparative studies on the pathways for serine biosynthesis in animal tissues. J. Biol. Chem. 241: 4068-4076. Willis, J. E., and H. J. Sallach, 1964. The occurrence of D-3-phosphoglycerate dehydrogenase in animal tissues. Biochim. Biophys. Acta, 81: 39-54.
Department of Poultry Science, Texas Agricultural Experiment Station, Texas A&M College Station, Texas 77843
University,
(Received for publication April 4, 1974)
POULTRY SCIENCE 54: 116-118, 1975
INTRODUCTION
T
HE capacity for glycine synthesis has been reported to be greater in mature male chickens than in adult rats (Sugahara and Ariyoshi, 1967). The requirement of glycine or serine in broiler rations have been consistently shown for optimum growth. In addition to the synthesis of serine from glycine in the chicken liver (Scrimgeour and Huennekens, 1960), serine can be formed from amino nitrogen and glucose via a "phosphorylated" or a "nonphosphorylated" pathway (Walsh and Sallach, 1966). In chicken liver both pathways exist but the enzymes of the phosphorylated pathway have a higher activity. In other vertebrates, the workers have found that the relative activities of the two pathways differed in various organs. The purpose of this investigation was to evaluate each target organ for glycine and serine metabolic enzymatic activity. MATERIALS AND METHODS Twenty crossbred day-old broiler chicks were placed in electrically heated chick batteries with raised screen floors and fed a *Presently with the Department of Poultry Nutrition, University of Maryland, College Park, Maryland, 20742.
chick starter (Table 1) and water ad libitum for three weeks. The chicks were then sacrificed by cervical dislocation and liver, kidney, heart, spleen and brain removed for enzyme determination. The extracted organs from ten chicks were placed in iced 0.1 M potassium phosphate buffer and the remaining chick organs were frozen in dry ice and reagent grade acetone at —60° C. The tissues in cold 0.1 M potassium phosphate were prepared and assayed for D-3phosphoglycerate dehydrogenase (E.C. 1.1.l.s), serine dehydratase (E.C. 4.2.1.13), and phosphoserine phosphatase (E.C. 3.1.3.3) by the method of Fallon et al. (1966). The supernatant from the phosphate buffer preparations was utilized for the determination of glycerate dehydrogenase by the method of Willis and Sallach (1964). Acetone powders of the organ tissues were prepared by freezing in dry ice and acetone by the procedure of Blakely (1954). Hydroxypyruvate-Prglutamate transaminase and serine transhydroxymethylase (E.C. 2.1.2.1) activities were determined from the powdered tissue by the method of Walsh and Sallach (1966) and Schirch and Mason (1962), respectively. Protein determinations were made according to the method of Lowry et al. (1951) using
116
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on November 8, 2014
ABSTRACT Day-old broiler type chicks were fed a practical starter ration for three weeks, sacrificed and the D-3-phosphoglycerate dehydrogenase (E.C. 1.1.1 .s), phosphoserine phosphatase (E.C. 3.1.3.3), glycerate dehydrogenase (E.C. 1.1.1.29), hydroxypyruvate-P:glutamate transaminase, serine transhydroxymethylase (E.C. 2.1.2.1), and serine dehydratase (E.C. 4.2.1.13) enzyme activities were determined in liver, kidney, heart, spleen and brain tissue in evaluating glycine and serine metabolism. The distribution of the various glycine and serine biosynthetic enzymes in the chick tissues studied show that the major portion of glycine and serine synthesis occurs in hepatic tissue via the "phosphorylated pathway."
117
GLYCINE AND SERINE ENZYMES
TABLE 1.—Composition of chick starter diet Ingredient
g-/kg. 260.0 268.2 323.2 61.5 48.5 25.0 2.5 0.3 0.3 0.5 10.0
1 The vitamin mixture fortified each kg. of diet with the following: 7 mg. thiamine • HC1, 7 mg. riboflavin, 8 mg. pyridoxine. HC1, 60 mg. niacin, 30 mg. D-calcium pantothenate, 2 mg. folic acid, 2 mg. menadione sodium bisulfite, 220 (i.g. vitamin B 12 , 9000 I.U. vitamin A palmitate, 450 I.C.U. vitamin D 3 , 44 I.U. dl-a-tocopherol acetate, and 1.65 gm. choline chloride.
crystalline bovine serum albumin as a standard. RESULTS AND DISCUSSION D-3-phosphoglycerate dehydrogenase activity was found in all organs studied (Table
TABLE 2.—Organ distribution of glycerate dehydrogenase, hydroxypyruvate-P:glutamate transaminase, serine transhydroxymethylase, serine dehydratase, D-3-phosphoglycerate dehydrogenase and phosphoserine phosphatase activities in 3-week old chicks Enzymes Liver
Kidney
Specific activity' Brain Spleen
D-3-phosphoglycerate dehydrogenase mn.. Moles NAD/mg. pro9.7 ± .3 4.3 ± .3 tein/hour x 10 " 3 Phosphoserine phosphatase m(x. 62 ± 4 145 ± 11 Moles Pi/mg. protein/hour Glycerate dehydrogenase my.. Moles 259 ± 28 177 ± 16 NADH/mg. protein/hour Hydroxypyruvate-P:glutamate transaminase mpu Moles a-ketoglutarate/mg. protein/hour 120 ± 7 91 ± 5 Serine transhydroxymethylase m(ju Moles TPNH/mg. protein/hour x 205 ± 16 66 ± 5 10"' Serine dehydratase m|x. Moles 73 ± 5 242 ± 19 NAD/mg. protein /hour 'Means of two observations/tissue ± SEM. Each observation T h e dash represents activity too low to measure.
3.4 ± .2 34 ± 4
2.2 ± .1
*
100 ± 11
63 ± 6
86 ± 4
7±1 41 ± 3
consisted of five chicks.
Heart 1.7 ± .1 18 ± 2
*
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on November 8, 2014
Corn Milo Soybean meal (50% protein) Fish meal (65% protein) Soybean oil Defluorinated rock phosphate Salt Zinc oxide Manganese sulfate Sulfaquinoxoline Vitamin mixture'
2). The activity decreased progressively in liver, kidney, brain, spleen and heart tissues. The first enzyme in the ' 'nonphosphorylated'' pathway, glycerate dehydrogenase (E.C. 1.1.1.29), is found in the same relative proportions in the various tissues as is D-3phosphoglycerate dehydrogenase (Table 2). There was no measurable activity of this enzyme in the heart tissue (Table 2). A general observation in comparing the first enzyme of the "nonphosphorylated" and "phosphorylated" pathways is that glycerate dehydrogenase has 26.7 fold higher activity than D-3-phosphoglycerate dehydrogenase in chick liver. Hydroxypyruvate-P:glutamate transaminase is the second enzyme in the "phosphorylated" pathway for serine biosynthesis. The activity for this enzyme was highest in hepatic tissue; kidney and brain each had approximately 2 / 3 of the activity of the liver (Table 2). Hydroxypyruvateialanine transaminase was not determined because previous reports have shown avian hepatic tissue contained only minor amounts (Walsh and Sallach, 1966).
118
C. N. COON AND J. R. COUCH
Phosphoserine phosphatase and serine dehydratase activity in chick tissues was 2 to 3-fold higher in kidney as compared to activity in the liver (Table 2). The spleen was the only organ studied which contained no measurable phosphoserine phosphatase activity. Serine dehydratase activity was not observed in the brain, spleen or heart (Table 2). Serine transhydroxymethylase, the reversible enzyme for glycine and serine intercon-
found in kidney and spleen tissues (Table 2). The activity for this enzyme was not evident in brain and heart tissue. The distribution studies of the various glycine and serine biosynthetic enzymes in chicks demonstrate that the major portion of glycine and serine synthesis occurs in the hepatic tissue
via the
"phosphorylated"
pathway. The catabolism of serine to pyruvate occurs primarily in the kidney. REFERENCES Blakely, R. L., 1954. The interconversion of serine
NEWS AND NOTES (Continued from page 115)
necessary expertise to local people. ' 'The Shaver Poultry Breeding Farms make an award annually to a graduate student in the Department of Animal and Poultry Science, Ontario Agricultural College, to assist in research and training. A similar fellowship is awarded to a graduate student in a department of the Ontario Veterinary College who is pursuing studies related to poultry diseases. In addition, generous contributions of stock for experimental purposes have been made. "It is fitting that Donald McQueen Shaver be awarded an O.A.C. Centennial Medal for his contributions to the poultry industry throughout the World, and to this campus."
D. C. WARREN HONORED Dr. D. C. Warren, Fremont, California, received two honours in 1972, a D.Sc. degree at Indiana University, and an Award for Distinguished Service in the Field of Agriculture at Kansas State University. The citation for the Doctor of Science degree read, in part: "An eminent geneticist who was named to the Agricultural Hall of Fame in 1971 for his contributions in the field of poultry breeding, Dr. Don C. Warren grew up on a farm in eastern Indiana near Saratoga, where his chores included helping tend the family's flock of chickens. After graduation from high school he spent a summer at Indiana State Normal College and, thus equipped, became a teacher of a
(Continued on page 154)
Downloaded from http://ps.oxfordjournals.org/ at Library, University of Guelph on November 8, 2014
version, activity was determined to be the highest in liver tissue, with moderate activity
and glycine:Role of pteroylglutamic acid and other cofactors. Biochem. J. 58: 448-462. Fallon, H. J., E. J. Hackney and W. L. Byrne, 1966. Serine biosynthesis in rat liver. J. Biol. Chem. 241: 4157-4166. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265. Schirch, L., and M. Mason, 1962. Serine transhydroxymethylase: Spectral properties of the enzymebound pyridoxal-5-phosphate. J. Biol. Chem. 237: 2578-2581. Scrimgeour, K. G., and F. M. Huennekens, 1960. Serine hydroxymethylase. Methods in Enzymology, Volume V, (editors S. P. Colawick and N. O. Kaplan) Academic Press, Inc., New York, pp. 838-843. Sugahara, M., and S. Ariyoshi, 1967. Metabolic amount of glycine in the rat. Agr. Biol. Chem. 31: 106-114. Walsh, D. A., and H. J. Sallach, 1966. Comparative studies on the pathways for serine biosynthesis in animal tissues. J. Biol. Chem. 241: 4068-4076. Willis, J. E., and H. J. Sallach, 1964. The occurrence of D-3-phosphoglycerate dehydrogenase in animal tissues. Biochim. Biophys. Acta, 81: 39-54.