THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, [ne.

Vol. 262, No. 24, Issue of August 25, pp. 11514-11518,1987 Printed in U.S.A.

Purification and Characterization of Aquacobalamin Reductase (NADPH) from EugZena gradis*

(Received for publication, July 29, 1986)

Fumio Watanabe, Yuji Oki, Yoshihisa NakanoS, and Shozaburo Kitaoka From the Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 591, Japan

Euglena aquacobalamin reductase (NADPH EC 1 .6 .99~) was purified, and its subcellular distribution was studied to elucidate the mechanism of the conver- sion of hydroxocobalamin to 6'-deoxyadenosylcobal- amin. The enzyme was found in the mitochondria. It was purified about 150-fold over the Euglena mito- chondrial extract in a yield of 38%. The purified en- zyme was homogeneous in polyacrylamide gel electro- phoresis. Spectra of the purified enzyme showed that it was a flavoprotein. The molecular weight of the enzyme was calculated to be 66,000 by Sephadex G- 100 gel filtration and 65,000 by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis. The enzyme was specific to NADPH with an apparent K , of 43 PM and to hydroxocobalamin with an apparent K , of 55 PM. The enzyme did not require FAD or FMN as a cofactor. The optimum pH and temperature were 7.0 and 40 "C, respectively.

The conversion of OH-Cbll to Ado-Cbl in bacteria (1, 2) probably involves three enzymatic steps: the reduction of Co3+ in OH-Cbl to Co2+ by aquacohalamin reductase (EC 1.6.99.8), the reduction of Go2+ to Co" by cob(I1)alamin reductase (EC 1.6,99.9), and the adenosylating reaction of Co' to Ado-Cbl by cob(1)alamin adenosyltransferase (EC 2.5.1.17). This ad- enosyltransferase has been found in bacterial (2, 3) and ani- mal cells (4, and was purified several hundredfold from Clostridium tetanomorphum (5). However, the activities of the two other enzymes involved in the reduction of OH-Cbl have been detected only in C. t e t a n o m o ~ ~ u m (1).

Euglena gracilis has well-deve~oped organelles like those of higher animals and requires Cbl for growth (6); it gives readily Cbl-limited cells (7). Therefore, for the elucidation of the physiological function of Cbl and the mechanism of coenzyme Cbl synthesis, E. gracilis is an organism suitable for use as a model of higher animal systems.

Here, we describe the purification of aquacobalamin reduc- tase (NADPH: EC 1.6.99.") from E. gracilis and some of its properties. We also identified the subcellular distribution of the enzyme and discuss the mechanism of the synthesis of coenzyme Cbl in this protozoan.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. The abbreviations used are: Cbl, cobalamin; OH-Cbl, hydroxo-

cobalamin; Ado-Cbl, 5'-deoxyadenosylcobalamin; Mops, S-fN-mor- pho1ino)propanesulfonic acid; DTT, dithiothreitol; HPLC, high-per- formance liquid chromatography; CN-Cbl, cyanocobalamin; Me-Cbl, methylcobalamin; SDS, sodium dodecyl sulfate.

EXPERIMENTAL PROCEDURES

Organism and Culture-E. gracilis SM-ZK, a streptomycin- bleached mutant of E. gracilis z, which lacks chloroplasts without any change in other cellular components (8), was cultured for 5 days at 27 "C with illumination (2000 lux) in Koren-Hutner medium (9). We used Cbl-limited (0.05 pglliter medium) medium for experiments with subcellular fractionation.

Assay of A q u a c o ~ ~ m i n Reductase ~ N A ~ ~ H ~ and Other Enzymes- The aquacobalamin reductase (NADPH) activity was assayed by spectrophotometrical estimation of the amount of OH-Cbl converted to cob(I1)alamin at 40 "C (Hitachi 200-10 spectrophotometer). The assay mixture (1.0 ml) contained 50 &mol of Tris-acetate buffer, pH 7.0, 0.1 wmol of OH-Cbl, and 0.2 pmol of NADPH. The amount of cob(I1)alamin formed was assayed by measurement of the decrease in absorbance of OH-Cbl at 525 nm and calculated on the basis of the differential molecular extinction coefficient of OH-Cbl to cob(I1)alamin (5.57 X lo3 M" cm" at 525 nm). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the reduc- tion of OH-Cbl at the rate of 1 gmol/min.

Succinate-semialdehyde dehydrogenase (EC 1.2.1.16) (lo), a mi- tochondrial marker enzyme, glutamate dehydrogenase (EC 1.4.1.4) (111, a marker enzyme for the cytosol in Eugkna, and glucose-6- phosphatase (EC 3.1.3.9) (12), a microsomal marker enzyme, were assayed by the methods of the references cited.

Isolation of Eugkna Mitochondria-All procedures were done at 0- 4 "C. A cell homogenate of E. gracilis SM-ZK was obtained by partial trypsin digestion of the pellicles followed by mild mechanical disrup- tion by a modification of the method of Tokunaga et al. (ll), and the homogenate was fractionated by differential centrifugation. Eugknu cells (ahout 6 g, wet weight) grown for 5 days in the Cbl-limited medium were washed twice with 10 mM Mops in KOH buffer, pH 7.5, containing 0.3 M sucrose, and suspended in 5 ml of the same buffer, Trypsin (30 mg; bovine pancreas, Sigma type 111) was added to the suspension. The mixture was gently stirred in an ice bath for 45 min and centrifuged at 2,000 X g for 5 min to remove trypsin and broken cells. The treated cells were suspended in 5 ml of 10 mM Mops-KOH buffer, pH 7.5, containing 0.25 M sucrose and 3 mg of trypsin inhibitor (egg white, Sigma type 11-0) to inhibit the action of trypsin remaining in the cell suspension: within 2 min, the mixture was centrifuged at 2,000 X g for 5 min to remove trypsin inhibitor and broken cells. The partly digested, unbroken cells that were on the bottom of the tube were suspended in 10 mM Mops-KOH buffer, pH 7.5, containing 0.25 M sucrose and stirred gently for 10 min to cause the cells to burst. The cell homogenate was centrifuged at 2,000 X g for 5 min to remove the pellicle, paramylum, and undisrupted cells. The supernatant was centrifuged at 10,000 X g for 10 min. The pellet was washed twice and suspended in the same buffer and the suspension was used as the mitochondrial preparation.

Purification of Aquucobalamin Reductase (NADPH)--Purification procedures were at 0-4 "C unless otherwise specified. Partial trypsin digestion of Eugkm cells (62 g, wet weight) was used to obtain mitochondria. Mitochondria of E. gracilis (986 mg of protein) were suspended in 33 ml of 10 mM Tris-acetate buffer, pH 7.0, containing 1 mM EDTA and 1 PM DTT, disrupted by sonic oscillation (10 kHz, 20 s X 4), and centrifuged at 100,000 X g for 60 min to remove the

column (2 X 20 cm) of DEAE-Bio-Gel A equilibrated with 10 mM membrane fraction. The supernatant fraction (32 ml) was put on a

Tris-acetate buffer, pH 7.0, containing 1 mM EDTA and 1 pM DTT, and eluted at the flow rate of 21 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

11514

Euglena Aquacobalumin Reductase INADPHI 11515

active fractions (27 ml) were combined, dialyzed overnight against the same buffer (2 liters), and put on a column (1.5 X 5 cm) of DEAE- Bio-Gel A equilibrated with the same buffer. The column was washed with 30 ml of the same buffer and eluted with 100 ml of a linear gradient (0-0.3 M) of potassium chloride in the same buffer at the flow rate of 13 ml/h. The active fractions (11 ml) were combined and concentrated in a Centricon-30 microconcentrator (Amicon Corp.). The concentrated solution was put on a column (1 X 90 cm) of Sephadex G-100 equilibrated with 100 mM Tris-acetate buffer, pH 7.0, containing 1 D M EDTA and 1 p~ DTT, and eluted with the same buffer at the flow rate of 3 ml/h. The active fractions (8 ml) were combined and dialyzed against 10 mM Tris-acetate buffer, pH 7.0, containing 1 mM EDTA and 1 p~ DTT (1 liter). The dialyzed solution was put on an affinity column (1.5 x 10 cm) of Affi-Gel blue equilibrated with the same buffer a t the flow rate of 0.7 ml/h. The column was washed with 20 ml of the same buffer and eluted with 50 ml of a linear gradient (0-0.5 M) of potassium chloride in the same buffer. The active fractions (15 ml) were combined, concentrated to a final volume of 2 ml in the Centricon-30, and stored at -20 "C.

Assay of Ado-Cbl-Ado-Cbl was measured by HPLC (Japan Spec- troscopic Co., Ltd; UV spectrophotometer UVIDEC-100-V, HPLC pump BIP-I). Cbl was extracted with 80% ethanol at 90 "C for 30 min twice from a cell homogenate of Euglena disrupted by sonic oscillation (10 kHz, 20 s X 5). The extract was left to evaporate, and the residue was dissolved in distilled water. This solution was used as the sample for HPLC analysis. All procedures described above were done in the dark to protect the extracted Cbl from photolysis. The samples were put on a Unisif QC 18 column (7.21 X 300 mm) equilibrated with 0.04 M tartaric acid-sodium phosphate buffer, pH 3.0, containing 25% methanol at 40 "C. The flow rate was 1 ml/min. Cbl analogues were eluted with 30 ml of a linear gradient (25-75%) of methanol in the same buffer and assayed by measurement of absorbance at 350 nm. The retention times of OH-Cbl, CN-Cbl, Ado- Cbl, and Me-Cbl were 9.0, 13.5,18.0, and 21.0 min, respectively.

Assay of Overall Conversion of OH-Cbl to Ado-Cbl-The activity of the overall conversion of OH-Cbl to Ado-Cbl was measured by the assay of Ado-Cbl by HPLC. The assay of mixture (1.0 mi) contained 50 mM Tris-acetate buffer, pH 7.0, 2 mM MgCL, 3 mM ATP, 1 pM OH-Cbl, 0.01 p~ [G-3HjOH-Cbt (6.8 pCi), 0.4 mM NADPH, and enzyme. The reaction was started by the addition of NADPH, allowed to proceed for 60 min at 30 "C, and stopped by the addition of 4 ml of ethanol; then, the Cbl in the reaction mixture was extracted with 80% ethanol under the same conditions as described above. The Ado- Cbl formed was separated from OH-Cbl by HPLC and the Ado-Cbl fractions were collected and counted with a liquid scintillation counter (Aloka, LSC 903). (G-3H]OH-Cbl was prepared from [G-3H]CN-Cbl (9.2 Ci/mmol) by the method of Weissbach et al. (13).

Sucrose Linear Gradient Centrif~ation-The following procedures were done at about 4 "C. A portion (1.0 ml) of Euglena cell homog- enate prepared by the modified method of Tokunaga et al. (11) described above was layered on top of a 9.0-ml linear sucrose gradient (20-60%, w/w) and centrifuged at 100,000 X g for 2.5 h on a Hitachi 55P centrifuge with a swing-out bucket rotor (RPS-4OT). Fractions (0.6 ml) were collected from the bottom of the centrifuge tube.

Absorption Spectra of the Enzymtic Products-Spectra of OH- Cbl, c o b ( i I ) a ~ ~ i n , and the enzymatic products were measured with a Shimadzu multipurpose recording spectrophotometer (MSP-5000). Authentic OH-Cbl (0.2 pmol) was dissolved in 2.0 ml of 50 mM Tris- acetate buffer, pH 7.0, and authentic c o b ~ I I } a l ~ i n was prepared from this OH-Cbl solution by the method of Vitols et al. (5). Spectra of the enzymatic products were measured against a control mixture, which was the reaction mixture without the enzyme.

Polyacrylamide Gel Electrophoresis-Electrophoresis with 7.5% polyacrylamide disc gel was done as described by Davis (14) a t pH 9.4. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis with 10% acrylamide was done by the method of Weber and Osborn (15). Phosphorylase b from rabbit muscle (M, 94,000), albumin from bovine serum (M. 67,000), ovalbumin (Mr 43,000), carbonic anhydrase from bovine erythrocytes (M, 30,000), trypsin inhibitor from soybean (M, 20,100), and a-lactalbumin from bovine milk (M, 14,400) were used as standard proteins. Proteins in the gel were stained with Coomassie Brilliant Blue R-250 and destained with 7% acetic acid.

Estimation of M,--The M, of aquacobalamin reductase (NADPH) was estimated with use of a column (1 X 90 cm) of Sepbadex G-100 equilibrated with 100 mM Tris-acetate buffer, pH 7.0, and calibrated with blue dextran (average M,, 2,MK1,000), lactate dehydrogenase from pig heart ( M , 135,000) (16), malate dehydrogenase (NAD') from pig heart ( M , 70,000) (17), ovalbumin (M, 43,000), trypsin inhibitor from

soybean (M, 20,100), and cytochrome c from horse heart (M, 12,400). The activities of these enzymes were assayed in the references cited. Blue dextran and cytochrome c were assayed by measurement of the absorbance at 650 and 550 nm, respectively. Ovalbumin and trypsin inhibitor were assayed by the method of Bradford (18).

Protein Assay-Protein was measured by the method of Bradford (18) with ovalbumin as the standard protein.

Materiuh-"H-Cbl, CN-Cbl, Me-Cbl, and Ado-Cbl were obtained from Sigma. FAD and FMN were purchased from Wako Pure Chem- ical Industries, Ltd. An electrophoresis calibration kit was obtained from Pharmacia P-L Biochemicals. DEAE-Bio-Gel A and Affi-Gel blue were purchased from Bio-Rad. Centricon-30 was from the Ami- con Corp. Unisil QC 18 was obtained from the Gaskuro Kogyo Co., LM. [G-3H]CN-Cbl (9.2 Ci/mmol) was obtained from Du Pont-New England Nuclear.

RESULTS AND DISCUSSION

The enzymatic products of the Euglena aquacobalamin reductase (NADPH) were identified by measurement of their absorption spectrum. The absorption maximum of authentic OH-Cbl and cob(1I)alamin was at 525 and 475 nm, respec- tively, and that of the enzymatic product was at 475 nm (Fig. 1). Nonenzymatic reduction of OH-Cbl by reducing sub- stances of either high or low molecular weight in the Euglena crude homogenate were not detected. The results indicate that aquacobalamin reductase (NADPH) occurs in E. gracilis and that OH-Cbl is reduced to cob(I1)alamin by this enzyme.

Fluctuations in aquacobalamin reductase (NADPH) activ- ity and the amount of Ado-Cbl during Euglena cell growth are shown in Fig. 2. The activity of the enzyme reached a maximum (2.0 nmol/min/106 cells) in the early logarithmic growth phase and decreased thereafter to a constant activity of about 0.6 nmol/min/106 cells. The cellular concentration of Ado-Cbl also reached a maximum (1.1 fmol/106 cells) in the logarithmic phase. The results suggest that the two other enzymes involved in the synthesis of coenzyme Cbl, cob(1I)alamin reductase, and cob(1)alami~ adenosyltransfer- ase also are active in the early logarithmic phase and that the Ado-Cbl synthesized acts as a coenzyme of ribonucleotide

Wavelength (nm)

FIG. 1. Absorption spectra of the enzymatic products. De- tails of experimental procedures are described in the text. The enzy- matic reaction mixture contained 50 mM Tris-acetate buffer, pH 7.0, 0.1 mM OH-Cbl, 0.2 mM NADPH, and the crude enzyme (0.87 mg of protein). The reaction was started by addition of the enzyme, allowed to proceed for 10 min a t 40 "C, and stopped by being cooled to 0- 2 "C. The nonenzymatic reduction of OH-Cbl by reducing substances in the Euglena crude homogenate was measured in the same reaction mixture without the NADPH, as described above. Spectra of the enzymatic and nonenzymatic products were immediately scanned from 400 to 600 nm at the scanning speed of 400 nm/min. - - - -, authentic OH-Cbl; - - - ,authentic cob(I1)alamin; - - - -, nonen- zymatic products; and -, enzymatic products.

11516 Euglena Aquacobalamin Reductase (NADPH) .

W if I,., z I, 3 0 2 4 6 8

Day FIG. 2. Aquacobalamin reductase (NADPH) activity and the

amount of Ado-Cbl in cells during their growth. E. gracilis (8.5 X IO6 cells), which was first cultured for 5 days in Koren-Hutner medium, was inoculated in new medium (150 ml) and cultured for 8 days with illumination (2000 lux) at 27 "C. On the days indicated, 10 mi of Euglena cell culture was sampled. A portion (1.0 ml) of the sample was used to count cell numbers in a hematometer and the remaining 9.0 ml was washed twice with 10 mM Tris-acetate buffer, pH 7.0. Euglena cells were suspended in the same buffer (2.0 ml), disrupted by sonic oscillation (10 kHz, 20 s X 5), and centrifuged at 3000 X g for 5 min to remove the paramyion. The supernatant was used as the crude enzyme. Aquacobalamin reductase (NADPH) activ- ity was assayed in this preparation. The assay mixture (1.0 m1) of the enzyme contained 50 mM Tris-acetate buffer, pH 7.0, 0.1 mM OH- Cbl, 0.2 mM NADPH, and crude enzyme (0.1-0.5 mg of protein). Cbl was extracted by the addition of 4.0 ml of ethanol to 1.0 ml of the crude homogenate; the mixture was shaken vigorously and heated at 90 "C for 30 min in the dark. The suspension was centrifuged at 5000 X g for 10 min, and the Cbl remaining in the pellet was extracted with 80% ethanol (2.0 ml) under the same conditions. The combined supernatant fraction was allowed to evaporate and the residue was dissolved in 1.0 ml of distilled water. Abo-Cbl in this solution was assayed by HPLG. Refer to "Experimental Procedures" for other details. The data are represented by the mean of values from four experiments. A, cell numbers; e, aquacobalamin reductase activity; and ., amount of Ado-Gbl.

reductase (19), a CbI-dependent enzyme, which functions in DNA synthesis during that phase.

A cell homogenate of E. gracilis was separated by linear sucrose gradient (20-60%, w/w) centrifugation. The mito- chondria, microsomes, and cytosol were separated satisfactor- ily, since the activities of succinate-semialdehyde dehydrogen- ase, a mitochondrial marker enzyme, and glucose-6-phospha- tase, a microsomal marker enzyme, were found in peaks of about 1.18 and 1.12 g/cms, respectively, and that of glutamate dehydrogenase, a cytosolic marker enzyme in Euglena, was found in the top fraction (Fig. 3). The distribution profile of the activity of aquacobalamin reductase (NADPH) was iden- tical to that of succinate-semialdehyde dehydrogenase but not to that for glucose-6-phosphatase or glutamate dehyclrogen- ase, Most of the activity of the succinate-semialdehyde de- hydrogenase and aquacobalamin reductase (NADPH) in the homogenate was recovered in the mitochondrial fraction. The results show that aquacobalamin reductase (NADPH) of E. ~racilis is located in the mitochond~a.

The purification of the aquacobalamin reductase (NADPH) from the mitochondria of E. gracilis is summarized in Table I. The enzyme was purified about 150-fold over the mitochon- drial fraction in a yield of 38%. Disc polyacrylamide gel electrophoresis in 7.5% acrylamide at pH 9.4 for the final preparation gave a single protein band (Fig. 4). The purified enzyme was pale yellow. Absorption peaks of the purified native enzyme were at around 385 and 455 nm, and absorption disappeared completely on the addition of 0.2 mM NADPH under anaerobic conditions (Fig. 5), indicating that the Eu-

Fraction number FIG. 3. Subcellular distribution of aquacobalamin reduc-

tase (NADPH). Partial trypsin digestion of Euglena cells (wet weight, about 5 g) grown for 5 days in Cbl-Iimi~d Koren-Hunter medium (Cbl; 0.05 pg/liter medium) was used to obtain a cell homog- enate as described in the text. A portion (1.0 ml) of the cell homog- enate was layered on 9.0 ml of a linear sucrose gradient (20-60%, w/ w) and centrifuged at 100,000 X g for 2.5 h. Fractions were collected from the ~ t t o m of the tube. e, a q u a ~ b a I ~ ~ n reductase ( N A ~ P ~ ) , or AR; 0, succinate-semialdehyde dehydrogenase (NADP+), or SSADH, a, glutamate dehydrogenase (NADP+), or GDE, 0, glucose- 6-phosphatase, or G-6-Pase; and -, sucrose density. The activities of these enzymes were assayed as described under "Experimental Procedures." The sucrose density was found by measure men^ of the refractive index.

TABLE I P ~ r ~ f i ~ a ~ ~ n of ~ ~ c o ~ ~ ~ r n ~ n reductase [ N A ~ P H ~ from Euglena

gracilis Details of the purification are described under "Experimental

Procedures."

Protein activity activity

protein

Total Specific Yie,d

mg units milliunits/mg %

Mitochondria 986 14.7 14.9 100 Ultracentr~~gation 424 12.6 29.7 85.7 First DEAE-Bio-Gel A 29.7 12.1 407.4 82.3 Second DEAE-Bo-Gel A 15.9 12.2 767.3 83.0 Sephadex G-100 6.6 7.9 1197 53.7 Affi-Gel blue 2.5 5.6 2240 38.1

glena aquacobalamin reductase (NADPH) is a flavoprotein. Some properties of the Euglena aquacobalamin reductase

(NADPH) were studied. The optimum pH for activity was 7.0. The enzyme, when treated a t various pH for 10 min at 55 "C, was stable between pH 6.0 and 8.0 but completely lost its activity at below pH 5.0. The optimum temperature was 40 "C. The enzyme, when incubated at various temperatures for 10 min a t pH 7.0, was stable up to 50 "C; activity was completely lost a t 60 "C. The activation energy of the enzyme was calculated to be 2.7 kcal/mol from the Arrhenius plots. The enzyme reaction followed Michaelis-Menten kinetics to- ward NADPH and OH-Cbl. The apparent ICm values were 43 PM for NADPH in the presence of 0.2 mM OH-Cbl and 55 gM for OH-Cbl in the presence of 0.2 mM NADPH. The Euglena aquacobalamin reductase (NADPH) was specific to OH-Cbl, but not to CN-Cbl, and NADH could not replace NADPH. The enzyme of C. t e ~ a n ~ ~ o ~ ~ ~ ~ m (1) and the overall conver- sion reaction of OH-Cbl to Ado-Cbl in some organisms (4,20) are specific to NADH. The enzyme of Clostridium (1) requires FAD or FMN as cofactors for the reduction of OH-Cbl, but the Euglena enzyme did not. The Ms of the Euglena enzyme was estimated to be 66,000 by Sephadex G-100 gel filtration and 65,000 by SDS-polyacrylamide gel electrophoresis. The enzyme activity was inhibited 73,87, and 100% by incubation for 10 min with 5,5'-dithio-~is-(Z-nitrobenzoic acid), N-ethyl- maieimide, or mersalyl at 1 mM, respectively. The results

Euglena Aquacobalamin Reductase (NADPH) 11517

A B

,+ + FIG. 4. Polyacrylamide gel electrophoresis of aquacoba-

lamin reductase (NADPH) from the final purification step. A, the purified enzyme (10 pg of protein) was treated by electrophoresis on a 10% acrylamide gel in the presence of SDS, at constant current (6 mA/gel), with bromphenol blue as a migration marker. Proteins in the gel were stained continuously with 0.005% Coomassie Brilliant Blue R-250 containing 25% isopropanol and 10% acetic acid, with the same solution containing 10% isopropanol and 10% acetic acid, and then with the solution containing 10% acetic acid, and destained with 7% acetic acid. B, the purified enzyme (10 pg of protein) was treated by electrophoresis with 7.5% acrylamide gel in the absence of SDS, at constant current (2 mA/gel), with bromphenol blue as a migration marker. Proteins in the gel were stained with 0.005% Coomassie Brilliant Blue R-250 containing 10% acetic acid and destained with 7% acetic acid.

Wavelength Inm)

FIG. 5. Absorption spectra of purified aquacobalamin re- ductase (NADPH). Spectra of the purified native and reduced (0.2 mM NADPH) enzymes were made by measurement of absorbance of 260-650 nm at a scanning speed of 400 nm/min. The reduced enzyme was prepared as follows: the vessel containing the native enzyme (2.0 ml) was evacuated and flushed several times with oxygen-free argon gas and then 100 mM NADPH (2 pl) was added under a stream of argon gas. The protein concentration of the native enzyme was 1 mg/ ml. -, - - -, spectrum of the purified native enzyme; - - - - -, spectrum of the enzyme reduced by 0.2 mM NADPH under anaerobic conditions.

indicate that an SH group is in the active center of the enzyme. The activity was inhibited 49 and 81% by 1 mM Zn2+ or Al"', respectively; other metal ions (Na+, K', NiZ+, M$', Mn2+, Co2+, and Ca2+, all at 1 mM) and 1 mM EDTA did not cause inhibition.

Walker et al. (1) have suggested that the Clostridium en- zyme may be identical to NADH-FAD reductase and that the FADH, or FMNH, formed by the enzyme reduce OH-Cbl nonenzymatically to cob(II)alamin, since FAD or FMN is essential in the first reduction step of the bacterial system. The Euglena enzyme did not require these compounds as cofactors, and spectral experiments with the purified Euglena

enzyme confirmed that the enzyme contained 1 molecule of FAD or FMN as a prosthetic group (calculated on the basis of the molecular extinction coefficients of FAD and FMN at 375 nm). The results indicate that in Euglena, OH-Cbl was directly reduced to cob(I1)alamin by the enzyme for the first reduction step.

Ohta et al. (2) have reported that cob(1)alamin adenosyl- transferase is located in the ribosomes of Lactobacillus leich- mannii. In E. gracilis SM-ZK, overall conversion activity of OH-Cbl to Ado-Cbl was not detected in the microsomes but only in the mitochondria (Table 11). In the wild-type E. gracilis z, the activities of aquacobalamin reductase (NADPH) and the overall conversion reaction were detected only in the mitochondria, not in the chloroplasts or the microsomes (data not shown). The results indicate that all three enzymes in- volved in Ado-Cbl synthesis are located in the mitochondria in Euglena.

The Euglena enzyme was specific to OH-Cbl with an ap- parent K, of 55 PM, which is high considering the physiolog- ical concentration of Cbl in Euglena cells (about 2 PM, as calculated in our laboratory). However, the overall conversion rate of OH-Cbl to Ado-Cbl in the mitochondria was low (1.38 pmol/h/mg protein) (Table 11). The results suggest that Eu- glena aquacobalamin reductase (NADPH) can reduce OH-Cbl efficiently a t cellular concentrations of Cbl since the rate of the overall conversion is high enough to maintain the cellular Ado-Cbl concentration required for growth (1.1 fmol/1Ofi cells) (Fig. 2).

Isegawa et al. (21) have reported that after Euglena was labeled with radioactive CN-Cbl for 2 h, 16% of the CN-Cbl taken up by the cells (1.02 fmol/lO" cells) is converted to Ado- Cbl, and some 68 and 20% of the Ado-Cbl is located in the cytosol and the mitochondria, respectively. However, we found that aquacobalamin reductase (NADPH) and the sys- tems for the conversion of OH-Cbl to Ado-Cbl were located in the mitochondria, not in the cytosol (Table 11). Ado-Cbl would accumulate in the cytosol in vivo experiments because it was formed in the mitochondria but transferred into the cytosol by a counter-transport system, since the mitochon- drial conversion rate of OH-Cbl to Ado-Cbl (1.38 pmol/h/mg protein) was high enough to maintain the rate of Ado-Cbl synthesis in living Euglena cells (about 2.04 fmol/h/mg pro- tein, as calculated from the data reported by Isegawa et al.). Euglena mitochondria had a rapid Cbl-uptake system (about 30 fmol/min/mg protein), and some Cbl-binding proteins, probably involved in the membrane transport of Cbl, occurred in the membrane fraction of Euglena mitochondria.2

Some studies have indicated that OH-Cbl is a better sub- strate for Ado-Cbl synthesis than CN-Cb1(4,13,20). Probably these observations result from the substrate specificity of the aquacobalamin reductase (NADPH), which is specific to OH- Cbl but not to CN-Cbl in many organisms, including E. gracilis. An enzyme involved in the decyanation of CN-Cbl has been found in mammalian tissues (22). The same enzy- matic activity has been detected in Euglena homogenates.'

Walker et al. (1, 5) have suggested that cob(I1)alamin re- ductase and cob(1)alamin adenosyltransferase form a struc- tural and functional complex in C. tetanomorphum, since these enzymes are found together when purified. The Euglena aquacobalamin reductase (NADPH) was eluted as a single peak on first and second DEAE-Bio-Gel A ion-exchange chromatography and on Sephadex G-100 gel filtration. The peaks did not show activity for the overall conversion of OH- Cbl to Ado-Cbl (data not shown). These results suggest that

* F. Watanabe, Y. Oki, Y. Nakano, and S. Kitaoka, unpublished data.

11518 ~ u g ~ e n a Aquacobalam~~ Reductase t ~ A ~ P H ~ TABLE I1

D ~ ~ r i b u ~ i o n in the s u ~ e l ~ u l a r fractions of E. grucik of the ~ ~ i v i t ~ s of the o v e ~ l l conuersion of OH-Cbt to A ~ ~ - C ~ l , aquucobalamin reductase (NADPH), and marker enzymes

Partial trypsin digestion of Euglena cells (wet weight, about 6 g) grown for 5 days in Cbl-limited Koren-Hutner medium (Cbl; 0.05 pgfliter medium) was used to obtain a cell homogenate as described under ‘‘Exper~ment~ Procedures.” The cell homogenate was centrifuged at 10,000 X g for 10 min. The pellet was washed twice and suspended in 10 mM Mops-KOH buffer, pH 7.5, containing 0.25 M sucrose, and the suspension was used as the m i t ~ h o n ~ ~ a l fraction. The supernatant was centrifuged at 100,000 X g for 60 min. The pellet was washed and suspended in the same buffer; the suspension was used as the microsomal fraction. The supernatant was used as the cytosolic fraction. The marker enzymes, aquacobalamin reductase (NADPH), and the conversion of OH-Cbl to Ado-Cbl were assayed as described in the text. The data are typical distribution data from the three experiments.

Enzyme activities (percentage of the activities in the crude extract)

Homogenate Cvtosol Mitochondria Microsomes

Overall conversion of OH-Cbl to Ado-Cbl 100 (0.38)’’ 0 94.9 (1.381” 0 Aquacobalamin reductase (NADPH) 100 (4.11Ib 4.8 (0.42)6 97.2 (13.0)b 0 Glutamate dehydrogenase 100 97.3 0 6.8 Succinate-semialdehyde dehydrogenase 100 4.8 95.5 0 Glucose-6-phosphatase 100 21.1 5.9 64.9

Specific activity (pmol/h/mg protein) given in parentheses. Specific activity (nmol/min/mg protein) given in parentheses.

the Euglena aquacobalamin reductase (NADPH) does not 8. Buetow, D- E. ( 1 9 s ) in The B i & Y Of E U g k m (Buebw, D. E-, bind to the two Other enzymes‘ ‘Owever, 9. Koren, L., and Hutner, S. H. (1967) J. Protozool. 14, (Suppl.) 17 and cob(1)a~a~in are labile and are spontaneously reoxidized 10. Tokunaga, M., Nakano, y., and Kitaoka, s. (1976) B ~ h ~ m . to OH-Cbl under aerobic conditions, so the three enzymes Biophys. Acta 429,55-62 may form a structural complex in vivo or they may be contig- 11. Tokunaga, M., Nakano, Y., and Kitaoka, S. (1979) J. Protozd. uous in the mitochondria (or both). 12. De Duve, C., Pressman, B. C., Gianetto, R., Wattiaus, T. G., and

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