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Comparative enzymolog of serine biosynthesis in mammalian systems
418
SHORT COMMUNICATIONS
BBA 23 254
Comparative enzymology of serine biosynthesis in mammalian systems Enzymic reactions that could provide two independent pathways for serine biosynthesis have been described in mammalian tissues. The first of these pathways involves the compounds D-3-phosphoglycerate, hydroxypyruvate-P and 1.-serine-P (phosphorylated pathway) 1. The second pathway involves the non-phosphorylated analogues, D-glycerate and hydroxypyruvate (non-phosphorylated).,,8.The availability of two pathways could be an important factor in regulating the celt's supply of serine. As an extension of a previous study ~ we have compared serine synthesis v i a both routes in cell-free extracts of cultured human cells (KB) and mammalian liver. The methods for the cultivation of KB cells and the preparation of cell-free extracts have been previously described 4. For the preparation of cell-free extracts from mammalian livers the organs were rapidly removed, frozen in solid CO,,, and stored at --6o °. When required, approx. I g of tissue was thawed and homogenized in 5 ml of o.5 M Tris-HC1 buffer (pH 7.5) with a Virtis homogenizer. In certain preparations Io mM dithiothreitol was included in the buffer. The subsequent steps in the preparation were performed as described for KB cells 4. D-~14C]3-phosphoglycerate synthesized according to PARK el al.a, was a gift from Dr. N. PoI~. D-[l~CJglycerate was prepared from D-~14CJ3-phosphoglycerate by enzymatic hydrolysis of the phosphate group with purified bacterial phosphatase (Worthington Biochemical Corp.). After the hydrolysis glycerate was purified by chromatography on a Dowex-I (formate) column. The column was eluted with a linear gradient of formic acid (o-o.25 M) and the D-[14C]glycerate eluted as a discrete peak when the formic acid concentration reached approx, o.Io M. Standard procedures were used for the measurement of protein 6 and radioactivity 4. Amino acid production was measured by incubating Ix~C 3-phosphoglycerate or D@4Clglycerate with cell-free extracts of the various tissues and fractionating the radioactive products by chromatography on Dowex-5o (H+). The amino acids produced were adsorbed to the resin at an acid pH and subsequently eluted with NH~OH. The chromatograt)hic procedure of ROBERTS et al. 7 was used for the fractionation of the amino acids and the identification of the radioactive components. The amino acids eluted from the original Dowex-5o (H +) column with NH4OH were evaporated to dryness, dissolved in o.5 ml of I. 5 M HC1, and added to a I cm i 5o cm column of Dowex-5o (H~), 2oo-4oo mesh. They were then eluted with I. 5 M HC1. Paper chromatography with phenol, saturated with water, confirmed the identity of the amino acids in the column fractions and separated serine from glutamate. The capacity to synthesize amino acids from phosphoglycerate and glycerate was measured in cell-free preparations from KB cells, two foetal guinea-pig livers, the corresponding maternal livers, and an adult rat liver. As shown in Table I all the tissues examined produced appreciable quantities of Ft4C]amino acids from L14Cphosphoglycerate, but failed to produce more than 3 re>motes of amino acid/mg of protein from [14CJglycerate. Characterization of the radioactive amino acids by column chromatograt)hy (Figs. IA, B) demonstrated that the preparations from KB cells and the foetal guineapig liver synthesize only serine, while that from the adult guinea pig synthesized both serine and alanine in equal amounts. The recoveries from the columns were 8o %,. Biochim. Biophys. Acta, 124 (1966) 418-42o
419
SHORT COMMUNICATIONS
88 % and 95 % for the material synthesized by the KB cells and the foetal and adult liver preparations, respectively. The column fractions containing the radioactive product of the KB cell extract were pooled and subjected to paper chromatography. Serine and glutamate were present with the radioactivity migrating solely as serine.
80C E
40( o
v
ADULT300 B 200 SerineA Alarinei\-
KB
120C Serine
~ 1oo
j
&
i
, oo ~
FOETAL-
~ 40c
Tube number
u
c
"G 2oc
.\
jo
Alaninell | n-
10
2o
3o
Tube number
Tube number Fig. I. C h r o m a t o g r a p h y of the amino acids formed from [14C]phosphoglycerate. A, P r o d u c t of K B cell extract; B, p r o d u c t of guinea-pig liver extract; C, p r o d u c t of rat-liver extracts. The flow rates of the different columns varied between 0.25 and o.3o ml/min. The fraction n u m b e r at which an amino acid eluted depends on the flow rate since fractions were always collected at I o - m i n intervals.
TABLE
I
AMINO ACID F O R M A T I O N
BY CELL-FREE
PREPARATIONS
FROM
MAMMALIAN
TISSUE
The reaction mixture (o.5 ml) contained 2.5 m M phosphoglycerate (2oooo counts/rain per #mole) or 2.5 mM glycerate (16000 c o u n t s / m i n per #,mole); 50 mM p h o s p h a t e buffer (pH 7.5); 20 mM g l u t a m a t e or L-alanine; 2.8 mM D P N ; 5 mM MgSO4; and 4 mM reduced glutathione. G l u t a m a t e was used w h e n phosphoglycerate was the s u b s t r a t e and alanine w h e n glycerate was the substrate. The s u b s t i t u t i o n of g l u t a m a t e for alanine did not increase the conversion of glycerate to amino acids. The reaction m i x t u r e was incubated for 3° rain at 37 °. After t e r m i n a t i o n of the reaction with 0. 5 ml of io % trichloroacetic acid, 5/*moles of L-serine was added as carrier and the protein was r e m o v e d b y centrifugation. The s u p e r n a t a n t fluid was placed on a i cm x 3 cm c o l u m n of Dowex-5o (H +) lOO-2OO mesh. The column was washed with 18 ml of o.oi M HC1 and 5 ml of HsO. The amino acids were eluted b y w a s h i n g the column with 7.5 ml of 1. 5 M NH4OH.
Tissue
[14C]Amino acid formed from (mlzmoles/mg protein) [laC]Phosphoglycerate D-El~C]Glycerate
KB* KB 47-day Foetal guinea pig* 59-day Foetal guinea pig Adult guinea pig I Adult guinea pig I I Adult rat
66 lo9"* 35 26** 23** 18 85**
3
* P r e p a r a t i o n s made w i t h o u t dithiothreitol in the buffers. ** The average of duplicate determinations, The experimental values agreed to within 5 %*** Assay not run.
Bioehim. Biophys. Aeta, 124 (1966) 418-42o
42o
SHORT COMMUNICATION b
While the activities shown in Table I could represent yields rather than initial biosynthetic rates, the capacity for serine synthesis per mg of protein appears greatest in KB cells and higher in foetal than in adult guinea-pig liver. When the rat liver extracts were incubated with [14C~phosphoglycerate and the products fractionated on a Dowex-5o (H +) column, over 5 ° % of the recovered radioactivity was eluted with the amino acids (Table I). The addition of ~o ~moles of serine-P or glycerate to the incubation did not reduce the yield of this material indicating that it was not a product of either the phosphorylated or the non-phosphorylated pathway. Column chromatography (Fig. IC) showed one component with the elution characteristics of alanine. The recovery of radioactivity in the alanine peak was 85 % and radioactive serine was not detected. Paper chromatography of the radioactive product either before or after column chromatography showed one component with the mobility of alanine. Our results indicate that the phosphorylated pathway is the major pathway to serine in polyploid (KB) and diploid (guinea-pig liver) cells. The failure to detect the phosphorylated pathway with the preparations from rat liver may be explained by the recent report that rats fed a commercial diet were deficient in phosphoglycerate dehydrogenase and serine-forming capacity, whereas these activities were present in animals fed a synthetic diet lacking serine and glycine 8. These experiments extend to whole animals the observations made with cultured cells that exogenous serine reduces the capacity for serine synthesis4, 9. The phosphorylated pathway has also been observed in preparations from rat brain ~°. The rat-liver preparation that failed to convert D-glycerate to serine contained 6 units of D-glycerate dehydrogenase (EC 1.1.1.29) per mg of protein when assayed according to WILLIS AND SALLACH3. It appears that the presence of enzyme activity alone is insufficient evidence that the non-phosphorylated pathway is functional and caution is required in interpreting variations in enzyme levels in terms of serine synthesis. This is particularly true if the animal had been provided with an exogenous source of serine. This investigation was supported by Research Grant AI-o3957 from the U.S. Public Health Service. We wish to acknowledge the technical assistance of Mrs. M. POTOCHNY and thank Dr. A. NEMETH for the guinea-pig livers.
Department of Microbiology, School of Medicine University of Pennsylvania, Philadelphia, Pa. (U.S.A.)
LEWIS I. PIZER
I A. ICHIHARA AND D. M. GREENBERG, J . Biol. Chem., 224 (I957) 331. 2 H . J. SALLACH, J. Biol. Chem., 223 (1956) t l O I . 3 J. E. VqlLLIS AND H. J. SALLACH, J. Biol. Chem., 237 (1962) 91o. 4 L. I. PIZER, J. Biol. Chem., 239 (1964) 4219. 5 R. B. PARK, N. G. [)ON, 1~. P. LOUWRIER AND ~{. CALVIN, Biochim. Biophys. Acta, 42 (196o) 27. 6 0 . H. LOWRY, N. J. ROSEBROUGH, A. 1~. FARR AND I{,. J. RANDALL, .]. Biol. Chem., 193 (1951) 265 . 7 R. ]3. ROBERTS, P. H. ABELSON, i). g . COWRIE, E. T. BOLTON AND R. J. BRITTEN, Carnegie Inst. Wash. Publ., 6o 7 (1955) 5 TM 8 T. TANAKA, Protein, Nucleic Acid, Enzyme, Tokyo, lO (1965) 068. 9 U . EAGLE, C. L. "WASHINGTON AND M. LEVY, J . Biol. Chem., 240 (1965) 3944. i o \ ¥ . F. BRIDGERS, J. Biol. Chem., 240 (1965) 4591.
Received March i7th, 1966 Biochim. Biophys. Acta, 124 (1966) 4 1 8 - 4 2 o
SHORT COMMUNICATIONS
BBA 23 254
Comparative enzymology of serine biosynthesis in mammalian systems Enzymic reactions that could provide two independent pathways for serine biosynthesis have been described in mammalian tissues. The first of these pathways involves the compounds D-3-phosphoglycerate, hydroxypyruvate-P and 1.-serine-P (phosphorylated pathway) 1. The second pathway involves the non-phosphorylated analogues, D-glycerate and hydroxypyruvate (non-phosphorylated).,,8.The availability of two pathways could be an important factor in regulating the celt's supply of serine. As an extension of a previous study ~ we have compared serine synthesis v i a both routes in cell-free extracts of cultured human cells (KB) and mammalian liver. The methods for the cultivation of KB cells and the preparation of cell-free extracts have been previously described 4. For the preparation of cell-free extracts from mammalian livers the organs were rapidly removed, frozen in solid CO,,, and stored at --6o °. When required, approx. I g of tissue was thawed and homogenized in 5 ml of o.5 M Tris-HC1 buffer (pH 7.5) with a Virtis homogenizer. In certain preparations Io mM dithiothreitol was included in the buffer. The subsequent steps in the preparation were performed as described for KB cells 4. D-~14C]3-phosphoglycerate synthesized according to PARK el al.a, was a gift from Dr. N. PoI~. D-[l~CJglycerate was prepared from D-~14CJ3-phosphoglycerate by enzymatic hydrolysis of the phosphate group with purified bacterial phosphatase (Worthington Biochemical Corp.). After the hydrolysis glycerate was purified by chromatography on a Dowex-I (formate) column. The column was eluted with a linear gradient of formic acid (o-o.25 M) and the D-[14C]glycerate eluted as a discrete peak when the formic acid concentration reached approx, o.Io M. Standard procedures were used for the measurement of protein 6 and radioactivity 4. Amino acid production was measured by incubating Ix~C 3-phosphoglycerate or D@4Clglycerate with cell-free extracts of the various tissues and fractionating the radioactive products by chromatography on Dowex-5o (H+). The amino acids produced were adsorbed to the resin at an acid pH and subsequently eluted with NH~OH. The chromatograt)hic procedure of ROBERTS et al. 7 was used for the fractionation of the amino acids and the identification of the radioactive components. The amino acids eluted from the original Dowex-5o (H +) column with NH4OH were evaporated to dryness, dissolved in o.5 ml of I. 5 M HC1, and added to a I cm i 5o cm column of Dowex-5o (H~), 2oo-4oo mesh. They were then eluted with I. 5 M HC1. Paper chromatography with phenol, saturated with water, confirmed the identity of the amino acids in the column fractions and separated serine from glutamate. The capacity to synthesize amino acids from phosphoglycerate and glycerate was measured in cell-free preparations from KB cells, two foetal guinea-pig livers, the corresponding maternal livers, and an adult rat liver. As shown in Table I all the tissues examined produced appreciable quantities of Ft4C]amino acids from L14Cphosphoglycerate, but failed to produce more than 3 re>motes of amino acid/mg of protein from [14CJglycerate. Characterization of the radioactive amino acids by column chromatograt)hy (Figs. IA, B) demonstrated that the preparations from KB cells and the foetal guineapig liver synthesize only serine, while that from the adult guinea pig synthesized both serine and alanine in equal amounts. The recoveries from the columns were 8o %,. Biochim. Biophys. Acta, 124 (1966) 418-42o
419
SHORT COMMUNICATIONS
88 % and 95 % for the material synthesized by the KB cells and the foetal and adult liver preparations, respectively. The column fractions containing the radioactive product of the KB cell extract were pooled and subjected to paper chromatography. Serine and glutamate were present with the radioactivity migrating solely as serine.
80C E
40( o
v
ADULT300 B 200 SerineA Alarinei\-
KB
120C Serine
~ 1oo
j
&
i
, oo ~
FOETAL-
~ 40c
Tube number
u
c
"G 2oc
.\
jo
Alaninell | n-
10
2o
3o
Tube number
Tube number Fig. I. C h r o m a t o g r a p h y of the amino acids formed from [14C]phosphoglycerate. A, P r o d u c t of K B cell extract; B, p r o d u c t of guinea-pig liver extract; C, p r o d u c t of rat-liver extracts. The flow rates of the different columns varied between 0.25 and o.3o ml/min. The fraction n u m b e r at which an amino acid eluted depends on the flow rate since fractions were always collected at I o - m i n intervals.
TABLE
I
AMINO ACID F O R M A T I O N
BY CELL-FREE
PREPARATIONS
FROM
MAMMALIAN
TISSUE
The reaction mixture (o.5 ml) contained 2.5 m M phosphoglycerate (2oooo counts/rain per #mole) or 2.5 mM glycerate (16000 c o u n t s / m i n per #,mole); 50 mM p h o s p h a t e buffer (pH 7.5); 20 mM g l u t a m a t e or L-alanine; 2.8 mM D P N ; 5 mM MgSO4; and 4 mM reduced glutathione. G l u t a m a t e was used w h e n phosphoglycerate was the s u b s t r a t e and alanine w h e n glycerate was the substrate. The s u b s t i t u t i o n of g l u t a m a t e for alanine did not increase the conversion of glycerate to amino acids. The reaction m i x t u r e was incubated for 3° rain at 37 °. After t e r m i n a t i o n of the reaction with 0. 5 ml of io % trichloroacetic acid, 5/*moles of L-serine was added as carrier and the protein was r e m o v e d b y centrifugation. The s u p e r n a t a n t fluid was placed on a i cm x 3 cm c o l u m n of Dowex-5o (H +) lOO-2OO mesh. The column was washed with 18 ml of o.oi M HC1 and 5 ml of HsO. The amino acids were eluted b y w a s h i n g the column with 7.5 ml of 1. 5 M NH4OH.
Tissue
[14C]Amino acid formed from (mlzmoles/mg protein) [laC]Phosphoglycerate D-El~C]Glycerate
KB* KB 47-day Foetal guinea pig* 59-day Foetal guinea pig Adult guinea pig I Adult guinea pig I I Adult rat
66 lo9"* 35 26** 23** 18 85**
3
* P r e p a r a t i o n s made w i t h o u t dithiothreitol in the buffers. ** The average of duplicate determinations, The experimental values agreed to within 5 %*** Assay not run.
Bioehim. Biophys. Aeta, 124 (1966) 418-42o
42o
SHORT COMMUNICATION b
While the activities shown in Table I could represent yields rather than initial biosynthetic rates, the capacity for serine synthesis per mg of protein appears greatest in KB cells and higher in foetal than in adult guinea-pig liver. When the rat liver extracts were incubated with [14C~phosphoglycerate and the products fractionated on a Dowex-5o (H +) column, over 5 ° % of the recovered radioactivity was eluted with the amino acids (Table I). The addition of ~o ~moles of serine-P or glycerate to the incubation did not reduce the yield of this material indicating that it was not a product of either the phosphorylated or the non-phosphorylated pathway. Column chromatography (Fig. IC) showed one component with the elution characteristics of alanine. The recovery of radioactivity in the alanine peak was 85 % and radioactive serine was not detected. Paper chromatography of the radioactive product either before or after column chromatography showed one component with the mobility of alanine. Our results indicate that the phosphorylated pathway is the major pathway to serine in polyploid (KB) and diploid (guinea-pig liver) cells. The failure to detect the phosphorylated pathway with the preparations from rat liver may be explained by the recent report that rats fed a commercial diet were deficient in phosphoglycerate dehydrogenase and serine-forming capacity, whereas these activities were present in animals fed a synthetic diet lacking serine and glycine 8. These experiments extend to whole animals the observations made with cultured cells that exogenous serine reduces the capacity for serine synthesis4, 9. The phosphorylated pathway has also been observed in preparations from rat brain ~°. The rat-liver preparation that failed to convert D-glycerate to serine contained 6 units of D-glycerate dehydrogenase (EC 1.1.1.29) per mg of protein when assayed according to WILLIS AND SALLACH3. It appears that the presence of enzyme activity alone is insufficient evidence that the non-phosphorylated pathway is functional and caution is required in interpreting variations in enzyme levels in terms of serine synthesis. This is particularly true if the animal had been provided with an exogenous source of serine. This investigation was supported by Research Grant AI-o3957 from the U.S. Public Health Service. We wish to acknowledge the technical assistance of Mrs. M. POTOCHNY and thank Dr. A. NEMETH for the guinea-pig livers.
Department of Microbiology, School of Medicine University of Pennsylvania, Philadelphia, Pa. (U.S.A.)
LEWIS I. PIZER
I A. ICHIHARA AND D. M. GREENBERG, J . Biol. Chem., 224 (I957) 331. 2 H . J. SALLACH, J. Biol. Chem., 223 (1956) t l O I . 3 J. E. VqlLLIS AND H. J. SALLACH, J. Biol. Chem., 237 (1962) 91o. 4 L. I. PIZER, J. Biol. Chem., 239 (1964) 4219. 5 R. B. PARK, N. G. [)ON, 1~. P. LOUWRIER AND ~{. CALVIN, Biochim. Biophys. Acta, 42 (196o) 27. 6 0 . H. LOWRY, N. J. ROSEBROUGH, A. 1~. FARR AND I{,. J. RANDALL, .]. Biol. Chem., 193 (1951) 265 . 7 R. ]3. ROBERTS, P. H. ABELSON, i). g . COWRIE, E. T. BOLTON AND R. J. BRITTEN, Carnegie Inst. Wash. Publ., 6o 7 (1955) 5 TM 8 T. TANAKA, Protein, Nucleic Acid, Enzyme, Tokyo, lO (1965) 068. 9 U . EAGLE, C. L. "WASHINGTON AND M. LEVY, J . Biol. Chem., 240 (1965) 3944. i o \ ¥ . F. BRIDGERS, J. Biol. Chem., 240 (1965) 4591.
Received March i7th, 1966 Biochim. Biophys. Acta, 124 (1966) 4 1 8 - 4 2 o