Biochimica et Biophysica Acta, 1! 19 (1992) 175- !77 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00

175

BBAPRO 34107

NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes Fumio Watanabe 1, Yoshihisa Nakano 2, Hisako Saido 2, Yoshiyuki Tamura i and Hiroyuki Yamanaka 1 I Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, Osaka (Japan) and 2 Department of Agricldtural Chemistry, Unirersity of Osaka Prefectare, Osaka (Japan) (Received 7 August 1991)

Key words: Vitamin B-12; Cobalamin; Aquacobalamin reductase; NADPH-cytochrome c (P-450) reductase; (Rat liver microsome)

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79000. The purified aquacobalamin reductase showed a high specific activity (about 5 5 / z m o i / m i n per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobolamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.

Introduction

The bacterial system for the synthesis of a cobalamin (Cbl) coenzyme, 5'-deoxyadenosylcobalamin (AdoCbl), involves three distinct enzymes, aquacobalamin (AqCbl) reductase (EC 1.6.99.8), cob(ll)alamin reductase (EC 1.6.99.9) and eob(l)alamin adenosyltransferase (EC 2.5.1.17) [1-3]. Mammalian cells are assumed to have the identical system found in the bacteria. The synthetic activity of hydroxocobalamin (OH-Cbl) or AqCbl to AdoCbl is found in the mitochondrial fraction of mammalian cells [4,5]. AqCbl reductase catalyzes the reduction of AqCbl to cob(II)alamin. The enzyme has been reported to occur in some microorganisms [1,6] and mammalian tissues [7]. Clostridium tetanomorphum and Euglena gracilis contain NADH-linked (EC 1.6.99.8) and NADPH-linked (EC 1.6.99.11)AqCbl reductases, respectively, both

Abbreviations: AdoCbl, 5'-deoxyadenosylcobalamin; Cbl, cobalamin; OH-Cbi, hydroxocobalamin. Correspondence: F. Watanabe, Laboratory of Nutrition and Food Science, Hagoromo-gakuen College, 1-89-1 Hamadera-minamimachi, Sakai, Osaka 592, Japan.

of which have been purified and characterized [1,8]. While mammalian liver contains both [7], which are distributed in both mitochondrial (approx. 40%) and microsomal (60%) membranes of rat or human liver [9,10]. Information concerning the enzymological properties or physiological roles of each of the mammalian enzymes is not available at present. Here we describe characterization of NADPH-linked AqCbl reductase from rat liver microsomes. Materials and Methods

Animal. Male Wister-strain rats (body weight; about 250 g) fed a commercially available rat diet, CE-2 (Clea, Tokyo, Japan), ad libitum were used. Preparation of rat liver microsomes. Livers were obtained from rats starved for 24 h, washed with chilled 0.9% (w/v) NaCI solution, cut into small pieces with a razor blade and homogenized in about 10 vols. of 10 mM Tris-HCl buffer (pH 7.5), containing 0.25 M sucrose, 50 mM KCI, 2 mM MgC! 2 and 0.1 mM ethylenediaminetetraacetic acid (EDTA) by using a glass homogenizer with a Teflon pestle (two strokes at 1000 rpm). The homogenate was filtered through a double layer of gauze to remove unbroken tissucs and then centrifuged at 500 × g for 10 min to remove unbroken cells. The supernatant, after centrifugation at 12 000 x g

176 for 20 min, was further centrifuged at 100000 × g for 60 min. The precipitate was washed twice with 10 mM Tris-HCl buffer (pH 7.0) containing 1 mM EDTA and 0.1 mM dithiothreitol (DTI'), suspended with the washing buffer and used as the microsomal fraction. All procedures were done at 4°C. Enzyme assay. NADPH-linked AqCbl reductase activity was assayed by spectrophotometric estimation of the amount of AqCbl to cob(ll)alamin at 45°C as described previously [9]. The reaction mixture (1.0 ml) contained 50 mM Tris-HC! buffer (pH 7.5), 0.1 mM OH-Cbl, 0.2 mM NADPH and enzyme. The cob(ll)alamin formed was assayed by measuring the decrease in absorbance of AqCbl at 525 nm (E525 = 5.57 X 10 3 M - t cm-t). NADPH-cytochrome c (P-450) reductase activity was assayed by the method of Masters et al. [11]. Solubilization and purification of NADPH-linked AqCb! reductase. Solubilization mixture for the rat liver microsomal NADPH-linked AqCbl reductase contained 50 mM Tris-HC! buffer (pH 7.5), 20% (w/v) ethylene glycol, 1% (w/v) Triton X-100, 1% (w/v) sodium deoxycholate and the rat liver microsomal fraction, 4 mg of protein/ml of mixture. The enzyme was solubilized with stirring at 0°C for 60 min. The mixture was centrifuged at 100000× g for 60 min and the supernatant was subjected to the following purification. Purification procedures were performed at 0-:¢°C unless otherwise specified. The solubilized fraction (250 ml) was put on a column (3 × 10 cm) of DEAE-cellulose equilibrated with 10 mM Tris-HC! buffer (pH 8.0), containing 20% (w/v) ethylene glycol, 0.2% (w/v) Triton X-100, 1 mM EDTA and 0.1 mM DTI" and eluted at a flow rate of 20 ml/h. The column was washed with 100 ml of the same buffer and then eluted with 300 ml of a linear gradient (0-0.5 M) of potassium chloride in the same buffer. The active fractions (34 ml) were combined, dialyzed overnight against the same buffer (2 !), and put on a column (1 × 5 cm) of 2'5'ADP-Sepharose 4B equilibrated with the same buffer. The column was washed with 100 ml of the same buffer and eluted with 30 ml of 1 mM NADPH in the same

buffer. The active fractions were combined and concentrated with poly(ethylene glycol) 20000. The concentrated solution was put on a column (1 × 100 cm) of Toyopearl HW55 equilibrated with 100 mM Tris-HCI buffer (pH 8.0) containing 20% (w/v) ethylene glycoi, 0.2% (w/v) Triton X-100, 1 mM EDTA and 0.1 mM DTT and eluted with the same buffer at a flow rate of 20 ml/h. The active fractions were combined, concentrated to a final vol. of 1 ml with poly(ethylene glycol) 20000 and stored at -20°C until experimental use. Estimation of monomeric molecular weight by SDSPAGE. SDS-polyacrylamide gel electrophoresis was done in 5-20% (w/v) linear gradient polyacrylamide slab gels as described by Laemmli [12]. Electrophoresis was carried out at a constant current (10 mA/gel), with Bromophenol blue as a migration marker. Proteins in the gel were stained with Coomassie brilliant blue R-250 and destained in 7% (v/v) acetic acid. The monomeric molecular weight of the enzyme was calibrated with an electrophoresis calibration kit (Pharmacia LKB Biotechnology, Uppsala, Sweden). Protein assay. Protein was assayed by the method of Bradford [13] with ovalbumin as a standard. Chemicals. AdoCbl, CN-Cbl, OH-Cbl and methylcobalamin (CH3-Cbl) were obtained from Sigma. 2'5'ADP-Sepharose 4B was obtained from Pharmacia LKB Biotechnology. Toyopeari HW55 was obtained from Tosoh, Japan. Results and Discussion

The NADPH-linked AqCbl reductase was completely solubilized from rat liver microsomes by use of a 1% (w/v) Triton X-100/l% (w/v) sodium deoxycholate system. Other detergents (cholic acid, digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, all at 1% (w/v)) only partially solubilized the enzyme. The purification of the NADPH-linked AqCbl reductase from rat liver microsomes is summarized in Table I. The enzyme was purified about 534-fold over the microsomal fraction in a yield of about 32%. SDS-

TABLE I

Purification of NADPH-linked AqCbl reductase from rat liver microsomes The detailed purification procedures are described in the text. Steps

Microsomal membrane Solubilization DEAE-cellulose 2'5'-ADP-Sepharose Toyopearl HW55

Protein

NADPH-linked AqCbl reductase

NADPH-cytochrome c reductase

Activity ratio

(mg)

specific activity (~ tool/ rain per mg protein) 0.021 0.021 0.031 !.345 11.220

yield (%)

specificactivity (/zmoi/ min per mg protein)

yield (%)

Cytochrome c reductase/ AqCbl reductase

100 97.9 68.8 37.1 32.1

0.099 0.099 0.146 6.673 54.865

100 97.9 68.8 39.1 33.2

4.71 4.71 4.71 4.96 4.89

1000 978.4 466.4 5.8 0.6

177

Mr

1

2

94000 6 7000 43000 t

30000

201 O0 .......... 14400

Fig. 1. SDS-polyacrylamide gel electrophoresis of rat liver microsomal NADPH-linked AqCbl reductase from the final purification step. The purified enzyme (4/~g of protein) and molecular weight standard proteins were subjected to electrophoresis with a 5-20% (w/v) linear gradient of acrylamide slab gel in the presence of SDS. The details are described in the text. 1, molecular weight (M r) standard proteins; 2, the purified enzyme.

polyacrylamide electrophoresis in 5-20% (w/v) linear gradient polyacrylamide slab gel for the final preparation gave a single protein band, of which a monomeric molecular weight was estimated to be 79000 (Fig. 1). The purified enzyme had the ability to reduce cytochrome c (54.8 /xmol/min per mg protein), potassium ferricyanide (107.7/xmol/min per mg protein) and 2,6-dichlorophenolindophenol (27 # m o l / m i n per mg protein) as well as AqCbl (11.2/~mol/min per mg protein). About 33% of the NADPH-cytochrome c reductase activity which was found in rat liver microsoreal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked AqCbl reductase was about 5.0 through the purification steps. Mammalian NADPH-cytochrome c reductase has been reported to be a flavoprotein with a monomeric molecular weight of 78000-79000 [14,15]. These results indicate that rat liver microsomal NADPH-linked AqCbl reductase is the NADPH-eytochrome c reductase, which has been purified and well characterized [14,15,16]. It is possible that the NADPH-c3'tochrome c reductase functions in the synthesis of the Cbl coenzymes in the microsomes.

Some properties of the enzyme as the microsomai NADPH-linked AqCbl reductase were determined. The optimum pH and temperature for activity was 7.5 and 45°C, respectively. The enzyme reaction followed Michaelis-Menten type kinetics toward NADPH and AqCbi. The apparent K,, values were 58 /zM for AqCbl in the presence of 0.2 mM NADPH and 14/xM for NADPH in the presence of 0.1 mM AqCbl. The enzyme was specific for AqCbl, but not for CN-Cbl, AdoCbl and CH 3-Cbl. The NADPH-linked AqCbl reductase has been reported to occur in mitochondrial membrane as well as in microsomes of rat liver [9]. The mitochondrial enzyme, which has been purified and characterized (H. Saido, F. Watanabe and Y. Nakano, unpublished data), was different from the microsomal enzyme, judging from their enzymologicai properties. The results of the characterization of the mitochondria! enzyme will be reported elsewhere.

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