J. Biochcm., 69, 325-330 (1971, On Functional Role of Cytochrome b5 II. NADH-linked Ascorbate Radical Reductase Activity in Microsomes Toshihiko HARA* and Shigeki MINAhAMI* *The Biochemistry Laboratory , National Nakano Chest Hospital, Tokyo and **the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka Received for publication, Mat 28. 1970 1) :Microsomes catalyze the reduction of ascorbate radical by NADH . The enzyme which functions in this reaction is identified as the microsomal NADH-cytochrome c reductase, the electron transfer system involving cytochrome b5. Reduced cytochrome h5, whether bound to or isolated from the microsomal membrane, reacts rapidly with the ascorbate radical to form ascorbate. With intact microsomes , the cytochrome is maintained in the fully reduced state in the presence of NADH. This condition favors the electron transfer from NADH to ascorbate radical as catalyzed by micro somes. 2) The non-ionic detergent, Emalgen 810, which inhibits the reduction of micro somal bound cytochrome b5 by NADH, also inhibits the NADH-ascorbate radical reductase activity of microsomes. 3) Cytochrome b5 is reduced by ascorbate very slowly but not reduced by ascorbate radical. Under aerobic condition, the cytochrome appears to be only partially reduced by ascorbate due to the autoxidation of the reduced cytochrome. 4) Microsomes with NADH generating system prevent the oxidation of ascorbate. This effect is inhibited by Emalgen 810, the inhibitor of NADH-cytochrome b, reduc tase system of microsomes. Role of NADH-cytochrome b5 reductase system to main tain intracellular level of ascorbate is discussed. It has been demonstrated that oxidation of of an ascorbate oxidizing enzyme in micro NADH in microsomes is activated by catalytic somes. Formation of ascorbate radical as an amount of ascorbate which cannot be replaced intermediate form was indicated by Z amazaki et by dehydroascorbate (1, -2). For the inter al. (1) in their study on plant ascorbate oxidase pretation of such reaction, a "radical-like" [EC 1. 10. 3. 3]. Staudinger ct al. ( ~)_)by using semidehydroascorbate was proposed: the ex the enzyme for generating ascorbate radical, istence of which has been confirmed by direct have demonstrated that ascorbate radical may measurement in electron spin resonance spec be the true electron acceptor from NADH in trometry (3 ). microsomes and that microsomes contain an The generation of ascorbate radical in enzyme system which transfer electron from microsomes needs to postulate the existence NADH to ascorbate radical. On the other Vol. 69, No. 2, 1971 325 326 T. HARA and S. MINAKAMI hand, it has been demonstrated that oxidation the partially reduced cytochrome b5 was autox of ascorbate to dehydroascorbate by ascorbate idized under aerobic condition following a oxidase is inhibited if both NADH and micro first order reaction. The rate constant was somes are present (5), about 0.3 min-1 at 20°C in 0.1M phosphate We are interested in the NADH-ascorbate buffer, pH 7.2. When NADH was added to radical reductase activity in microsomes and microsomes excessively, the NADH oxidation have demonstrated that microsomal NADH proceeded more rapidly than it could be ascribed cytochrome b5 reductase system may function to the autoxidation of cytochrome b5. Under as the catalyst for reducing ascorbate radical such a condition, the increase in the reoxi by NADH. dation rate of cytochrome b5 will accelerate the oxidation velocity of NADH, as observed in the following experiments. The remaining MATERIALS AND METHODS portion of the NADH oxidation may be due to Preparation-Microsomes were prepared as the cyclic reduction-oxidation of cytochrome described previously (6). Ascorbate oxidase P-450. was purified from the cortex of Japanese cu Effect of Non-ionic Detergent on NADH cumbers by the method of Tokuyama ct al. Oxidation by Microsomes-Previously, we have (7 ). The preparation contained copper in the reported that the electron transfer from NADH amount of 1.1 ,ƒÊg/mg protein, having an en to cytochrome b5 in intact microsomes is in zymatic activity of 1,200 units/mg protein by hibited by the non-ionic detergent, Emalgen expressing its unit according to Lovett-Janison 810 ((5). On the other hand, the detergent and Nelson (8). Initial velocity of oxygen showed an inhibitory effect of NADPH oxidase uptake in the presence of 1 mm ascorbate and without showing significant inhibition of NADH 1 unit/ml of ascorbate oxidase was 0.38 mm/ oxidation. This observation may be explained min at 20°C in 0.1 M phosphate buffer, pH 7.0, as follows. The oxidation of NADH is mainly saturated with air. due to the reaction of cytochrome b5 where Analytical Methods-Oxygen uptake was the reoxidation rate of reduced cytochrome b5 measured by a Clark oxygen electrode. The is an important determinant for the overall activity of ascorbate oxidase was estimated as NADH oxidation rate. In this case, the inhi described by Sack et al. (9). The copper bition of intramicrosomal electron transfer by content of the enzyme was measured with the detergent does not necessarily reflect on bathocuproin reagent (10 ). Ascorbate was the overall oxidation rate. For the oxidation determined by the reduction of 2, 6-dichloro of NADPH, the contribution of P-450 system phenol indophenol and subsequent extraction is most probable, in which the rate deter with amyl alcohol. mining step in the sequence is the step to Other spectrophotometrical and chemical reduce P-450. As the reduction of P-450 by analyses were performed in the same way as NADPH is inhibited by the detergent (un described previously (G). published observation), it may depress the rate of NADPH oxidation. The characteristics of the detergent discussed above were used for RESULTS the study of NADH-ascorbate radical reductase Relation between Oxidation of NADH and in microsomes. Oxidation of cytochrome b5 in Microsomes Kinetics of NADH-Ascorbate Radical Re Oxidation of NADH by liver microsomes is ductase Activity of Microsemes in the Presence mediated by cyclic oxidoreduction of cyto and Absence of the Detergent-NADH oxidase chrome b5 or cytochrome P-450. When micro activity of microsomes was activated by the somes were treated with a small amount of presence of ascorbate and this activation was NADH less than necessary to reduce cyto far more pronounced on addition of ascor chrome b5 completely, NADH reduced only the bate oxidase (Fig. 1), as originally observed stoichiometric amount of cytochrome b5. Then by Staudinger et al. (1 ). Under our J. Biochem. ON FUNCTIONAL ROLE OF CYTOCHROME b5 327 Fig. 1. Oxidation of NADH and NADPH by micro Fig. 2. Oxidation of NADH and NADPH by micro somes. The assay mixture contained in 3 ml: 3_mg somes in the presence of Emalgen 810. 0.8`c Emalgen of microsomal protein, 10 mm Amytal, 0.1 M phos 810 was previously added to the assay mixture. The phate buffer, pH 7.2. conditions were the same as in Fig. 1. experimental conditions, the ratio of the ac tivation was more than 25 fold when both ascorbate and ascorbate oxidase were added. On the other hand, the NADPH oxidase ac tivity of microsomes was only slightly activated by ascorbate radical. These results indicate that the ascorbate-dependent (or ascorbate radical-dependent) reduced nicotinamide co enzyme oxidase activity of microsomes is more specific for NADH. The effect of Emalgen 810 on the ascor bate radical-dependent NADH (or NADPH) oxidase activity of microsomes is shown in Fig. 2. By the addition of ascorbate in the Fig, 3. Effect of ascorbate and ascorbate oxidase on presence of the detergent, the NADH oxidase microsomal cytochrome b;, reduced by NADH. The activity was enhanced in the same manner as assay mixture contained in 3 ml: 2.4 mg of micro observed without the detergent. This suggests somal protein, 0.1 M phosphate buffer pH 7.2. that the ascorbate oxidase activity intrinsic to microsomes is not affected by the deter (Fig. 3). In the presence of ascorbate, the re gent. When ascorbate oxidase was further added oxidation of cytochrome b5 was accelerated in to the system, the oxidation of both NADH agreement with the observation by Blume et and NADPH increased, although the activa al. (11), and further addition of ascorbate tions were not so large as observed in the oxidase caused more activation. This obser system without the detergent. Under the vation indicates that the activating effect of condition when the reduction of cytochrome ascorbate on NADH oxidase activity of micro b5 was inhibited by the detergent, the reduc somes can be ascribed to the activating effect tion rate of cytochrome b5 became rate-limit of ascorbate radical on the oxidation of cyto ing in the overall oxidative sequence of chrome b5. The prior additions of ascorbate NADH. and ascorbate oxidase followed by NADH ad Effect of Ascorbate Radical on Microsomal dition did not change the autoxidation rate. Bound Cytochrome b5-The addition of ascor When microsomes were treated with bate or subsequent addition of ascorbate oxidase NADPH aerobically, cytochrome b5 was par to microsomes changed the rate of reoxidation tially reduced to the point where the rates of of cytochrome b5 which was reduced NADH reduction and reoxidation were in balance. Vol. 69, No. 2, 1971 328 T. HARA and S. MINAKAMI When ascorbate was added to microsomes cytochrome b5. This observation also indicates under this condition, the steady state level of that the oxidation of cytochrome b5 by ascor reduced cytochrome b5 fell to a lower level. bate radical, as shown in intact microsomes, This indicates an increase in the reoxidation is due to the direct electron transfer between rate of the cytochrome by the presence of them. Furthermore, the effect of ascorbate ascorbate. to activate the oxidation of microsomal bound Similar phenomenon could be observed cytochrome b5 and to increase the NADH with a use of NADH as a reductant in the oxidizing activity of microsomes, which can presence of Emalgen 810, when the reduction be observed even without adding ascorbate rate of cytochrome b5 was depressed by the oxidase exogenously, seems to have occurred detergent to be comparable to the autoxidation by the cooperation of some endogenous ascor rate of the cytochrome.