- Email: [email protected]
Enzymatic synthesis and purification of l -pyrroline-5-carboxylic acid
ANALYTICAL
BIOCHEMISTRY
82,
170-176 (1977)
Enzymatic Synthesis and Purification L-Pyrroline+Carboxylic Acid
of
ROBERT J. SMITH, SYLVIA J. DOWNING, ANDJAMES M. PHANG Endocrine Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Received February 22, 1977; accepted May 16, 1977 AVyrroline-Scarboxylic acid (PSC) is an intermediate in the metabolism of proline, omithine, and glutamic acid. It has been obtainable by chemical synthesis only as a mixture of the D- and L-stereoisomers. We report a method for the enzymatic synthesis of P5C on a preparative scale. The P5C is formed from L-omithine by purified omithine aminotransferase and then isolated by Dowex50W cation-exchange resin chromatography. The purified compound exists entirely as the biologicahy active L-stereoisomer. With [Ylomithine as precursor, high specific activity uniformly labeled [Y]PSC can be obtained.
A’-Pyrroline-Scarboxylic acid (PSC) is the common intermediate in the interconversions of proline, ornithine, and glutamic acid in mammalian tissues (Fig. 1). Its role in the regulation of interchange between the urea cycle and the tricarboxylic acid (TCA) cycle and in supplying proline for protein synthesis has been only partially elucidated (1). Although specific radioisotopic assays have been developed for the enzymes which form and degrade P5C (2-9, pure L-PSC has remained difficult to obtain. Previously described methods (6,7) have employed laborious chemical syntheses and have the disadvantage of producing mixtures of the D- and L-stereoisomers. We now describe a relatively simple enzymatic method for synthesizing gram quantities of P5C. Using purified omithine aminotransferase, L-ornithine is converted to P5C with the simultaneous conversion of a-ketoglutarate to L-glutamic acid (Fig. 2). The PSC produced exists entirely as the L-stereoisomer and can be highly purified. In addition, radioisotopically labeled P5C can be prepared from commercially available labeled ornithine. METHODS
Materials o-Aminobenzaldehyde, pyridoxalJ’-phosphate, a-ketoglutarate, /3nicotinamide adenine dinucleotide, reduced form (NADH), and Dowex170 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN ooO3-2697
L-PYRROLINE-5-CARBOXYLIC
ACID
171
FIG. 1. Schematic diagram of metabolic pathways involving PSC. The asterisk denotes enzyme(s) that have not been detined in mammalian cells.
5OW (hydrogen form, strongly acid cation-exchange resin, 8% crosslinked, 100-200 mesh) were obtained from Sigma Chemical Company. L-Omithine monohydrochloride was from Nutritional Biochemicals. Uniformly labeled L-[14C]ornithine and r.,-[14C]glutamic acid were from New England Nuclear. Porcine insulin was from Schwarz-Mann. Baker-flex cellulose thin-layer chromatography sheets were from J. T. Baker Chemical Company. Sprague-Dawley rats were obtained from Zivic-Miller Company. PSC synthesized by a chemical method (6) from y,y-dicarbethoxy-y-acetamidobutyraldehyde was the generous gift of Dr. A. Baich. General Omithine aminotransferase activity in the enzyme preparations was measured by the method of Phang et al. (4). A unit was defined as the amount of enzyme which would form 1 pmol of PSC/hr under standard assay conditions of 0.7 mM ornithine, 0.7 mM a-ketoglutarate, and incubation at 37°C. Protein concentration was determined by the method of Lowry et al. (8) with porcine insulin as a standard. PSC was measured calorimetrically with o-aminobenzaldehyde by the method of Strecker (9). Omithine L-ORNITHINE-
Aminopansferase
/L-PYRROLINE-SCARBOXYIATE
CMETOGLUTARATE-L-GLUTAMATE
FIG. 2. Diagram showing conversion of L-omithine to L-PSC. a-Ketoglutarate accepts the &amino group from omithine to form L-glutamate. When uniformly labeled L-[T]omithine is used as a substrate, L-PSC is labeled but not glutamate.
172
SMITH,
DOWNING,
AND PHANG
Ascending thin-layer chromatography was performed in one dimension in a variety of solvent systems on Baker-flex cellulose sheets in a Gelman chromatography chamber. Chromatograms were developed by spraying with a solution of 0.3 g of ninhydrin in 100 ml of n-butanol and 3 ml glacial acetic acid and heating at 110°C for 10 min. Ornithine Aminotransferase Purijcation
The method for purification of ornithine aminotransferase was derived from that of Sanada et al. (10). Unless otherwise specified, all operations were performed at 0-5°C. In a Waring Blendor 25 g of liver from 150-g male Sprague-Dawley rats was homogenized for 2 min in 126 ml of water containing 8 mg of pyridoxal-5’-phosphate. The homogenate was sonicated for 2 min at a setting of 40 with a Branson Sonifier and centrifuged for 30 min at 25,000 g. The supernatant was decanted, its volume was measured, and 1.0 M potassium phosphate buffer, pH 6.0, and 0.5 M a-ketoglutarate were added to give final concentrations of 0.05 M and 0.005 M, respectively. The mixture was immersed in a 75°C water bath until it reached 6O”C, kept at 60°C for 1 min, cooled in an ice bath, and centrifuged for 10 min at 20,000 g. Ammonium sulfate was added to the supernatant to a final concentration of 20%, w/v, and the precipitate after a lo-min centrifugation at 10,000 g was discarded. Ammonium sulfate then was added to the supernatant to a final concentration of 30%, w/v; it was divided equally among four 1 x IO-cm tubes and centrifuged for 10 min at 16,000 g. The supernatant was completely removed by decanting and by carefully swabbing the walls of the tubes. Two hundred microliters of cold water was added to each tube, and the precipitate was suspended by stirring. The contents of the four tubes were combined, centrifuged for 10 min at 16,000 g . and the supernatant was discarded. The precipitate was dissolved in 1.4 ml of 0.2 M potassium phosphate buffer, pH 8.0, and centrifuged for 30 min at 100,000 g. The purified enzyme, which remains in the supernatant, was dialyzed against 0.2 M potassium phosphate buffer, pH 8.0, and was stored at -25°C. Preparation of PSC
In a volume of 250 ml, a reaction mixture was prepared which contained 5 mmol of L-ornithine monohydrochloride, 5 mmol of a-ketoglutarate, and 1 mg of pyridoxal-5’-phosphate in 0.1 M potassium phosphate buffer, pH 8. Solutions of omithine and a-ketoglutarate were adjusted to pH 8 prior to use. Approximately 100 units of purified ornithine aminotransferase were added, and the mixture was incubated for 8 hr at 37°C in a Dubnoff shaker. It was then acidified with 50 ml of 6 N HCl, deproteinized, and the supernatant was lyophilized. Twelve milliliters of normal HCl was added to the white, oily-appearing residue; it was stirred and allowed to settle for several hr at 4°C. Two milliliters of the supernatant was then applied to a 45-ml bed volume, 11-mm diameter column of
L-PYRROLINE-SCARBOXYLIC
173
ACID
Dowex-SOW and eluted with normal HCl. The effluent was collected in 5-ml fractions, and PSC was identified in milliliters 190 to 240. These fractions were pooled and stored at 4°C. The remainder of the supernatant was purified similarly in 2-ml aliquots. Preparation of Radioisotopically
Labeled P5C
The same principle was used to prepare radioisotopically labeled L-PSC. In this case, 150 &i of L-[14C]ornithine was combined with 26 pmol of a-ketoglutarate, 150 pg of pyridoxal-5’-phosphate, and 100 units of purified ornithine aminotransferase in 37.5 ml of 0.1 M potassium phosphate buffer at pH 8.0. The mixture was incubated at 37°C for 8 hr, acidified with 7.5 ml of 6 N HCI, deprote‘inized, lyophilized, redissolved in 2.0 ml normal HCl, and purified over Dowex as described above. Preparation of Xiphoid Cartilage Homogenate
Three xiphoid cartilages were removed from 400-g male SpragueDawley rats, cleaned of connective tissue, and ground to a fine powder in liquid nitrogen with a porcelain mortar and pestle. The powder was transferred to a Potter-Elvehjem homogenizer, thoroughly homogenized in 0.75 ml of 0.1 M potassium phosphate buffer, pH 8.0, sonicated for 40 set, and centrifuged at 25,000 g for 30 min. The supernatant was dialyzed against 250 ml of 0.1 M potassium phosphate buffer for 90 min with three buffer changes and used on the same day to convert P5C to proline as described below. RESULTS Ornithine Aminotransferase Purification
Typical results of ornithine aminotransferase purification are summarized in Table 1. Twenty-five grams of rat liver provided approximately 200 units of 400- to 600-fold purified enzyme. TABLE PURIFICATION
Preparation Crude sonicate Sonicate supematant Heat step supernatant 20% Ammonium sulfate supematant Final enzyme
Total volume (ml) 144 122 101 10.5 0.54
OF ORNITHINE
1 AMINOTRANSFERASE
Protein bWm1)
Specific activity (units/mg)
Purification
Total units
Yield (So)
16.78 12.02 3.28
0.588 0.760 2.685
1 1.3 4.6
1415 1115 885
loo 79 63
1.77 1.47
2.254 229.8
3.8 391
419 183
30 13
174
SMITH,
DOWNING,
AND PHANG
P5C Preparation The recovery of P5C was determined calorimetrically with o-aminobenzaldehyde using chemically synthesized PSC (6) as a standard. A 250-ml reaction mixture containing 5 mmol of L-ornithine produced 1.7 mmol of PSC, which represented 34% conversion to P5C. The final yield of PSC after Dowex purification was 1 mmol. When 150 &i of L-[14C]ornithine was reacted with ornithine aminotransferase as described above, approximately 105 $Zi was recovered as pure L-[14C]P5C. P5C Purity Each mole of P5C formed by ornithine aminotransferase from ornithine and a-ketoglutarate was accompanied by the production of 1 mol of glutamic acid. Ornithine and a-ketoglutarate are well separated from PSC by Dowex-50 column chromatography. P5C and glutamic acid, however, appear in discrete but closely associated peaks (Fig. 3), making it important to measure the amount of glutamic acid remaining in the purified P5C. When a known quantity of L-[14C]glutamic acid was added to the P5C prior to purification and the PSC was purified over Dowex-SOW in the usual manner, only 0.5% of the total glutamic acid radioactivity was recovered in the effluent fraction containing P5C. The purified PSC was also chromatographed on ascending thin-layer cellulose sheets in several solvent systems (Table 2). In each case a prominent .-
4
250
Effluent
Volume
(ml)
FIG. 3. Elution profiles of L-glutamic acid and L-PK. To 2 ml of impure enzymatically synthesized P5C, 2.5 &i of L-[14Clglutamic acid was added. The mixture was applied to a 45-ml bed volume Dowex-SOW column, eluted with normal HCI, and collected in 5-ml fractions. The closed circles (0) denote L-glutamic acid measured in counts per minute in a 0.025-m] aliquot of each fraction. The open circles (0) denote P5C measured by the standard o-aminobenzaldehyde spectrophotometric assay.
L-PYRROLINE-5CARBOXYLIC TABLE THIN-LAYER
175
ACID
2
CHROMATOGRAPHY
OF
PSC” R, Values*
Solvent system I-PropanoVwater (7:3) Ethanol/water (77:23) n-Butanoliacetic acid/water (4:l:l)
P5C
Second ninhydrin spot
Glutamic acid control
59 91 45
78 -
57 81 31
a Approximately 20 nmol of P5C in 10 ~1 of N HCI was applied to each chromatogram. Approximately 20 nmol of glutamic acid standard was also applied in 10 ~1 of N HCI. The solvent front moved approximately 20 cm in 3 hr. *R, = (distance of sample from origin/distance of solvent from origin) x 100.
PSC spot with a characteristic yellow color appeared immediately on staining with ninhydrin. In the ethanol/H,0 system, which produced the highest R, for PK, a second, faint lavender spot appeared only after prolonged (overnight) development. This spot had the migration and staining characteristics of glutamic acid. No other ninhydrin-reactive material was demonstrable, thus verifying the absence of amino acid contaminants other than glutamic acid. P5C Optical Conjiguration
The amount of PSC existing as the L-stereoisomer was measured by determining the maximum percentage of P5C that could be converted to proline by PSC reductase, since P5C reductase reacts only with the Lstereoisomer (11). A homogenate of rat xiphoid cartilage (see Methods) was used as the source of PSC reductase because this tissue has high P5C reductase activity and lacks proline oxidase. Thus it can convert PSC to proline but cannot oxidize the proline back to PSC (12). Approximately 0.1 $Zi of [14C]P5C prepared as described above was incubated with 200 pg of xiphoid homogenate protein and 0.3 mg of NADH in 0.1 M potassium phosphate buffer, pH 6.8, at 37°C for 2 hr. The reaction was stopped by the addition of o-aminobenzaldehyde and HCI, final concentrations of 5 mg/ml and 1 N, respectively. When this mixture was applied to a Dowex50 column, the o-aminobenzaldehyde-PSC complex was strongly bound by the resin and thus was readily separated from proline (2). Of the initial PSC radioactivity, 95.4% was recovered in a single, symmetrical peak with migration characteristics identical to proline, confirming that at least 95% of the P5C existed as the L-stereoisomer.
176
SMITH,
DOWNING,
AND PHANG
DISCUSSION
P5C is an intermediate in the interconversions of proline, glutamic acid, and ornithine and, thus, may provide an important link between the urea cycle, the TCA cycle, and proline. It has previously been synthesized by the hydrolysis of yy-dicarbethoxy-y-acetamidobutyraldehyde with hydrochloric acid (6). This produces a relatively pure compound which is a mixture of the D- and L-stereoisomers. Another chemical method employing oxidation of hydroxylysine to P5C has been reported recently (7), but it most probably produces a similar mixture of the D- and L-stereoisomers. It has been suggested that L-proline might be converted to P5C in large quantities by proline oxidase (13), but successful application of this approach has not been reported. The enzymatic conversion of ornithine to P5C by purified ornithine aminotransferase described here provides a relatively simple method for obtaining large amounts of P5C. Chromatographic analysis has shown it to be approximately 99% free of impurities after a single purification step on Dowex-SOW cation-exchange resin. The P5C exists entirely as the Lstereoisomer, which is the actual biological metabolite in mammalian tissues. In studies of mammalian enzymes, the use of pure L-PSC will allow for accurate kinetic measurements and will avoid the possible problem of D-P5C in racemic mixtures serving as a substrate for D-amino acid oxidase. A final advantage of the enzymatic synthetic method is that it can be used to produce 14C-labeled PSC for tracer studies. REFERENCES 1. Adams, E. (1970) Znt. Rev. Connect. Tissue Res. 5, 2-91. 2. Phang, J. M., Downing, S. J., and Valle, D. (1973) Anal. Biochem. 55, 266-271. 3. Phang, J. M., Downing, S. J., Valle, D. L., and Kowaloff, E. M. (1975) .Z. Lab. Clin. Med. 85, 312-317. 4. Phang, J. M., Downing, S. J., and Valle, D. (1973) Anal. Biochem. 55, 272-277. 5. Valle, D., Phang, J. M., and Goodman, S. I. (1974) Science 185, 1053-1954. 6. Vogel, H. J., and Davis, B. D. (1952) J. Amer. Chem. Sot. 74, 109-112. 7. Wu, G. Y., and Seifter, S. (1975) Anal. Biochem. 67, 413-421. 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 9.
10. 11. 12. 13.
Strecker, H. J. (19.57) J. Biol. Chem. 225, 825-834. Sanada, Y., Suemori, I., and Katunuma, H. (1970) Biochim. Biophys. Actu 220,42-50. Strecker, H. J. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17B, p. 261, Academic Press, New York. Smith, R. J., Downing, S. J., and Phang, J. M. (1976) C/in. Res. 24, 371A. Strecker, H. J. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17B, p. 251, Academic Press, New York.
BIOCHEMISTRY
82,
170-176 (1977)
Enzymatic Synthesis and Purification L-Pyrroline+Carboxylic Acid
of
ROBERT J. SMITH, SYLVIA J. DOWNING, ANDJAMES M. PHANG Endocrine Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Received February 22, 1977; accepted May 16, 1977 AVyrroline-Scarboxylic acid (PSC) is an intermediate in the metabolism of proline, omithine, and glutamic acid. It has been obtainable by chemical synthesis only as a mixture of the D- and L-stereoisomers. We report a method for the enzymatic synthesis of P5C on a preparative scale. The P5C is formed from L-omithine by purified omithine aminotransferase and then isolated by Dowex50W cation-exchange resin chromatography. The purified compound exists entirely as the biologicahy active L-stereoisomer. With [Ylomithine as precursor, high specific activity uniformly labeled [Y]PSC can be obtained.
A’-Pyrroline-Scarboxylic acid (PSC) is the common intermediate in the interconversions of proline, ornithine, and glutamic acid in mammalian tissues (Fig. 1). Its role in the regulation of interchange between the urea cycle and the tricarboxylic acid (TCA) cycle and in supplying proline for protein synthesis has been only partially elucidated (1). Although specific radioisotopic assays have been developed for the enzymes which form and degrade P5C (2-9, pure L-PSC has remained difficult to obtain. Previously described methods (6,7) have employed laborious chemical syntheses and have the disadvantage of producing mixtures of the D- and L-stereoisomers. We now describe a relatively simple enzymatic method for synthesizing gram quantities of P5C. Using purified omithine aminotransferase, L-ornithine is converted to P5C with the simultaneous conversion of a-ketoglutarate to L-glutamic acid (Fig. 2). The PSC produced exists entirely as the L-stereoisomer and can be highly purified. In addition, radioisotopically labeled P5C can be prepared from commercially available labeled ornithine. METHODS
Materials o-Aminobenzaldehyde, pyridoxalJ’-phosphate, a-ketoglutarate, /3nicotinamide adenine dinucleotide, reduced form (NADH), and Dowex170 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN ooO3-2697
L-PYRROLINE-5-CARBOXYLIC
ACID
171
FIG. 1. Schematic diagram of metabolic pathways involving PSC. The asterisk denotes enzyme(s) that have not been detined in mammalian cells.
5OW (hydrogen form, strongly acid cation-exchange resin, 8% crosslinked, 100-200 mesh) were obtained from Sigma Chemical Company. L-Omithine monohydrochloride was from Nutritional Biochemicals. Uniformly labeled L-[14C]ornithine and r.,-[14C]glutamic acid were from New England Nuclear. Porcine insulin was from Schwarz-Mann. Baker-flex cellulose thin-layer chromatography sheets were from J. T. Baker Chemical Company. Sprague-Dawley rats were obtained from Zivic-Miller Company. PSC synthesized by a chemical method (6) from y,y-dicarbethoxy-y-acetamidobutyraldehyde was the generous gift of Dr. A. Baich. General Omithine aminotransferase activity in the enzyme preparations was measured by the method of Phang et al. (4). A unit was defined as the amount of enzyme which would form 1 pmol of PSC/hr under standard assay conditions of 0.7 mM ornithine, 0.7 mM a-ketoglutarate, and incubation at 37°C. Protein concentration was determined by the method of Lowry et al. (8) with porcine insulin as a standard. PSC was measured calorimetrically with o-aminobenzaldehyde by the method of Strecker (9). Omithine L-ORNITHINE-
Aminopansferase
/L-PYRROLINE-SCARBOXYIATE
CMETOGLUTARATE-L-GLUTAMATE
FIG. 2. Diagram showing conversion of L-omithine to L-PSC. a-Ketoglutarate accepts the &amino group from omithine to form L-glutamate. When uniformly labeled L-[T]omithine is used as a substrate, L-PSC is labeled but not glutamate.
172
SMITH,
DOWNING,
AND PHANG
Ascending thin-layer chromatography was performed in one dimension in a variety of solvent systems on Baker-flex cellulose sheets in a Gelman chromatography chamber. Chromatograms were developed by spraying with a solution of 0.3 g of ninhydrin in 100 ml of n-butanol and 3 ml glacial acetic acid and heating at 110°C for 10 min. Ornithine Aminotransferase Purijcation
The method for purification of ornithine aminotransferase was derived from that of Sanada et al. (10). Unless otherwise specified, all operations were performed at 0-5°C. In a Waring Blendor 25 g of liver from 150-g male Sprague-Dawley rats was homogenized for 2 min in 126 ml of water containing 8 mg of pyridoxal-5’-phosphate. The homogenate was sonicated for 2 min at a setting of 40 with a Branson Sonifier and centrifuged for 30 min at 25,000 g. The supernatant was decanted, its volume was measured, and 1.0 M potassium phosphate buffer, pH 6.0, and 0.5 M a-ketoglutarate were added to give final concentrations of 0.05 M and 0.005 M, respectively. The mixture was immersed in a 75°C water bath until it reached 6O”C, kept at 60°C for 1 min, cooled in an ice bath, and centrifuged for 10 min at 20,000 g. Ammonium sulfate was added to the supernatant to a final concentration of 20%, w/v, and the precipitate after a lo-min centrifugation at 10,000 g was discarded. Ammonium sulfate then was added to the supernatant to a final concentration of 30%, w/v; it was divided equally among four 1 x IO-cm tubes and centrifuged for 10 min at 16,000 g. The supernatant was completely removed by decanting and by carefully swabbing the walls of the tubes. Two hundred microliters of cold water was added to each tube, and the precipitate was suspended by stirring. The contents of the four tubes were combined, centrifuged for 10 min at 16,000 g . and the supernatant was discarded. The precipitate was dissolved in 1.4 ml of 0.2 M potassium phosphate buffer, pH 8.0, and centrifuged for 30 min at 100,000 g. The purified enzyme, which remains in the supernatant, was dialyzed against 0.2 M potassium phosphate buffer, pH 8.0, and was stored at -25°C. Preparation of PSC
In a volume of 250 ml, a reaction mixture was prepared which contained 5 mmol of L-ornithine monohydrochloride, 5 mmol of a-ketoglutarate, and 1 mg of pyridoxal-5’-phosphate in 0.1 M potassium phosphate buffer, pH 8. Solutions of omithine and a-ketoglutarate were adjusted to pH 8 prior to use. Approximately 100 units of purified ornithine aminotransferase were added, and the mixture was incubated for 8 hr at 37°C in a Dubnoff shaker. It was then acidified with 50 ml of 6 N HCl, deproteinized, and the supernatant was lyophilized. Twelve milliliters of normal HCl was added to the white, oily-appearing residue; it was stirred and allowed to settle for several hr at 4°C. Two milliliters of the supernatant was then applied to a 45-ml bed volume, 11-mm diameter column of
L-PYRROLINE-SCARBOXYLIC
173
ACID
Dowex-SOW and eluted with normal HCl. The effluent was collected in 5-ml fractions, and PSC was identified in milliliters 190 to 240. These fractions were pooled and stored at 4°C. The remainder of the supernatant was purified similarly in 2-ml aliquots. Preparation of Radioisotopically
Labeled P5C
The same principle was used to prepare radioisotopically labeled L-PSC. In this case, 150 &i of L-[14C]ornithine was combined with 26 pmol of a-ketoglutarate, 150 pg of pyridoxal-5’-phosphate, and 100 units of purified ornithine aminotransferase in 37.5 ml of 0.1 M potassium phosphate buffer at pH 8.0. The mixture was incubated at 37°C for 8 hr, acidified with 7.5 ml of 6 N HCI, deprote‘inized, lyophilized, redissolved in 2.0 ml normal HCl, and purified over Dowex as described above. Preparation of Xiphoid Cartilage Homogenate
Three xiphoid cartilages were removed from 400-g male SpragueDawley rats, cleaned of connective tissue, and ground to a fine powder in liquid nitrogen with a porcelain mortar and pestle. The powder was transferred to a Potter-Elvehjem homogenizer, thoroughly homogenized in 0.75 ml of 0.1 M potassium phosphate buffer, pH 8.0, sonicated for 40 set, and centrifuged at 25,000 g for 30 min. The supernatant was dialyzed against 250 ml of 0.1 M potassium phosphate buffer for 90 min with three buffer changes and used on the same day to convert P5C to proline as described below. RESULTS Ornithine Aminotransferase Purification
Typical results of ornithine aminotransferase purification are summarized in Table 1. Twenty-five grams of rat liver provided approximately 200 units of 400- to 600-fold purified enzyme. TABLE PURIFICATION
Preparation Crude sonicate Sonicate supematant Heat step supernatant 20% Ammonium sulfate supematant Final enzyme
Total volume (ml) 144 122 101 10.5 0.54
OF ORNITHINE
1 AMINOTRANSFERASE
Protein bWm1)
Specific activity (units/mg)
Purification
Total units
Yield (So)
16.78 12.02 3.28
0.588 0.760 2.685
1 1.3 4.6
1415 1115 885
loo 79 63
1.77 1.47
2.254 229.8
3.8 391
419 183
30 13
174
SMITH,
DOWNING,
AND PHANG
P5C Preparation The recovery of P5C was determined calorimetrically with o-aminobenzaldehyde using chemically synthesized PSC (6) as a standard. A 250-ml reaction mixture containing 5 mmol of L-ornithine produced 1.7 mmol of PSC, which represented 34% conversion to P5C. The final yield of PSC after Dowex purification was 1 mmol. When 150 &i of L-[14C]ornithine was reacted with ornithine aminotransferase as described above, approximately 105 $Zi was recovered as pure L-[14C]P5C. P5C Purity Each mole of P5C formed by ornithine aminotransferase from ornithine and a-ketoglutarate was accompanied by the production of 1 mol of glutamic acid. Ornithine and a-ketoglutarate are well separated from PSC by Dowex-50 column chromatography. P5C and glutamic acid, however, appear in discrete but closely associated peaks (Fig. 3), making it important to measure the amount of glutamic acid remaining in the purified P5C. When a known quantity of L-[14C]glutamic acid was added to the P5C prior to purification and the PSC was purified over Dowex-SOW in the usual manner, only 0.5% of the total glutamic acid radioactivity was recovered in the effluent fraction containing P5C. The purified PSC was also chromatographed on ascending thin-layer cellulose sheets in several solvent systems (Table 2). In each case a prominent .-
4
250
Effluent
Volume
(ml)
FIG. 3. Elution profiles of L-glutamic acid and L-PK. To 2 ml of impure enzymatically synthesized P5C, 2.5 &i of L-[14Clglutamic acid was added. The mixture was applied to a 45-ml bed volume Dowex-SOW column, eluted with normal HCI, and collected in 5-ml fractions. The closed circles (0) denote L-glutamic acid measured in counts per minute in a 0.025-m] aliquot of each fraction. The open circles (0) denote P5C measured by the standard o-aminobenzaldehyde spectrophotometric assay.
L-PYRROLINE-5CARBOXYLIC TABLE THIN-LAYER
175
ACID
2
CHROMATOGRAPHY
OF
PSC” R, Values*
Solvent system I-PropanoVwater (7:3) Ethanol/water (77:23) n-Butanoliacetic acid/water (4:l:l)
P5C
Second ninhydrin spot
Glutamic acid control
59 91 45
78 -
57 81 31
a Approximately 20 nmol of P5C in 10 ~1 of N HCI was applied to each chromatogram. Approximately 20 nmol of glutamic acid standard was also applied in 10 ~1 of N HCI. The solvent front moved approximately 20 cm in 3 hr. *R, = (distance of sample from origin/distance of solvent from origin) x 100.
PSC spot with a characteristic yellow color appeared immediately on staining with ninhydrin. In the ethanol/H,0 system, which produced the highest R, for PK, a second, faint lavender spot appeared only after prolonged (overnight) development. This spot had the migration and staining characteristics of glutamic acid. No other ninhydrin-reactive material was demonstrable, thus verifying the absence of amino acid contaminants other than glutamic acid. P5C Optical Conjiguration
The amount of PSC existing as the L-stereoisomer was measured by determining the maximum percentage of P5C that could be converted to proline by PSC reductase, since P5C reductase reacts only with the Lstereoisomer (11). A homogenate of rat xiphoid cartilage (see Methods) was used as the source of PSC reductase because this tissue has high P5C reductase activity and lacks proline oxidase. Thus it can convert PSC to proline but cannot oxidize the proline back to PSC (12). Approximately 0.1 $Zi of [14C]P5C prepared as described above was incubated with 200 pg of xiphoid homogenate protein and 0.3 mg of NADH in 0.1 M potassium phosphate buffer, pH 6.8, at 37°C for 2 hr. The reaction was stopped by the addition of o-aminobenzaldehyde and HCI, final concentrations of 5 mg/ml and 1 N, respectively. When this mixture was applied to a Dowex50 column, the o-aminobenzaldehyde-PSC complex was strongly bound by the resin and thus was readily separated from proline (2). Of the initial PSC radioactivity, 95.4% was recovered in a single, symmetrical peak with migration characteristics identical to proline, confirming that at least 95% of the P5C existed as the L-stereoisomer.
176
SMITH,
DOWNING,
AND PHANG
DISCUSSION
P5C is an intermediate in the interconversions of proline, glutamic acid, and ornithine and, thus, may provide an important link between the urea cycle, the TCA cycle, and proline. It has previously been synthesized by the hydrolysis of yy-dicarbethoxy-y-acetamidobutyraldehyde with hydrochloric acid (6). This produces a relatively pure compound which is a mixture of the D- and L-stereoisomers. Another chemical method employing oxidation of hydroxylysine to P5C has been reported recently (7), but it most probably produces a similar mixture of the D- and L-stereoisomers. It has been suggested that L-proline might be converted to P5C in large quantities by proline oxidase (13), but successful application of this approach has not been reported. The enzymatic conversion of ornithine to P5C by purified ornithine aminotransferase described here provides a relatively simple method for obtaining large amounts of P5C. Chromatographic analysis has shown it to be approximately 99% free of impurities after a single purification step on Dowex-SOW cation-exchange resin. The P5C exists entirely as the Lstereoisomer, which is the actual biological metabolite in mammalian tissues. In studies of mammalian enzymes, the use of pure L-PSC will allow for accurate kinetic measurements and will avoid the possible problem of D-P5C in racemic mixtures serving as a substrate for D-amino acid oxidase. A final advantage of the enzymatic synthetic method is that it can be used to produce 14C-labeled PSC for tracer studies. REFERENCES 1. Adams, E. (1970) Znt. Rev. Connect. Tissue Res. 5, 2-91. 2. Phang, J. M., Downing, S. J., and Valle, D. (1973) Anal. Biochem. 55, 266-271. 3. Phang, J. M., Downing, S. J., Valle, D. L., and Kowaloff, E. M. (1975) .Z. Lab. Clin. Med. 85, 312-317. 4. Phang, J. M., Downing, S. J., and Valle, D. (1973) Anal. Biochem. 55, 272-277. 5. Valle, D., Phang, J. M., and Goodman, S. I. (1974) Science 185, 1053-1954. 6. Vogel, H. J., and Davis, B. D. (1952) J. Amer. Chem. Sot. 74, 109-112. 7. Wu, G. Y., and Seifter, S. (1975) Anal. Biochem. 67, 413-421. 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 9.
10. 11. 12. 13.
Strecker, H. J. (19.57) J. Biol. Chem. 225, 825-834. Sanada, Y., Suemori, I., and Katunuma, H. (1970) Biochim. Biophys. Actu 220,42-50. Strecker, H. J. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17B, p. 261, Academic Press, New York. Smith, R. J., Downing, S. J., and Phang, J. M. (1976) C/in. Res. 24, 371A. Strecker, H. J. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17B, p. 251, Academic Press, New York.