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Mar 28, 1985 - as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of ...
THE JOURNALOF BIOLOGICAL CHEMISTRY

Vol. 260. No. 24, Issue of October 25, pp. 12920-12926 1985 Printed in Ij.S.A.

0 1985 by The American Society of Biological Chemists, Inc.

Monodehydroascorbate Reductase from Cucumber Is a Flavin Adenine Dinucleotide Enzyme* (Received for publication, March 28,1985)

M. Anwar Hossain and Kozi Asada From The Research Institute for Food Science, Kyoto University, Uji, Kyoto61 1, Japan

Monodehydroascorbate reductase (EC 1.6.5.4) was peroxy radical with a-tocopherol (Packer et al., 1979; Niki et purified from cucumber fruit to a homogeneous state al., 1984). The lipid peroxide-dependent production of MDA as judged by polyacrylamide gel electrophoresis. The has been observed in mitochondria and microsomes (Green cucumber monodehydroascorbate reductase was a and O’Brien, 1973).Several other organic radicals also oxidize monomer with a molecular weight of 47,000. It con- ascorbate producing MDA, and MDA has been supposed to tained 1mol of FAD/mol of enzyme which was reduced be an intermediate in the transition metal-catalyzed autoxibyNAD(P)Handreoxidizedbymonodehydroascordation of ascorbate (Bielski, 1982; Scarpa et al., 1983). Thus, bate. The enzyme had an exposed thiol group whose the production of MDA radicals seems to be ubiquitous in blockage with thiol reagents inhibitedtheelectron cells. transferfromNAD(P)H to theenzymeFAD.Both MDA radicals have been shown to have cytotoxicity (Stich NADH and NADPHserved as electron donorswith K , values of 4.6 and 23 FM, respectively, and V ,, of 200 et al., 1976; Bram et al., 1980), and it seems likely that MDA mol of NADH and 150 mol of NADPH oxidized mol of radicals generated in cells are scavenged. MDA radical is enzyme-’ s-l. The K,,, for monodehydroascorbate was spontaneously disproportionated to ascorbate and dehydroas1.4 PI. The amino acid composition of the enzyme is corbate at anappreciable rate (-lo5 M-’s-l at pH7.0; Bielski, presented. In addition to monodehydroascorbate, the 1982). Even so, MDA radicals in cells seem to be reduced to enzyme catalyzed the reduction of ferricyanide and ascorbate by an NADH-dependent activity. In mammalian 2,6-dichloroindophenolbutshowed little reactivity cells, the activity is localized in the outer membranes of with calf liver cytochrome b5 and horse heart cytochrome e. The kinetic datasuggested a ping-pong mitochondria (It0 et al., 1981; Diliberto et al., 1982), micromechanism for the monodehydroascorbate reductase- somes (Schulze and Staudinger, 1971), coated vesicles, and catalyzed reaction. Cucumber monodehydroascorbate Golgi apparatus (Sun et al., 1983). Ehrlich ascites cells have reductase occurs in soluble form and can be distin- NAD(P)H-dependent MDA reducing activity (Pethig et al., 1985). The participation of cytochrome b, and its reductase guished from NADPH dehydrogenase, NADH dehydroin the MDA reducing activity has been shown by Hara and genase, DT diaphorase, microsome-bound NADH-cytochrome b6 reductase, and NADPH-cytochromec re- Minakami (1971). Reduction of MDA by purified NADHductase by its molecular weight, amino acid composi- cytochrome b5 reductase has been shown by Iyanagi and tion, andspecificity of electron acceptors and donors. Yamazaki (1969). On the other hand, Schulze et al. (1970) and Ponninghaus and Schulze (1972) have shown the occurrence of MDA reductase in microsomes which can be distinMonodehydroascorbate (MDA’) is produced by univalent guished from the cytochrome b5 reductase in immunological oxidation of ascorbate in enzymatic and nonenzymatic reac- properties and sensitivity toward inhibitors. Soluble MDA tions. It is the primary oxidation product of ascorbate in reductase purified from Neurospora crassa appears to contain reactions catalyzed by ascorbate oxidase, peroxidase (Yama- neither heme nor flavin (Schulze et d.,1972). MDA reductase has been found in many plants (Mathews, zaki and Piette, 1961), including an ascorbate-specific one (Hossain et al., 1984), and dopamine-&hydroxylase (Diliberto 1951; Beevers, 1954; Fujimura and Ikeda, 1957; Marr6 and and Allen, 1981). Its production has been shown by the Arrigoni, 1958; Arrigoni*etal., 1981), but little has been done interaction with ascorbate of ceruloplasmin (Lohmann et al., to purify and characterize it. The enzyme is localized in 1979) and of ferricytochromes b5 (Everling et al., 1969) and chloroplast stroma of spinach(Hossain et al., 1984) and (Njus et al., 1983). The MDA radical is also produced by cucumber fruit (Yamauchi et aL, 1984). It participatesin the univalent oxidation of ascorbate by superoxide and hy- ascorbate regeneration in spinach chloroplasts for scavenging droxyl radicals (Cabelli and Bielski, 1983). Furthermore, hydrogen peroxide by ascorbate peroxidase (Hossain et al., MDA is produced by the reaction of ascorbate with the a- 1984). Wepartially purified MDA reductase from spinach and chromanoxy radical which is formed by the reaction of lipid showed the involvement of thiol in the catalysis (Hossain et * This work was supported by research grants from the Ministry al., 1984). We report here the purification of soluble MDA reductase of Education, Science, and Culture of Japan and the Yamada Science Foundation. The costs of publication of this article were defrayed in from cucumber to a homogeneous state. We found it tobe an part by the payment of page charges. This article must therefore be FAD enzyme and describe its enzymatic properties. This is hereby marked “advertisement” in accordance with 18 U.S.C. Section the first report of isolation of a soluble flavin enzyme with a 1734 solely to indicate this fact. The abbreviations used are: MDA, monodehydroascorbate; substrate that is a radical, although several flavin enzymes can catalyze one-electron reduction of substrates. HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

12920

12921

Monodehydroascorbate Reductase Cucumber

- - -- l+NADH

FIG. 2. Absorption spectra of purified cucumber MDA reductase (83 NM) in 50 mM potassium phosphate, pH 7.0, under aerobic conditions.

-.“.-

Curve 1, oxidized enzyme; curve 2, fully reduced enzyme with its 1.5 molar equivalents of NADH (the same spectrum was observed when NADH was added under anaerobic conditions); curve 3, half reduced enzyme with its 0.5 molar equivalent of NADH; curve 4, fully reduced enzyme with dithionite; curve 5, reoxidized enzyme from the fully NADH-reduced species by MDAradical generated by ascorbate and ascorbate oxidase; curve 6, spectrum obtained 2 min after the enzyme was reduced with its 1.5 molar eqivalents of NADH. The scan time from 300 to 700 nm was 1 min.

Oxidiied enzyme (1) (2) 1 + 0 5 N A D H (3)

- 05

250300

400

500

700

Wavelength (nm) flavin enzymes which catalyze the univalent reduction of electron acceptors: NADPH dehydrogenase (EC 1.6.99.1), NADH dehydrogenase (EC 1.6.99.3), microsome-bound DISCUSSION NADPH cytochrome c reductase (EC 1.6.2.4), and NADHMDA reductase activity coupled to reduced pyridine nu- cytochrome b5 reductase (EC 1.6.2.2). These enzymes differ cleotides has been widely found in eukaryotes: animals, plants, in intracellular localization and flavin prosthetic groups, their fungi, algae (Arrigoni et al., 1981), and protozoa (Shigeoka et absorption peaks, molecular weight, or specificity to electron al., 1980). Because of the absence of ascorbate inprokaryotes, donors and acceptors. Cucumber MDA reductase also can be this enzyme is thought to be absent from bacteria. The cuc- distinguished from the FAD enzyme menadione reductase umber MDA reductase isolated here was found to be an FAD (DT diaphorase, EC 1.6.99.2) because it does not catalyze the enzyme having a molecular weight of 47,000. Both NADH reduction of menadione (Table 111) and is not inhibited by and NADPH serve as electron donors, and the enzyme is dicumarol, a specific inhibitor of menadione reductase. Plant inhibited by thiol reagents, which are similar to those of menadione reductase (Spitzberg and Coscia, 1982) isnot partially purified plant enzymes (Beevers, 1954; Fujimura and inhibited by p-chloromercuribenzoate, unlike MDA reductase Ikeda, 1957; Man6 and Arrigoni, 1958; Hossain et al., 1984). (Table IV). We found the menadione reducing activity in the MDA reductase from Neurospora is a simple protein, and 250 mMKC1 fraction of DEAE-Sephacel chromatography no prosthetic group has been found. Its molecular weight is (data notshown). 66,000, and only NADH serves as theelectron donor (Schulze The contents of MDA reductase in intact spinach chloroet al., 1972). Thus, the fungal enzyme appears to be different from the plant one. In mammalian tissues, MDA reducing plasts are estimated to be 2-10 p~ from its activity on the activity has been found in microsomes (Lumper et al., 1967), chlorophyll basis (Hossain et al., 1984), if we assume that the and microsome-bound NADH-cytochrome b5 reductase cata- spinach enzyme has the same molecular weight and specific lyzes the reduction of MDA in addition to cytochrome b5 activity as thecucumber enzyme and thechloroplast volume (Iyanagi andYamazaki, 1969). However, the molecular weight is 26 p1 mg of chlorophyll-’ (Heldt et al., 1973). The spinach of NADH-cytochrome b5 reductase (43,000) is smaller than enzyme is localized in the stroma in a soluble form (Hossain that of cucumber MDA reductase, and themicrosomal enzyme et al., 1984), and this is also the case for cucumber fruit uses only NADH, although both enzymes contain FAD. In (Yamauchi et al., 1984). The enzyme plays a role in the addition to microsomes, mammalian tissues have MDA re- generation of ascorbate from MDA which is produced by ducing activity in mitochondria. The outer mitochondrial ascorbate peroxidase for scavenging hydrogen peroxide (Hosmembrane cytochrome b participatesin the reaction, and sain et aL, 1984).In cucumber fruit, MDA reductase is thought to participate in the regeneration of ascorbate from MDA NADH serves as theelectron donor (Ito et al., 1981). Cucumber MDA reductase is, to our knowledge, the first produced by ascorbate peroxidase and ascorbate oxidase. The known flavin enzyme with a physiological electron acceptor flavin enzymes glutathione reductase (Foyer and Halliwell, that is a radical. It can be distinguished from the following 1976) and ferredoxin-NADP reductase (Shin, 1971) are localized in chloroplasts. Crystalline ferredoxin-NADP reductase purified from spinach (Asada and Takahashi, 1971) did not Portions of this paper (including “Materials and Methods,” “Recatalyze the oxidation of NADH byMDA under the standard sults,” Figs. 1 and 3-6, and Tables I-IV) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a assay conditions (data not shown). GSSG could not serve as standard magnifying glass. Full size photocopies are available from the electron acceptor of cucumber MDA reductase (Table 111). the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Thus, MDA reductase is the thirdflavin enzyme isolated from MD 20814. Request Document No. 85M-973, cite the authors, and include a check or money order for $8.80 per set of photocopies. Full chloroplasts. The reaction kinetics of MDA reductase (Fig. 4) seems to size photocopies are also included in the microfilm edition of the show that the reaction proceeds by a ping-pong mechanism. Journal thatis available from Waverly Press. MATERIALS AND METHODS AND RESULTS’

12922

Cucumber Monodehydroascorbate Reductase

Hossain, M. A,, and Asada, K. (1984) Plant Cell Physwl. 2 5 , 12851295 Hossain, M. A., Nakano, Y., and Asada, K. (1984) Plant Cell Physiol. 25,385-395 Ito, A., Hayashi, S., and Yoshida, T. (1981) Biochem. Biophys. Res. Commun. 101,591-598 Iyanagi, T., and Yamazaki, I. (1969) Biochim.Biophys.Acta 1 7 2 , 370-381 Koziol, J. (1971) Methods Enzymol. 18B, 253-285 Kubota, S., Yoshida, Y., and Kumaoka, H. (1977) J . Biochem. (Tokyo) 81,187-195 E-FAD NADH + E-FADH,-NAD+ Laemmli, U. K., and Favre, M. (1973) J. Mol. Bid. 80,575-599 E-FADH,-NAD+ + MDA -+ E-FADH. -NAD+ ascorbate Laroff, G. P., Fessenden, R. W., and Schuler, R. H. (1972) J. Am. chem. Soc. 94,9062-9073 E-FADH. -NAD+ + MDA -+ E-FAD NAD+ ascorbate Lohmann, W., Schreiber, J., and Greulich, W. (1979) Z. Naturforsch. Sect. C Biosci. 3 4 , 550-554 Thus, the reaction mechanism of MDA reductase is similar Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) to thatof NADH-cytochrome hs reductase (Rogers and StrittJ. Bwl. Chem. 193, 265-275 matter, 1974). MDA occurs in an anionic form at physiological Lumper, L., Schneider, W., and Staudinger, H. (1967) Hoppe-Seyler’s pH; therefore, the suppression of MDA reductase activity by 2. Physiol. Chem. 348, 323-328 salts (Fig. 6) see,msto be caused by competition between MDA MarrB, E., and Arrigoni, 0.(1958) Biochim. Biophys. Acta 3 0 , 453457 anion radicals and theanions, but the final conclusion awaits M. B. (1951) J . Biol. Chem. 189,695-704 the determination of the salteffect on the partialreactions of Mathews, Moore, S. (1963) J. Biol. Chem. 2 3 8 , 235-237 MDA reductase. Niki, E., Saito, T., Kawakami, A., and Kamiya, Y. (1984) J. Biol. Chem. 259,4177-4182 Acknowledgments-We are indebted to the Enzyme Factory, To- Nishikimi, M. (1975) Bwchem. Biophys.Res. Commun. 63,463-468 yobo Co., Ltd., Tsuruga, Japan, for the purification of the enzyme a t Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J . Biol. Chem. the first step. We thank Dr. M. Takahashi of this laboratory for his 258,27-30 helpful suggestions. We also thank Dr. Y. Sugiura of The Faculty of Packer, J. E., Slater, T. F., and Willson, R. L. (1979) Nature 2 7 8 , 737-738 Pharmacology and Dr. B. Mikami of this Institute, Kyoto University, for their help with theEPR determination andthe amino acid Panke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. Biochem. 6 0 , 45-50 analysis. Pethig, R., Gascoyne, P. R. C., McLaughlin, J. A., and Szent-Gyorgyi, A. (1985) Proc. Natl. Acud. Sci. U. S. A. 8 2 , 1439-1442 REFERENCES Ponnighaus, J. M., and Schulze, H. U. (1972) Hoppe-Seyler’s Z. Andrews, P. (1964) Biochem. J. 9 1 , 222-233 Physiol. Chem. 353,815-824 Arrigoni, O., Dipierro, S., and Borraccino, G. (1981) FEBS Lett. 1 2 5 , Rogers, M. J., and Strittmatter, P. (1974) J. Biol. Chem. 2 4 9 , 895242-244 900 Asada, K., and Takahashi, M. (1971) Plant Cell Physiol. 12,361-375 Scarpa, M., Stranato, R., Viglino, P., and Rigo,A. (1983) J. Biol. Beevers, H. (1954) Plant Physiol. (Bethesda) 2 9 , 265-269 Chem. 258,6695-6697 Bielski, B. H. J. (1978) Photochem. Photobiol. 2 8 , 645-649 Schuler, R. H. (1977) Radiut. Res. 69,417-433 Bielski, B. H. J. (1982) in Ascorbic Acid: Chemistry, Metabolism, and Schulze, H. U., and Staudinger, Hj. (1971) Hoppe-Seyler’s2. Physiol. Uses (Seib, P. A., and Tolbert, B. M., eds) pp. 81-100, American Chem. 352,309-317 Chemical Society, Washington, D.C. Schulze, H. U., Gallenkamp, H., and Staudinger, Hj. (1970) HoppeBielski, B. H. J., Comstock, D. A., and Bowen, R. A. (1971) J. Am. Seyler’s Z. Physwl. Chem. 351,809-817 93,5624-5629 Chem. SOC. Schulze, H. U., Schott, H.H., and Staudinger, Hj. (1972) HoppeBram, S., Froussard, P., Guichard, M., Jasmin, C., Augery, Y., SinSeyler’s 2. Physwl. Chem. 353,1931-1942 oussi-Barre, F., and Wray, W. (1980) Nature 284,629-631 Shigeoka, S., Nakano, Y., and Kitaoka, S. (1980) Biochem. J. 186, Cabelli, D. E., and Bielski, B. H. J. (1983) J. Phys. Chem. 87,1809377-390 1812 Shin, M. (1971) Methods Enzymol. 2 3 , 440-447 Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427 Spitsberg, V. L., and Coscia, C. J. (1982) Eur. J. Biochem. 127,67Diliberto, E. J., Jr., and Allen, P. L. (1981) J. Biol. Chem. 256,338570 3393 Stich, H. F., Karim, J., Koropatnick, J., and Lo, L. (1976) Nature Diliberto, E. J., Jr., Dean, G., Carte, C., and Allen, P. L. (1982) J. 260,722-724 Neurochem. 3 9 , 563-568 Strittmatter, P. (1967) Methods Enzymol. 10,553-556 Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 Strittmatter, P., and Velick, S. F.. (1956) J. Bwl. Chem. 2 2 1 , 253Ernster, L., Danielson, L., and Ljunggren, M. (1962) Biochim. Bw264 phys. Acta 5 8 , 171-188 Strittmatter, P., and Velick, S. F. (1957) J . Biol. Chem. 228, 785Everling, F. B., Weis, W., and Staudinger, Hj. (1969) Hoppe-Seyler’s 799 Z. Physwl. Chem. 350, 1485-1492 Sun, I. L., Crane, F. L., and Morrk, D. J. (1983) Biochem. Biophys. Faeder, E. J., and Siegel, L. M. (1973) Anal. Biochem. 53,332-336 Res. Commun. 115,952-957 Foyer, C. H., and Halliwell, B. (1976) Planta (Berl.) 1 3 3 , 21-25 Voordouw, G., Veeger, C., van Breemen, J. F. L., and van Bruggen, Fujimura, K., and Ikeda, S. (1957) Mem. Res. Znst. Food Sci. Kyoto E. F. J. (1979) Eur. J. Bioehem. 98,447-454 Uniu. 13,45-64 Williams, C . H., and Kamin, H. (1962) J. Biol. Chem. 237,587-595 Green, R. C., and OBrien, P. J. (1973) Biochim. Biophys. Acta 2 9 3 , Yagi, K. (1971) Methods Enzymol. 17B, 608-622 Yamauchi, N., Yamawaki, K., andUeda, Y. (1984) J.Jpn. SOC. Hortic. 334-342 Hara, T., and Minakami, S. (1971) J. Biochem. (Tokyo) 69, 325-330 Sci. 53,347-353 Heldt, H. W., Werdan, K., Milovancev, M., and Geller, G. (1973) Yamazaki, I., and Piette, L. H. (1961) Biochim. Biophys. Acta 5 0 , 62-69 Biochim. Biophys. Acta3 1 4 , 224-241

The enzymeFAD is reduced by NAD(P)H, and a charge transfer complex, E-FADH2-NAD+,is formed (Fig. 2 ) . When one exposed thiol group is blockedby thiol reagents, the electron transfer from NADH to the FAD is suppressed (Fig. 5 ) . The reduced enzyme then donates the electrons to MDA through two successive one-electron transfers, and the FAD semiquinone, E-FADH. -NAD+, is thought to be an intermediate.

+

+

+ +

Cucumber Momdehydroascorbate Reductase

1292.3

SUPPLEMENTAL MATERIAL TO rlonodehydroaacorbate Reductase from Cucumber Is a Flavln Adenlne Dlnucleotlde Enzyme by 14. Anwar I108(1aIn and lo11 Asada )UTERIALS AND YETHODS U e s - HDA reductase actlvlty Was asaayed BpECt~OphotometrlC.1ly by followlnq the decrease In absorbance at 140 nm due to NADH or NADPH oxldatlon by MOA generated wlth ascorbate oxldase, a s descrlbed prevlously Hoasaln E a L . 19841. The standard reactlon m l x t u r e contalned 5 0 mJ4 HEPESNaOII, pH 7.6, 0 . 1 mM NADH, 2.5 mJ4 ascorbate. the enzyme and ascorbate OhIdaso to qlvc about 3 UU IIDA. The steady state concentrarlon of HDA was detcrmlned from the absorbance at 160 nm IDLelskl %a?. 19711 uslnq an abaorbance coefflclent of 1.3 cm-' ISchuler 19771. Proteln was detbrmlned accordlnq to !awry 5t-A. 119511 wlth bovlne serum albumln as the standard. 2-M~rcaptoethanolv a s removed by psnslnq the sample through a column of Sephadex G-25 prlor to the determlnntlon. N a t l v c polyacrylamlde dlac-gel electrophoresis was performed wlth 7.58 qel uslnq the method of Davls 119641. Sodlum dod-cyl a u l f ~ t c - ~ l y a c r y l a m lslab d ~ qel elsctrophorssla was carrled out accordlnq to Lnemmll and Favre 119lll uslnq

mu-'

.15t gel.

AmlnO acid analysln was earrled out wlth a Hltachl 835 amlno acld analyzer. The enzyme I 4 6 UBIwas hydrolyzed I n 1 ml of 6 H HCl I n Evacuated and sealed tubes for 2 2 , 48. and 72 h at 110 OC. Half-cystlne was dotermtned by performlc acld oxldatlon IMwrc 19611 followed by hydrolysis In 6 P4 HC1 for 22 h at 110 C. Tryptophane was dett-rmlne.1 by the method of Panke %a>. 119741. The thlol qroup was assayed from the ~ n c r e a s e In absorbancp at 4 1 2 uelnq a n 1~ nm 1" the pr~senc(. of 5 , 5 ' - d l t h l o b l ~ I 2 - n l t ~ 0 b ~ n 2 0acid1

absorbance coefflclpnt of 11.6 m8-I cm-' IEllnan 19591. nsterlala- Cytochromc h was purlfled from calf llver nccordlnq to thc method of Strlttmnttrr 119671 wlth some modlf.catlone. A f t e r nolublllzatlon wlth pancrentlc llpase ISlqmnl In the p r s s r n c r of soybean trypsln Inhlbltor I S l q m a l and subscqucnt ammonlum sulfate frnctionntlon 1 s t ~ 11, ~ crude cytochrome b 5 was further purlfled by DEAE-Sephacel chromatoqraphy and flnnlly Cclluloflnr G-700 gel flltratlon. Purlfled cytochrome b5 lA41J/A275 2.1) was quantlfl~dfrom the nbsorbnncr of the dlthlonlte-reduced spccles at 421 nm uslnq a n absorbance Cocfflclrnt of 1 7 1 m" cm" Istrlttmatter and VclIck 19561. DEAE-Srphacal, blue Sepharorr C1.-68 and Sephadcx C - 1 0 0 W F r C products of Pharmncla Flne Chemlcnls ISwedenl. D - M k n o acid oxldase, cytochrome c lhorsc hrartl and m n k r vcnom I C ~ O I A I Y Sa d m n n f e u r l were obtalncd from Slqma. xanthine oxldnsr from Rorhrlngrr, Hannhrlm. and ascorbatr oxldasc from Toyobo Co., Ltd., Japan. ApoD-amlno a r l d oxldllse was prcpnrcd accordlnq to the method Of Ynql (19711. Ferredoxin pur1fle.l from spinach I A 4 2 0 ~ ~ 2 7 2 0.47. Asndn and Takahashl 19711 was used. Purlflcntlon of HDA reductase- Unlrss OthCrwLie stated, all OpcrdtIOnS were c n r r w d out at 0-4 ' c . Fresh cucumber fruits I C ~ ~ - ~O ~ ~A ' JIU $SI 1200 kg1 from n market w c r r homoqsnlrrd ln a larqs blender 'rlth 2 5 0 lltrrs of 0.2 H potasrlum phoaphatr. pH 7.8, supplemented wit, I O mN 2-mcrcaptocthanol and 0 . 5 mJ4 EDTA. The homqenate was p r ~ s sfrltvrrl throuqh canvas bnqs after m l x l n q wlth 4 kq of cellte. Ammonium nulfote .)ab nddrd to the palC qr(.*n flltratc l e x t r n c t . 480 1 l t ~ r s I to IO\ saturation. Thr mlxture was stlrrrd with 4 kq of crlltv for 10 m l n and then prr.6~ flltcred. The qrrenl9h preclpltate vhlch contalnsd about 3 1 of the actlvlty was discarded. The flltrata was brought to 158 aaturatlon by further addltlon of ammOnlum mlfste; 4 kq cellte was sqaln added. After atlrrlnq for 30 mln, the mlxturc vas pres. filtered. The preclpltste vas collected and stored a t - 2 0 OC. The purIfIcatIon untll thlB Step Was conducted for a half day at the Enzyme Factory of Toyobo Co., Ltd.. Tsuruqa. The amnonlum Bullate preflpltatc was suapndsd I n 6 lltars of 1 0 mll potasslum phosphate, pH 8 . 2 , contstnlnq 10 mn 2-mercaptcmthanol and 0 . 2 mM EDTA (buffer A I . The n u ~ p n . I o n was flltcred throuqh a BUChnCr funnel under low suction and the preclpltate v a s vsshsd vlth buffer A . The comblned buffer A for 2 . 5 days wlth flve flltrate ( 1 0 lltersl was dlalyzed aqs1n.t chsnqes of buffer. The dlalyzsd S01utIon I 1 2 llterml was clar1;Iad by cantrlfuqatlon at 1 3 , 0 0 0 X 9 for 30 mln and than loaded to a DEAE-Scphacal column I 1 0 X 30 cml equlllbratad wlth buffer A. About 2 0 1 of the actlvlty was found I n the wa.hlnq buffer. The enzyme va. eluted .tepvIse wlth 5 0 (4.500 all, 100 1 3 , 5 0 0 mll and 2 5 0 mM 12,500 all KC1 I n buffer A . MDA reductase actlvlty was found In each KC1-eluate. The active fractions were separately pooled and concentrated by ultraflltratlon throuqh a PM 10 membrana fllter Imlconl. Thus. ya obtalnad nonadsorbed, 5 0 m M , 100 m M , and 250 nu4 KC1-eluted fraction. oC U D A reductase ITable 1 1 . The 50 mM KC1 fractlon w . 3 ~ further purlfled because of I C s hlqh speclflc actlvlty. The concentrated yellow solutlon I20 all of the 50 .W KC1 frsctlon vam appllad t o u Ssphsdor C-100 column I 5 X 90 cml equlllbratsd vlth buffer B 110 mll potasslum phoaphnts, pH 7.0, contalnlnq 1 0 mll I-mrcsptwthanol and 0.1 mll EDTA) Supplemnted vlth 100 W KC1. Active fractlon. of the eluate were p l s d , concentrated and CqUlllbratsd vlth buff&* 8 by Ultcaflltratlon.

.

PurIfICaLlon step

Total

Total .aCtl"ltY

pro,."'"

T;pccIfi