Electron Transfer from a-Reduced Nicotinamide Adenine Dinucleotide to Flavoprotein , Cytochromes , and Mixed Function Oxidases of Rat Liver Microsomes

Cytochrome b5 of rat liver microsomes is reduced by a-NADH ; the extent of reduction is equal to that obtained with P-NADH. With both nucleotides, the rates of reduction of cytochrome b5 are very fast ; alternatively, rates of reduction of cytochrome c and dichloroindophenol have been measured. The rates observed with a+NADH are about 10% of the rates observed with P-NADH. Conditions have been established for the measurement of first order kinetic parameters in these reductions: K, of o(and /3-NADH are 13.0 and 3.3 pM for cytochrome c reductase activity and 5.1 and 6.7 PM, respectively, for dichloroindophenol reduction. Three lines of evidence suggest that LU-NADH and P-NADH may reduce cytochrome c and dichloroindophenol via the same microsomal flavoprotein. (a) There is competition between LYand /3-NADH. (b) The relative rates of cytochrome c reduction are diminished equally for o(and ,RNADH when the enzyme is inhibited or denatured. (c) The relative rates of reduction of both cytochrome c and dichloroindophenol are increased equally for czand /3-NADH when microsomal cytochrome b5 reductase is enriched. Both o(and /3-NADH are electron donors to cytochrome P-450 in microsomes. The initial rates of reduction of cytochrome P-450 by 01and P-NADH are equal (K = 0.126 min-I) and considerably slower than reduction by NADPH. In a second, slower phase of cytochrome P-450 reduction, the rate is somewhat less rapid with a-NADH (K = 0.030 min-‘) than with /3-NADH (K = 0.042 min-l). Compared to /3NADH and NADPH, a(-NADH is a more efficacious donor of electrons for microsomal mixed function oxidation of a methyl sterol intermediate of cholesterol biosynthesis. Furthermore, the substrate-independent rate of oxidation of LYNADH is very slow (10% of P-NADH). To date, only diaphorase-like activities have been reported


L. GAYLOR
From the Graduate School of Nutrition and the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca., New York 148~70 SUMMARY Cytochrome b5 of rat liver microsomes is reduced by a-NADH ; the extent of reduction is equal to that obtained with P-NADH.
With both nucleotides, the rates of reduction of cytochrome b5 are very fast ; alternatively, rates of reduction of cytochrome c and dichloroindophenol have been measured.
The rates observed with a+NADH are about 10% of the rates observed with P-NADH.
Conditions have been established for the measurement of first order kinetic parameters in these reductions: K, of o(-and /3-NADH are 13.0 and 3.3 pM for cytochrome c reductase activity and 5.1 and 6.7 PM, respectively, for dichloroindophenol reduction. Three lines of evidence suggest that LU-NADH and P-NADH may reduce cytochrome c and dichloroindophenol via the same microsomal flavoprotein. (a) There is competition between LY-and /3-NADH.
(b) The relative rates of cytochrome c reduction are diminished equally for o(-and ,R-NADH when the enzyme is inhibited or denatured. (c) The relative rates of reduction of both cytochrome c and dichloroindophenol are increased equally for cz-and /3-NADH when microsomal cytochrome b5 reductase is enriched. Both o(-and /3-NADH are electron donors to cytochrome P-450 in microsomes. The initial rates of reduction of cytochrome P-450 by 01-and P-NADH are equal (K = 0.126 min-I) and considerably slower than reduction by NADPH. In a second, slower phase of cytochrome P-450 reduction, the rate is somewhat less rapid with a-NADH (K = 0.030 min-') than with /3-NADH (K = 0.042 min-l). Compared to /3-NADH and NADPH, a(-NADH is a more efficacious donor of electrons for microsomal mixed function oxidation of a methyl sterol intermediate of cholesterol biosynthesis. Furthermore, the substrate-independent rate of oxidation of LY-NADH is very slow (10% of P-NADH).
To date, only diaphorase-like activities have been reported Japan.
for oxidation of (Y-NADH. Although the present report shows clearly that cr-NADH may function as an electron donor to microsomal mixed function oxidases, the physiological significance of these findings remains obscure because enzymic reduction of ok-NAD+ has not yet been reported.
Oxidation of (Y-SADH by broken cell preparations of rat liver is inhibited by cyanide and insensitive to inhibition by Antimycin A, rotenone, and Amytal. Furthermore, at least 50% of the cY-NADH-cytochrome c reductase activity of whole homogenate of liver is associated with the microsomal fraction (1). For some time we have been investigating a microsomal mixed function oxidase, methyl sterol oxidase, that accepts electrons from NADH; the oxidase is inhibited by cyanide (2). The multienzymic system is insensitive to inhibition by Antimycin A, rotenone, and Amytal.
Accordingly, we substituted a-NADH for P-NADH and observed that methyl sterol oxidase could be supported at maximal rates by the cr epimer of NADH as the sole source of exogenous reducing equivalents (see Fig. I).
In this report we show that in rat liver microsomes electrons are transferred from o(-NADH to cytochrome b, reductasel (Reaction A in Equation I), from a-NADH to microsomal cytochrome b5 and P-450 (A and B), and from a-KADH to oxygen and substrate (a, B, and C). Although the principal electron donor to oxidases in microsomes is NdDPH, the pathway from NADH also constitutes the over-all reaction (i.e. A, B, C) of a mixed function oxidase (3).
Although microsomal xenobiotic oxidation is supported mainly by NADPH, there is considerable current interest in the pathway of microsomal electron transport from K*4DH.
The particles contain cytochromc P-450, but NADPH-cytochrome P-450 reductase had been removed by 1  Proposed by Ichikawa and Loehr (4) and in this report. partial proteolysis.
The results presented in this report support the suggestion of Ichikawa and Loehr (4)  Liver was then removed and homogenized in 3 volumes of potassium phosphate buffer (pH 7.4 and containing 1 mM glutathione) . Mitochondria, cell debris, and nuclei were removed by centrifugation at 10,000 x g for 20 min. The resulting supernatant fraction was centrifuged again at 105,000 x g for 1 hour. The resulting microsomal pellets were washed one time with fresh buffer and collected by centrifugation.
The microsomes were diluted with buffer to a concentration of 10 to 20 mg of protein per ml. (Mitochondrial contamination was judged to be less than 50%.) Protein was assayed by the method of Lowry et al. (5).
In some experiments microsomes were treated with Subtilisin VII as described previously (6). The resulting preparation of microsomal subparticles was essentially free of KADPH-cytochrome c reductase and cytochrome bj.
NADH-cytochrome b5 reductasel was isolated by treatment of microsomes with detergent (7,8) or with lysosomes (9). Recent evidence suggests that partial proteolysis of the flavoprotein oc- curs during isolation by the latter method (7,8,10). Because the detergent-isolated enzyme contains residual detergent, an alternate procedure for obtaining the NADH-cytochrome bs reductase by treatment of rat liver microsomes with very small quantities of phospholipase A under mild conditions has been used in this laboratory (11).
Assays-Cytochrome P-450 was assayed by the method of Omura and Sato (12), and cytochrome b5 was assayed as described previously (13). NADH-dichloroindophenol reductase was measured at 25" by recording the change in absorbance at 600 nm of a l.O-ml solution containing the following: 45 nmoles of 2,6-dichloroindophenol; 70 nmoles of either o(-or P-NADH; 100 pmoles of phosphate buffer (pH 7.4) ; and approximately 40 pg of microsomal protein. A molar extinction coefficient of 21 x lo3 liters mole-l cm-l was used (14). Dicumarol was added to eliminate possible contribution of adsorbed soluble diaphorase (1) ; no effect of Dicumarol was observed.
NADH-cytochrome c reductase was measured similarly by substituting 30 nmoles of cytochrome c for DCIP2; the change in absorbance was measured at 550 nm. A difference molar extinction coefficient for reduced minus oxidized cytochrome c of 18.5 liters mole-l cm+ was used (15). Substrate-independent oxidation of NADH was measured at 25" as changes in absorbance at 340 nm for P-NADH (16) and at 344 nm for a-NADH (17). Molar extinction coefficients of 6.22 x lo3 for /3-NADH and 5.6 x lo3 liters mole-l cm+ for CX-NADH were used. Reaction vessels contained 100 nmoles of NADH; 100 nmoles of CN-, when added; and 100 pmoles of phosphate buffer (pH 7.4) in 1.0 ml final volume.
Anaerobic reduction of cytochrome P-450 was measured at 25" in Thunberg anaerobic cuvettes that contained, in 3 ml: either 420 nmoles of (-r-NADH, 360 nmoles of P-NADH, or 510 nmoles of NADPH; approximately 6.0 to 6.3 mg of protein; 60 mg of glucose; 1 mg of glucose oxidase; and 300 pmoles of phosphate buffer (pH 7.4). The cuvettes mere sealed with paraffin, and the gas phase was eschanged with 100% CO by several cycles of evacuation and filling.
The reaction was started by addition of reduced pyridine nucleotide from the side arm. Changes in the absorbance measured at 450 and 490 nm were measured in the dual wave length mode of a model 356 Perkin-Elmer spectrophotometer.
Because either commercial o(-NADH may contain small amounts of fl-NADH or microsomes may contain small amounts of endogenous /3-NADH (18), lactate dehydrogenase (0.02 mg) and 2 Mmoles of pyruvate were added for each milliliter of solution to cat.alyze oxidation of contaminating fi-NADH. Methyl sterol oxidase was measured with 4,4-dimethyl-5olcholest-7-eii-3,&ol as described previously (11) Rat liver microsomes (0.77 mg of protein per ml) were suspended in 1.0 ml of 0.1 M phosphate buffer, and the solutions were divided between two cuvettes.
Dicumarol and DL-isocitrate were purchased from Nutritional Biochemicals Corp. The nonionic detergents, Lubrol WX (13) and Triton WR-1339 (18), werepurchasedfrom ICI Organics and Ruger Chemical Co., respectively. Left, cytochrome c reduction was measured as described under "Experimental Procedure," except that the final volume was adjusted to 2.5 ml, and the protein concentration was 0.03 mg of protein per ml; right, DCIP reduction was measured as described under "Experimental Procedure" with 0.05 mg of protein per ml.

Reduction of ikficrosomal
Cytochrome b5 and Other Electron Acceptors by (w-NADH-In an unpublished observation, Okamoto et al. (1) indicated that microsomal cytochrome bS may be reduced by a-NADH.
The purpose of this subsection is to establish that both (r-NADH and @-NADH may supply reducing equivalents for reduction of microsomal cytochrome bs and that other electron acceptors, such as cytochrome c and DCIP, may be used to study reaction velocities.
Then, appropriate conditions were established to measure initial kinetic parameters with both 01-and @-NADH as the source of reducing equivalents and cytochrome c and DCIP as electron acceptors.
Addition of a-NADH to a suspension of rat liver microsomes yielded an immeasurably rapid reduction of cytochrome bs (Fig.  2). The extent of reduction achieved was about equal for the two epimers of KADH: 0.504 f 0.015 (S.D.) and 0.517 + 0.026 (S.D.) nmole per mg of protein for a-and P-NADH, respectively.
Because the rate of reduction of microsomal cytochrome bs was too rapid to measure under these conditions, rates of reduction of other electron acceptors, cytochrome c and DCIP, were measured under conditions of more dilute suspensions of microsomes. The rate of reduction of cytochrome c by a+?;ADH was also very rapid (Table I). The velocity was about 11 y0 of the rate measured with ,&NADH.
Similarly, the rate of reduction of DCIl' by LU-NADH was about 127, of the rate observed with P-NADH.
Although the rates of p-Xc'aDH oxidation using oxygen as the terminal electron acceptor were more rapid, in the presence of 0.1 mM CN-the rates of oxidation were equal for the two epimers (Table I). Thus, severe rate limitations exist in the latter process in contrast to cytochrome c reduction to the extent that the reaction velocities of a-and P-NADH oxidations were indistinguishable.
Furthermore, addition of pyruvate and lactate dehydrogenase (see "Experimental Procedure") did not alter rates of cr-NADH-dependent processes. Finally, addition for o(-and fl-ICADH in NADH-DCIP reductase was 5.1 and 6.7 pM, respectively (Fig. 4B).
Michaelis constants (graphs not shown) for the acceptors were 0.5 and 1.5 pM (c~-NADH) and 5.0 and 20.0 pM @-NADH) for cytochrome c and DCIP, respectively.
Thus, artificial electron acceptors, as well as cytochrome bg, may be reduced effectively by my-NADH in the microsomal system. Furthermore, rates of reduction can be measured accurately since conditions used yielded first order kinetic data for analysis. Establishment of these conditions was essential for the investigation of competition between CX-and P-NADH described below.
Competition between o(-and p-N&4 DH-Either one or more than one flavoprotein of microsomes may accept electrons from the NADHs (see Equation  1). To distinguish between these two possibilities, three kinds of experimental evidence are reported in this section: the kinetic results showing competition between 01-  the effects of enzyme destruction on ratios of activity measured separately with 01-and P-NADH; and the effects of enzyme enrichment on ratios of activity measured separately with 01-and @-NADH.
With a low concentration of o(-NADH added to all samples (16.7 pM a-NADH), saturation of DCIP reductase by P-NADH was essentially normal, and there was no strong indication of competition (Fig. 5A). However, K, for a+ and P-NADH were too similar for observation of a marked effect with DCIP as the electron acceptor (see Fig. 4B). The competitive effect of including a much higher concentration of cr-NADH (94 PM) in the assay of &NADH-cytochrome c reductase was more pronounced (Fig. 5B). The velocity of cytochrome c reductase at saturation of P-NADH was maximal whether or not o(-NADH was added. In addition, the data were analyzed according to the method described by Dixon and Webb (19) for two sub-strates3 For each concentration of p-NADH, the value of K, for LY-NADH was calculated.
A relatively constant value was obtained, 13.3 pM, which was essentially identical with the value of K, for a-NADH obtained directly from double reciprocal plots (13.0 pM in Fig. 48).
Conversely, a fully additive effect (i.e. no competition) was observed when microsomal NADPHcytochrome c reductase was measured in the presence of (Y-NADH (Fig. 5C). Since electron transfer from NADPH to cytochrome c is catalyzed by a different flavoprotein (3) of microsomes, the additive rather than the competitive effect was expected.
The effects of different destructive treatments on rates of NADH-cytochrome c reductase activities were measured with (Y-and P-NADH. Several different treatments were used, including effects of different concentrations of both ionic and nonionic detergents, p-chloromercuriphenylsulfonic acid, and heat denaturation (Fig. 6). Parallel responses were observed between a-and /3-NADH-dependent cytochrome c reductase activities. For example, Lubrol WX was inhibitory at all concentrations tested, but treatment with Triton X-100, deoxycholate, and even p-chloromercuriphenylsulfonic acid led to modest increases in activity at low concentrations.
Triton WR-1339 was not toxic, which has been observed repeatedly (18). Even with slow heat denaturation, parallel changes were observed. The only exception was stimulation of cr-NADH-dependent reduction by intermediate concentrations of Triton X-100, which are just sufficient to clarify the microsomal suspension.
Thus, with the relatively minor exception that may result from a physical change in vesicles, decays of enzymic activity were equal and parallel for reactions supported by o(-and P-NADH. The NADH-cytochrome bg reductasc of liver microsomas was obtained by various treatments (Table II).
Particles similar to those obtained by partial proteolysis with trypsin (4) and supernatant fraction from phospholipase A treatment (11) contain the enzyme (Column I, Table II), but cytochrome bj is removed somewhat by each of these treatments. Thus, reduction of cytochrome c by electron transfer from cytochrome bs was diminished (Column II).
Furthermore, with enrichment of the reductase, the ratio of activity measured with DCIP and cytochrome c as the terminal elect,ron receptors remained essentially equal for (Y-and P-NADH (Columns III and IV, Table II). Thus, with greater than 40.fold enrichment of the enzyme (from 268 to 11,700 nmoles per min per mg of protein; Table II), as well as with destruction of the enzyme (Fig. 6), parallel changes were observed.
The change in ratio of rates measured with each acceptor (Columns V and VI) were parallel, too. However, since the ratio changes, the properties of the reductase may be altered.
For example, the detergent-solubilized enzyme still contained substantial amounts of residual detergent, and the enzyme tended to aggregate.4 3 The following values were substituted into the equation given for catalysis of two substrates: K,(p-NADH) = 3.3 ,UM; V,,, (ol-NADH) = 75.3 nmoles per min; V,,,@NADH) = 678.8 nmoles per min. In the data shown as Fig. 5B 6. Effect of various destructive treatments upon NADHcytochrome c reductase activity in microsomes. -Microsomes (0.01 to 0.05 mg of protein per ml) were suspended in buffer with 30 PM of cytochrome c, and the suspensions were divided between cuvettes. The indicated amount of detergent or other substance was added, and the mixtures were incubated for 2 min at 25". The reaction was initiated by the addition of 70 nmoles of either N-or B-NADH.
For heat denaturation, microsomes were susnended in buffer. and the mixture was maintained at 50" with gentle stirring fir the indicated length of time. Samples were withdrawn and cooled rapidly to 25" for enzyme assay.
Substitution of cu-NADH for /3-NADH in Microsomal Oxidases -Since o(-NADH can serve as a source of reducing equivalents for microsomal cytochrome bs reductase, it follows that ol-NADH could be substituted for P-NADH in NADH-dependent microsomal oxidases. Accordingly, electron transfer from ol-NADH and P-NADH to two terminal oxidases was investigated: cytochrome P-450, which is the terminal oxidase of drug and xenobiotic metabolism (20) ; and methyl sterol oxidase, which is one component of the multienzymic microsomal system of cholesterol biosynthesis (21).
Cytochrome P-450 was reduced very rapidly by NADPH (Fig.  7A). Under these anaerobic conditions, the extent of reduction of cytochrome P-450 was about equal to that obtained with NADPH when microsomes were incubated for several minutes with either a-NADH or @-NADH (Fig. 7A). When experiments were carried out very carefully, we observed that the rate of reduction of cytochrome P-450 was clearly biphasic with either (Y-or @-NADH as the source of reducing equivalents (Fig. 7B). In the rapid phase of reaction the rates were identical with LY-and P-NADH, but a difference was noted in the second phase when LU-NADH was substituted for B-NADH. The component reduced in the rapid phase was equivalent to 30 to 407, of the total cytochrome P-450, which is very similar to the extent of fi-NADH-dependent cytochrome P-450 reduction reported by Ichikawa and Loehr (4), but, unfortunately, their results were reported for only the first few minutes of reduction (i.e. the rapid phase of k = 0.126 mi0).
For assay of methyl sterol oxidase, the microsomes were first treated with phospholipase A to remove contributions of en- dogenously generated fl-NADH from P-NAD+ (11). There was no detectable endogenous reduction of cu-NAD+. P-NAD+ was added for methyl sterol oxidase assay by coupling oxidase activity to the NAD-dependent decarboxylase (22) that catalyzes conversion of the product of oxidase action, 3@-hydroxy-4Pmethyl-5a-cholest-7-en-4a-oic acid, to carbon dioxide and 3-ketosteroid.
Addition of increasing amounts of a(-NADH yielded essentially saturation of enzymic activity (Fig. 8). With both /I-NADH and NADPH, only maximal rates were observed at intermediate concentrations and some inhibition was apparent when the concentration of reduced pyridine nucleotide was increased to 0.7 mM. Since W-NADH oxidase rates in the absence of substrate were relatively slow (0.8 nmole per min per mg of protein, Table  I), the increment of oxygen-dependent oxidation of o(-NADH produced upon addition of steroid substrate was significant ( Fig.   8 versus Table  I). Furthermore, (Y-NADH may appear more efficacious under these conditions because the substrate-independent aerobic loss of reduced pyridine nucleotide (Table I) was much slower than with @-NADH. The substrate-independent (Table I) and substrate-dependent ( Fig. 8) rates of ,& NADH oxidation were less favorable for direct assay of fi-NADH oxidation and oxygen uptake; the increment of oxygen uptake upon substrate addition could not be measured directly with the /3 epimer.5

DISCUSSION
To date, essentially diaphorase-like activities have been reported for enzymic oxidation of (r-NADH. That is, with the use of alternate electron acceptors, such as oxidation-reduction dyes and cytochrome c (I), oxidation of cr-NADH by microsomal enzymes had been observed, but no metabolic functions could be ascribed to the enzymic oxidation.
In the present report, we have shown that a-NADH may serve as electron donor to cytochrome bs, which is a natural electron acceptor in microsomes (Fig. 2). Furthermore, (Y-NADH is an efficacious electron donor for reduction of cytochrome P-450 (Fig. 7), and it functions as an electron donor to microsomal mixed function oxidation of a cholesterol biosynthetic intermediate (Fig. 8). However, the physiological significance of these latter observations is still not obvious since enzymic reduction of cr-NAD+ would have to occur in vivo. Because the evidence supports the suggestion that both cy-and /3-NADH may serve as electron donors to the same 6 Tinpublished observations, this laboratory. microsomal flavoprotein, cytochrome bg reductase (Table II; Figs. 5 and 6), transhydrogenation from P-NADH to a-NAD+ may be possible.
For example, transhydrogenation from NADH to NADP+ has been shown for the same microsomal cytochrome bs reductase (23). Under conditions used in this work, however, we were unable to detect formation of a-NADH by the analogous process.5 Thus, since reduction of the same flavoprotein by (Y-and P-NADH was indicated by several lines of evidence, transhydrogenation would have been expected. Three rather independent lines of evidence (Table II; Figs. 5 and 6) support the conclusion that the same flavoprotein may accept electrons from (Y-and P-NADH.
However, demonstration of cr-NADH-dependent reduction of cytochrome bs reductase will have to be carried out with enzyme purified to homogeneity without either undergoing partial proteolysis or with residual detergent.
Until this is achieved, the alternate proposal that microsomes contain more than one NADH-selective flavoprotein capable of reducing cytochrome bs must be considered (24).
The metabolic significance of the NADH-dependent pathways of microsomal electron transport is just now emerging.
Other workers reported a synergistic effect of NADH on NADPH reduction of cytochrome P-450 (25). More recently, Ichikawa and Loehr (4) observed that cytochrome P-450 could be reduced by a NADH-dependent system in rabbit liver microsomes from which cytochrome b5 and the NADPH-specific flavoprotein were removed.
In the scheme of electron flow from pyridine nucleotide to cytochrome P-450 postulated by Ichikawa and Loehr, cytochrome b5 is indicated as an alternate electron acceptor for electrons from cytochrome b5 reductase.
This conclusion is consistent with our results (6,26) and with the suggestion that, although cytochrome bS may be in oxidation-reduction equilibrium, it may not be an obligatory electron carrier from pyridine nucleotide to cytochrome P-450 and the terminal oxidases.
An experimental approach to the study of the contribution of NADH to microsomal mixed function oxidases is now possible with the use of o(-NADH.
Since increments of cr-NADH oxidation rates due to addition of substrate can be observed because the basal rate of oxidation of o(-NADH in the absence of substrate is very slow ( Table I), participation of the NADH pathway in mixed function oxidases may be studied without resorting to partial proteolysis (4,6), detergent treatment (13,26), or other chemical and physical means that have been used to remove the cytochrome 65 from the multienzymic system. Furthermore, neither o(-NAD+ nor ar-NADH produced inhibition of methyl Assay of reduction of cytochrome P-450 was carried out as described under "Experimental Procedure." In B, the total content of cytochrome P-450 was measured after 30 min of enzymic reduction by the addition of dithionite to the sample cuvette (12). Values were then expressed as the percentage of unreduced P-450 following incubation with c~-NADH (o---O) and &NADH (O-0). Calculated rate constants are indicated on the figure. sterol oxidase when added at concentrations that were inhibitory for /3-NADH and NADPH (Fig. 8). Indeed, maximal oxidase rates were obtained with (Y-NBDH. Thus, with or-NADH as the electron donor to the NADH-dependent process, significance of the reduction of cytochrome P-450 (Fig. 7) by NADH and rates of NADH-dependent drug oxidation (20) can be studied directly in untreated microsomes.
Finally, others have suggested that the methemoglobin reductase of erythrocytes is very similar to cytochrome bg reductase of tissue microsomes (27-29).
We studied substitution of (Y- Methyl sterol oxidase activity was assayed with phospholipase A-treated microsomes as described previously (11) with 100 nmoles of substrate and approximately 5 mg of microsomal protein in a final volume of 2.0 ml. Each incubation flask contained 0.5 Fmole of p-NAD" in addition to the indicated amounts of reduced pyridine nucleotides. Pyruvate and lactate dehydrogenase were omitted from the samples incubated with @-NADH.
NADH for P-KADH and found that methemoglobin was not reduced by either isomer when incubated with intact microsomal particles. 5 In addition, cytochrome b5 is reduced by incubation of the erythrocyte enzyme with o(-NADH. 5 These results are consistent with the observations of Hara and Minakami (29) who reported no reduction of methemoglobin by p-XADH unless microsomes were first disrupted by detergent treatment.
Subsequent work with CX-NADH in this enzymic process may facilitate the study of methemoglobin reductase in erythrocytes in a manner analogous to that described above for the study of NADH contributing to the microsomal system. Similarly, subsequent work with cu-NADH in microsomal ethanol oxidation described recently by Okamoto (30) may facilitate resolution of the controversy over the significance of this oxidative process.
The recent suggestion of Jacobson et al. (31) that a-NAD(H) may be produced as an artifact of isolation of /?-KAD(H) and not naturally occurring may be particularly interesting.
However, the use of the (r-NADH epimer for the purposes described above is as a model compound substituted for P-NADH.
Thus, our emphasis does not rest on the arguments for or against natural occurrence.