S-Carboxymethylcysteine Synthase from Escherichia Coli

Agric. Biol. Chem., 53 (9), 2481 -2487, 1989 2481

S-Carboxymethylcysteine Synthase from Escherichia coli Hidehiko Kumagai,* Hideyuki Suzuki, Hiroki Shigematsu and Tatsurokuro Tochikura Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan Received April 14, 1989

An enzyme that catalyzes the synthesis of S-carboxymethyl-L-cysteine from 3-chloro-L-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-L-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzymehas a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the ^-replacement reactions between 3-Cl-Ala and various thiol compounds. The apparent Kmvalues for 3-Cl-Ala and thioglycolic acid were 40mMand 15.4mM.The enzyme showedvery low activity as to the a,/^-elimination reaction with 3-Cl-Ala and L-serine. It was not inactivated on the incubation with 3-Cl-Ala. Theabsorption spectrum of the enzymeshows a maximum at 412nm, indicating that it contains pyridoxal phosphate as a co factor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.

3-Chloro-L-alanine (3-Cl-Ala) acts as a so- called suicide substrate for some pyridoxal- Materials and Methods phosphate (PLP) dependent enzymes, such Reagents. Casamino acids and Tryptone were obtained as glutamate-oxaloacetic acid transminase,1} from Difco Laboratories, yeast extract from Oriental glutamate-pyruvic acid transaminase2) and Yeast Co., amido black 10B from E. Merck AGand phenylmethylsulfonyl fluoride from Calbiochem-Behring. amino acid racemase.3) On the other hand, Sephadex G-150 and G-100, and blue dextran 2000 and someother PLPenzymes that catalyze the a,/?- molecular weight markers were from Pharmacia. DEAE- elimination and /^-replacement reactions were Cellulofine AHand Cellulofine GC-700-mwere from found to be insensitive to 3-Cl-Ala and to Chisso Co. SC-Cys was from Tokyo Kasei Co., 3-Cl-Ala utilize it as a preferable substrate.4~7) Fur- was a generous gift from ShowalDenko Co., and PLP was thermore, the development of chemical syn- kindly provided by Dainippon Pharmaceutical Co. Other thesis techniques has madeit possible to pro- chemicals were purchased from commercial sources. duce 3-Cl-Ala economically on a large scale.8) Bacterial strain and culture. Escherichia coli W3110 was These observations prompted us to inves- obtained from the stock culture of the Laboratory of tigate and to develop an enzymatic method Industrial Microbiology, Department of Food Science and for the synthesis of useful amino acids from 3- Technology, Faculty of Agriculture, Kyoto University. Cl-Ala. Cells were picked up from an agar plate containing LB medium,10) inoculated into a tube containing 5ml of LB This paper reports the purification and prop- broth and then grown at 37°C overnight with reciprocal erties of the enzyme responsible for the syn- shaking. This preculture was transferred to a 2-1 Sakaguchi thesis of S-carboxymethyl-L-cysteine (SC-Cys) flask containing 500ml of modified Vogel and Bonner from 3-Cl-Ala and thioglycolic acid (TGA). mediumn) containing 0.5% glucose, 0.2% citric acid - H2O, SC-Cys exhibits mucolytic activity and is use- 0.35% NaNH4HPO4-4H2O, 1% K2HPO4, 0.02% Mg- SO4 7H2O and 0.05% Casamino acids, and then grown ful for loosening mucus in the trachea.9) at 28°C for 24hr with reciprocal shaking. Two such sub- * Corresponding author. 2482 H. Kumagai et al. cultures were inoculated into a 30-1 jar fermentor (type Dowex 1x2 (OH form) column (3.6 by 34cm) and MSJ-U 301; Marubishi Co.) containing 201 of the elution was carried out with 0.2n acetic acid solution. modified Vogel and Bonner medium. Cultivation was carried out at 30°C for 24hr with aeration (0.51/1 of HPLC. HPLCwas performed with an M6000Apump, a medium per min) and agitation (150 rpm). The grown cells U6Kinjector and an M440 UVdetector (wavelength fixed were harvested with a refrigerated continuous flow at 280nm) (Waters Assoc, Milford, MA, U.S.A.). centrifuge (type GLE; Carl Padberg GmbH). Paper chromatography. Some products of the enzyme Enzymeassay. SC-Cys synthase activity was determined reaction were detected and determined by paper chroma- with an automatic amino acid analyzer (type K-101-AS; tography. Ten fil of the reaction mixture was applied on a Kyowa Seimitsu Co.; with Kyowa Gel 62210-S) by filter pater (Toyo No. 51A), followed by development in measuring the SC-Cys synthesized. The assay solution the descending modewith a solvent system of rc-butanol- contained 50 /miol of 3-Cl-Ala, 50 /miol ofTGA, 0.1 mmol acetic acid-water (4 : 1 : 1). The development was repeated of potassium phosphate (pH 7.8), 20nmol of PLP, to obtain a good separation. The products were detected 4.0/rniol of EDTAand the enzyme in a final volume of by spraying a 0.3% ninhydrin solution in a mixture of 1.0ml. The reaction was started by the addition of 3-C1- acetone and ethanol (3: 1). For determination of the Ala, and incubation was carried out at 37°C and stopped product, the spot was cut out and extracted with 5ml ofa by the addition of0.5ml of0.3n HCI. 75%ethanol solution in water containing one drop of 5% O-Acetylserine sulfhydrylase activity was determined CuSO4. The amount of the product was estimated spec- according to Kredick and Becker.12) trophotometrically by measuring the absorbance at The enzymatic a,/?-elimination reaction was assayed by measuring the amount of pyruvate by a spectrophotometric method with lactate dehydrogenase and NADH.13) 500Spectrophotometric measurement.nm.Spectrophotometric One unit of an enzyme is defined as the amount of determination and measurement of absorption spectra enzyme that catalyzed the formation of 1 jiimol of the were carried out with a Hitachi 150-20 spectrophotometer. product under the assay conditions. Specific activity is expressed as units per mgprotein. lH-NMRspectrometric analysis. The ^-NMRspec- trum was measured in D2Owith a Hitachi Model R-22 Protein determination. Protein concentrations were de- (90 MHz)spectrometer and chemical shifts were expressed termined by the method of Lowry et al.l4) with bovine relative to DSSas an internal standard. serum albumin as a standard. Mass spectrometric analysis. The mass spectrum was Electrophoresis. Electrophoresis and protein staining measured with a Hitachi Model M-80 mass spectrometer were carried out according to the guide book from with electron impact ion at an ionizing voltage of 20eV. Pharmacia (Polyacrylamide Gel Electrophoresis-Labora- tory Techniques). Proteins on native gel electrophoresis Optical rotatory dispersion analysis. Optical rotatory were stained with amido black 10B and those on SDS dispersion was measured with a JASCO J-5 ORD re- polyacrylamide gel electrophoresis were stained with Co- corder. SC-Cys was dissolved in 1n HC1 at the con- omassie blue R-250. centration of 20 mg/ml. Molecular weight estimation. The molecular weight of Amino acid sequence analysis. Amino acid sequence the enzyme was determined by gel filtration on a high analysis was carried out by automated Edman degra- performance liquid chromatograph (HPLC) equipped dation with an Applied Biosystems gas phase sequencer with a TSK-GEL3,000 SWcolumn. The molecular weight (Model 470A) equipped online with a PTH-amino acid of a subunit was estimated by SDS polyacrylamide electro- analyzer (Model 120A). phoresis, using an LMWmolecular weight calibration kit purchased from Pharmacia. It was also determined by Purification ofSC-Cys synthasefrom E. coli W3110. All SDS gel filtration on a HPLC equipped with a TSK-GEL operation were carried out at 0 to 5°C unless otherwise 2,000 SWcolumn. stated. The pH of the enzyme solution was adjusted to 7.0 with a 2.5% ammoniumhydroxide solution after each Ion-exchange column chromatographies. Ion exchange addition of ammoniumsulfate. column chromatography was performed for the purification of SC-Cys produced through the enzyme reaction. Purification steps. Cation-exchange columnchromatography was performed (i) Preparation ofa cell extract. Cells harvested from 40 1 on a Dowex 50x8 (H+ form) column (3.6 by 25cm) and of culture (wet weight, 230g) were suspended in 1,150ml elution was carried out with a 0.25n NH4OHsolution. of 0.1 m potassium phosphate buffer, pH 7.8, containing Anion-exchange chromatography was performed on a 10mM2-mercaptoethanol, 20jjm PLP and 4.0mM EDTA E. coli S-Carboxymethylcysteine Synthase 2483

(this solution was namedBuffer A) and 1.0mMphenyl- eluted at the concentration of about 80mM. The active methyl sulfonyl fluoride, and disrupted with a Dyno-Mill fractions were combinedand concentrated by the addition (W. A. Bachofen, Maschinenfabrik, Basel, Switzerland) at of ammoniumsulfate to 90%saturation. the agitation rate of 3,000 rpm using glass beads of0.25 to (vii) Sephadex G-100 gel filtration. The enzyme solution 0.50mmin diameter. The supernatant solution was ob- was applied to a Sephadex G-100 column (2.0 by 102cm) tained by centrifugation. equilibrated with 50 mMBuffer A. The enzyme was eluted (ii) First ammoniumsulfate fractionation. Solid am- with the same buffer in 5-ml fractions. The active fractions moniumsulfate was addedto the enzymesolution to 35% were combined and concentrated by the addition of saturation. After standing for more than 1 hr, the super- ammoniumsulfate to 90%saturation. natant was collected by centrifugation and brought to 70% (viii) Sephadex G-150 gel filtration. The enzyme solution saturation in the same manner. After standing overnight, was applied to a Sephadex G-150 column (2.0 by 100cm) the precipitate was collected by centrifugation, dissolved equilibrated with 50 mMBuffer A. The enzyme was eluted in a small amount of 50mMBuffer A and then dialyzed with the same buffer in 2-ml fractions. The active fractions against the same buffer for 2 days. were combined and precipitated by the addition of am- (iii) First DEAE-Cellulofine column chromatography. moniumsulfate to 90%saturation. The dialyzed enzyme solution was applied to a DEAE- (ix) Crystallization. The precipitated enzyme protein Cellulofine AHcolumn (6.0 by 53cm) equilibrated with (14.1 mg) was dissolved in a small amount (about 0.6ml) 50mMBuffer A. The column was washed with the same of Buffer A. Solid ammoniumsulfate was added to the buffer and then the enzymewas eluted in 20-ml fractions solution carefully until it became slightly turbid, and then with 70, 100 and 150mMBuffer A. The activity was eluted the mixture was placed in a refrigerator. Crystallization of with 70 and 100mM Buffer A. the enzyme was observed from the next day and was (iv) Second ammonium sulfatefractionation. The active completed within 1 week. fractions fromthe DEAE-Cellulofinecolumnwere combined and fractionated by the addition of solid ammonium sulfate. The activity was found in the fractions precipitated Results between 50 to 90% saturation. (v) Cellulofine GC-700-m gel filtration. The enzyme Formation of SC-Cys synthase by E. coli solution was applied to a Cellulofine column (4.0 by W311.0 90cm) equilibrated with 50mMBuffer A. The enzyme was E. coli W3110 produced SC-Cys synthase eluted with the same buffer in 20-ml fractions. The active constitutively in the LB broth. The activity fractions were combinedand concentrated by the addition of ammoniumsulfate to 90%saturation. After standing increased exponentially during the exponential overnight, the precipitate was collected by centrifugation, phase and then becameconstant at the maximum suspended in 50 mMBuffer A and then dialyzed against the level in the stationary phase. The addition ofl- same buffer. tryptophan or L-cysteine to the mediumdid (vi) Second DEAE-Cellulofine column chromatography. not affect the enzyme activity at all. The dialyzed active fraction was applied to a DEAE- Cellulofine AH column (2.8 by 38cm) equilibrated with 50mMBuffer A. The column was washed with the same Enzymepurification buffer and then the enzymewas eluted in 5-ml fractions The purification process (Table I), involving with an ascending linear gradient of the potassium phos- ammoniumsulfate fractionation and five col- phate in Buffer A, from 50 to 120mM.The activity was umn chromatographies, led to a 190-fold puri-

Table I. Purification of SC-Cys Synthase from E. coli

Total protein Total activity Specific activity Fraction (mg) (units) (units/mg)

Cell-free 1,960 100 Ammoniumsulfate (1) 1,030 53 DEAE-Cellulofine (1) 1,200 61 Ammonium sulfate (2) 646 33 Cellulofine GC-700-m 477 24 DEAE-Cellulofine (2) 252 13 Sephadex G-100 344 18 Sephadex G-150 200 10 2484 H. Kumagai et al.

Fig. 1. Crystals ofSC-Cys Synthase from E. coliW3110 The microphotograph was taken under a phase-contras microscope at the magnification of x 400. Fig. 2. Disc- and SDS-Polyacrylamide Gel Electro- phoresis of SC-Cys Synthase from E. coli W3110. fication with a recovery of 10.2%. The purifiec Electrophoresis and staining were carried out as described under Materials and Methods. The direction of migration SC-Cys synthase was crystallized as fine yellov is from top to bottom. Disc-polyacrylamide gel elec- needles by the addition of ammoniumsulfat< trophoresis (PAGE) was carried out with two kinds of (Fig. 1). The enzyme preparation gave a singl< buffer, pH 6.6 and 9.4, respectively. Fifteen fig of the band on SDS polyacrylamide gel electro enzymeprotein was applied to each column. For SDS- phoresis, though it showed a broad band or PAGE, molecular markers (lane 1) and 15/ig (lane 2) of disk polycrylamide gel electrophoresis at pF the enzyme were applied. The molecular markers used were phosphorylase b (molecular weight, 94,000), bovine 6.6 and 9.4 (Fig. 2). serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20, 100) and a-lactal- Molecular weight and subunit structure bumin (14,400). The molecular weight was estimated to b< 84,000 by HPLC gel filtration. SDS polyacryl 0.02% and 0.07% of the rate of SC-Cys syn- amide gel electrophoresis of the purified SC thesis. Pyruvate formation was not observed Cys synthase gave a single band of 37,000 anc with SC-Cys, L-cysteine, D-cysteine, D-serine SDS HPLC gel filtration gave a single peak o or 3-chloro-D-alanine. Incubation of the en- 42,000, indicating that the enzyme is i zyme with 3-Cl-Ala for 2hr did not cause any homodimer. loss of activity. The enzyme showed L-cysteine synthase, O-acetylserine sulfhydrylase, activity Catalytic properties corresponding to 4.1% of the rate of SC-Cys The apparent Kmvalues for 3-Cl-Ala anc synthesis. TGA were 40 and 15.4mM. The optimum pF for the SC-Cys synthase activity was 9 to 10.' Purification and identification of SC-Cys pro- and the stable pH range was 6.5 to 7.5. The duced through the enzyme reaction optimum temperature for SC-Cys synthesis Enzymatic synthesis of SC-Cys was carried was 50°C and the stable temperature range out under the assay conditions given under was up to 60°C, as judged when the enzyme Materials and Methods with 50ml of reaction was incubated at various temperatures foi mixture containing 100mM 3-Cl-Ala and lOmin. 200 mMTGA. The reaction was carried out for The purified SC-Cys synthase catalyzed py- 24hr and the synthesis of680mg (76% yield of ruvate formation from 3-Cl-Ala, L-serine anc 3-Cl-Ala) of SC-Cys was confirmed by amino L-cystathionine at the reletive rates of 1.0% acid analysis. The product was purified from E．coliS－CarboxymethylcysteineSynthase 2485

TableII．SUBSTRATESpECIFICITYOFSC－CysSYNTHASEFORTHIOLCoMPOUNDS

The amounts oftheproductsweredetermined asdescribedunderMaterialsandMethods．ActivltylS expressedrelativetothatfbundwithTGA（100％）．

Relativeactivlty Substrate Product （？′。）竺甲慧二霊蒜押二一 Thioglycolicacid S－Carboxymethyl－L－Cys Sodiumhydrosulfide L－Cys Methylmercaptan ふMethyトL－Cys Ethylmercaptan S－Ethyl－L－Cys n－Propylmercaptan S－Propyl－L－Cys iso－Propylmercaptan S－iso－Propyl－L－Cys Phenylmercaptan S－Phentyl－L－Cys Benzylmercaptan S－Benzyl－L－Cys 2－Mercaptoethanol S－2－Hydroxyethyl－L－Cys トThioglycerol S－2，3－Dihydroxypropyl－L－Cys LCys L－Lanthionine DL－Homocysteine Cystathionine

α－Mercaptoprop10nicacid S－1－Methylcarboxymethy1－L－Cys β－Mercaptopropionicacid S－2－Methylcarboxymethy1－L－Cys

a Not detected． b TheextinctioncoefhcientofSC－Cyswasusedfbrthedetermination・

thereactionmixturebychromatographieson TableII． Dowex50and Dowexl，aS described under MaterialsandMethods，andcrystallizedfrom ■仙…リ，J〃州叩（で什rJ WaterbyadjustlngthepH ofthesolutionto The enzyme solution exhibited an absorp－ 3．2．Purified SC－Cys，344．5mg，WaS Obtained tion maximum at412nm besides at280nm by this procedure．The structure of S－（Fig．3A，B）．Ontheadditionofasubstrate，3－ Carboxymethyl－L－CySteine was determined by Cl－Ala，the absorptlOn at412nm shifted to lH－NMR，maSS and ORD spectrometry，in 428nmandincreased，andanewabsorptlOn

COmParisonwiththeauthenticcompound． Peak appeared at343nm（Fig．3B－a）．This SPeCtrum did not change on standing fbr ∫JJ加J・rJJぐ叩くノ（竹中l、 lOmin．Further addition of TGA caused a Theβ－rePlacement reaction，SC－Cys syn－ decrease of428nm peak and anincreasein

thesis，and the γ－rePlacement reaction，S－ the343nmpeak（Fig．3B－b，C，d，e）． Carboxymethyl－hormocysteine synthesis，Were examineduslngTGAandthefbllowlngamino 一」川／〃りイ（ノ川血rJ／（〃〃汀 fJ。ん／．＼tWJ川。（ノ acid声aSthesubstrates：L－CySteine，L－Serine，L－ Theamino－terminalaminoacidsequenceof threonine，L－CyStine，3－Chloro－D－alanine，DL－ the enzyme was determined by automated homocysteine，L－methionine and L－homose－ Edman degradation．Thirty－Six amino acid rine．Butfbrmationoftheproductwasnotob－ residues，eXCePtthe33rdoneftomtheamino－ served． terminalend，Were determined，aS Shownin Theβ－rePlacementreactionwascarriedout Fig．4．Ahomologoussequencewassearched uslng3－CトAla and various thioIcompounds fbrintheProteinSequenceDataBaseofthe assubstrates．Theproductsweredetectedand National Biomedical Research Foundation， determined by paper chromatography，and but a sequence showlng high homology was Withanaminoacidanalyzer．Thefbrmationof not fbund．

VariousS－Substitutedaminoacidswasobserv＿ edandtherelativereactionratesareshownin 2486 H. Kumagai et al.

Fig. 3. Absorption Spectra of SC-Cys Synthase from E. coli W3110. The crystallized enzyme was dissolved in a small amount of K2HPO4-KH2PO4buffer, pH 7.8, containing 20yUMPLPand 4mMEDTA,and then dialyzed against the same buffer. The spectra of the solution was read against a blank containing the dialysis buffer. The spectra of the native enzyme are shown in (A) and (B). The concentration of the enzyme protein was 0.38mg/ml. The spectra were taken 1, 3, 5 and lOmin later after addition of50mM 3-Cl-Ala (a), and 1 (b), 3 (c), 5 (d) and 15hr (e) later after the further addition of 100mM TGA.

1 2 3 4 5 6 7 8 9 10 Ser-Lys- I I e-Phe-Gl u-Asp-Asn-Ser-Leu -Thr- during the purification of the enzyme (Table I. ll 12 13 14 15 16 17 18 19 20 Steps 3 and 7). I I e-Gl y-Hi s-Thr-Pro-Leu-Val -Arg-Leu-Asn- 21 22 23 24 25 26 27 28 29 30 The E. coli strain used in this work produced Arg- 1 1 e-Gl y-Asn-Gly-Arg- 1 1 e-Leu-Al a-Lys- the enzyme constitutively in the LB broth and 31 32 33 34 35 36 the addition of L-tryptophan or L-cysteine to Val-Glu-? -Arg-Asn-Pro- the medium did not affect the enzyme activity, Fig. 4. Amino-Terminal Amino Acid Sequence of SC- indicating that the enzyme is not involved in Cys Synthase from E. coli W3110. the synthesis of these amino acids, since the Sequence analysis was carried out with the lyophilized biosynthesis of these enzymesare knownto be crystalline preparation, as described under Materials and repressed by the final product of the synthesis Methods. pathway. The enzyme catalyzed the a,/?-elimination Discussion reaction of 3-Cl-Ala, L-serine and L-cystathionine, but at a very low rate compared with We found an enzyme that synthesizes S- SC-Cys synthesis. The enzyme was not inac- carboxymethyl-L-cysteine from 3-chloro-L-ala- tivated on incubation with 3-Cl-Ala. PLP-de- nine and thioglycolic acid in a wild type strain pendent enzymes which catalyze transamina- of E. coli, and named it S-carboxymethyl- tion, racemization or decarboxylation are cysteine synthase. The purification and char- knownto catalyze the a,/?-elimination and /?- acterization of the enzyme are reported in this replacement reactions of 3-Cl-Ala as second- paper. ary reactions, but most of the enzymes are On long-term storage of the enzyme in the gradually inactivated during the course of re- ammoniumsulfte precipitate, its activity de- action with 3-Cl-Ala.15) So the catalytic prop- creased but the activity was recovered and erties of the enzyme so far investigated in- occasionally enhanced on dialysis of the prep- dicate that it is a /^-replacement specific PLP aration against Buffer A. This was observed dependent enzvme. Wewere unable to find an E. coli S-Carboxymethylcysteine Synthase 2487 enzymewith similar molecular and catalytic 3) E. Wang and C. Walsh, Biochemistry, 17, 1313 (1978). properties amongother reported enzymes.So 4) H. Kumagai and E. W. Miles, Biochem. Biophys. Res. at present, the physiological reaction catalyzed Commun., 44, 1271 (1971). by this enzyme in E. coli cells remains obscure. 5) H. Yamada and H. Kumagai, Pure Appl. Chem., 50, Investigation of the reaction conditions with 1117 (1978). a cell-free extract or with E. coli cells is impor- 6) H. Kumagai, H. Tanaka, S. Sejima and H. Yamada, tant for the practical use of this enzyme. Agric. Biol. Chem., 41, 2071 (1977). 7) G. S. Dhillon, T. Nagasawa and H. Yamada, /. Furthermore, it is necessary to use racemic 3- BiotechnoL, 1, 47 (1984). chloro-alanine as the substrate and to eluci- 8) C. Inoue and S. Moriguchi, US Patent, 4,283,554 date the fate of D-form 3-chloro-alanine in the (1981). reaction mixture with a cell-free extract or 9) A. Quevauviller, Vu-Ngoc-Huyen, S. Garcet and L. cells. Lakah, Therapie, 22, 485 (1967). 10) J. H. Miller, "Experiments in Molecular Genetics," Acknowledgments. Wewish to thank Dr. S. Kuramitsu Cold Spring Harbor Laboratory, Cold Spring and Dr. H. Kagamiyama, Osaka Medical College, for the Harbor, N. Y., 1972. N-terminalaminoacidsequenceanalysisandDr. T. Ueno, ij) h. J. Vogel and D. M. Bonner, J. Biol Chem., 218, Kyoto University, for the !H-NMR and mass spec- 97 (1956). trometeric analysis, and the fruitful discussion. We also N. M. Kredick and M. A. Becker, in "Methods in wish to thank Mr. K. Nishikawa, Mrs. A. Hayashida Enzymology," Vol. 17B, ed. by H. Tabor and C. W. and Mrs. N. Yamanofor their excellent assistance. We Tabor, Academic Press, New York, 1971, pp. also acknowledge the support of Dr. C. Inoue and Dr. J. Kasai, ShowaDenkoCo., throughout the course of T. Nagasawa,459T. Ishii, H. Kumagai-464.and H. Yamada, this work. Eur. J. Biochem., 153, 5411 (1985). O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, /. Biol. Chem., 193, 265 (1951). ReferencesE. W. Miles, in "Vitamine B6, Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects," Part B 1) Y. Morino and M. Okamoto, Biochem. Biophys. Res. (Vol. IB), ed. by D. Dolphin, R. Poulson, O. Commun., 40, 600 (1970). Avramovi, J. Wiley & Sons, Inc., New York, 1986, 2) Y. Morino, H. Kojima and S. Tanase, J. Biol. Chem., pp. 253-310. 254, 279 (1979).