Reductase Activity In Liver Microsomes Research Articles

Acute P-407 Administration to Mice Causes Hypercholesterolemia by Inducing Cholesterolgenesis and Down-Regulating Low-Density Lipoprotein Receptor Expression

Poloxamer 407 (P-407) is a chemical that induces a dose-controlled dyslipidemia in mice. Our aim was to determine the acute effects of P-407 treatment on the mechanisms that influence hepatic cholesterol homeostasis. We measured lipid levels in plasma and liver samples from control and P-407-treated mice (24 h post-i.p. injection of 0.5 g kg(-1) of P-407 or saline for the control mice). We measured acyl-coenzyme A:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities in liver microsomes. The protein expression of ACAT2, scavenger receptor class B, type I (SR-BI), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G8 (ABCG8), low-density lipoprotein receptor (LDLr), and actin was measured by immunoblot. We found an increase in plasma cholesterol and triglyceride levels as well as increased hepatic cholesteryl esters (CE) in P-407-treated mice. The hepatic ACAT microsomal activity and ACAT2 protein expression were not altered by P-407. The protein expression of the LDLr was decreased in the livers of P-407-treated mice. This decrease was specific, because the expression of the SR-BI was unchanged. The P-407-induced hypercholesterolemia was accounted for by increased activity and protein expression of HMG-CoA reductase. ATP-binding cassette transporters A1 and G8 protein expression were not significantly different in P-407-treated mice compared to controls. The increased hepatic CE levels, following P-407 treatment, was neither related to an up-regulation of ACAT2 nor enhanced SR-BI expression. Hypercholesterolemia was associated with an up-regulation of both the protein expression and activity of HMG-CoA reductase and decreased LDLr expression.

Reductive metabolism of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one, by rat liver preparations.

The reduction of the triple bond and carbonyl group of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one (PBYO), by rat liver microsomes and cytosol was investigated. The triple-bond-reduced product trans-4-phenyl-3-buten-2-one (PBO) and the carbonyl-reduced product 4-phenyl-3-butyn-2-ol (PBYOL) were formed when PBYO was incubated with rat liver microsomes in the presence of NADPH. The triple bond of 1-phenyl-1-butyne, deprenyl, ethynylestradiol, ethinamate, and PBYOL, in which the triple bond is not adjacent to a carbonyl group, were not reduced by liver microsomes even in the presence of NADPH. PBO was further reduced to 4-phenyl-2-butanone (PBA) by liver cytosol with NADPH. PBYOL was also formed from PBYO by liver cytosol in the presence of NADPH or NADH. The microsomal triple-bond reductase activity was inhibited by disulfiram, 7-dehydrocholesterol, and 18 beta-glycyrrhetinic acid but not beta-diethylaminoethyldiphenylpropylacetate or carbon monoxide. The triple-bond reductase activity in liver microsomes was not enhanced by several inducers of the rat cytochrome P450 system. These results suggested that the triple-bond reduction is caused by a new type of reductase, not cytochrome P450. The microsomal and cytosolic carbonyl reductase activities were not inhibited by quercitrin, indomethacin, or phenobarbital. Only S-PBYOL was formed from PBYO by liver cytosol. In contrast, liver microsomes produced R-PBYOL together with the S-enantiomer to some extent. Ethoxyresorufin-O-dealkylase activity in rat liver microsomes was markedly inhibited by PBYO and PBO, partly by PBYOL, but not by PBA.

The in vitro effect of Helichrysi flos on microsomal lipid peroxidation

Helichrysum arenarium (L.) Moench has long been known as a medicinal plant in Europe for its cholagogue, choleretic, hepatoprotective and detoxifying activities. Antioxidant properties of its main phenolics, flavonoids may be supposed to be responsible for these effects. The aim of this study was to verify the antioxidant properties of lyophilized water extracts with different polyphenol and flavonoid contents from inflorescences. The effects of natural extracts on microsomal fraction of rat liver were examined. Enzymatically induced lipid peroxidation and NADPH cytochrome c reductase activity in liver microsomes were measured by spectrophotometric methods. Results were compared with the activity of silibinin flavonoid, the main agent of well-known milk thistle ( Silybum marianum L.). The natural plant extracts diminish the enzymatically induced lipid peroxidation in a concentration-dependent manner and reduce the cytochrome c dose dependently. The sample with higher polyphenol and flavonoid contents showed more stimulation of NADPH cytochrome c reductase. The lyophilized Helichrysi flos extracts proved to be more effective compared to silibinin in examined concentrations.

ケトン基を有する薬物の還元的代謝を制御する生理的, 遺伝的及び病的因子

In this review, we describe the physiological, genetic and pathological factors regulating the reductive metabolism of drugs with a ketone group. Acetohexamide (AH) was chosen as a model drug with a ketone group. Species differences of AH reductase activity were observed in liver cytosol and microsomes of animals tested. AH reductase activity in liver microsomes of rats was much higher in males than females. The activity was not detectable until 4 weeks of age after birth in both sexes, but increased markedly at puberty only in males. AH reductase activity in liver microsomes of male rats was decreased by testectomy, and restored by the treatment with testosterone propionate, indicating that the sex-related difference and postnatal development of the activity are regulated by androgens. There was a strain difference of AH reductase activity in liver microsomes of male rats. Of rat strains tested, only Wistar-Imamichi strain was found to lack male-specific microsomal enzyme activity. The inheritance pattern of AH reductase activity in liver microsomes of rats was determined by mating the genetic deficiency Wistar-Imamichi strain with Fischer-344 strain. Streptozotocin-induced diabetes significantly decreased AH reductase activity in liver microsomes of male rats. Furthermore, the physiological role of AH reductase present in liver microsomes of male rats was examined. We propose the possibility that the male-specific microsomal enzyme physiologically functions as a 20 beta-hydroxysteroid dehydrogenase.

Inheritance of acetohexamide reductase activities in liver microsomes and cytosol of rats.

The inheritance patterns of acetohexamide reductase activities in liver microsomes and cytosol of rats were determined by using the inbred Wistar-Imamichi and Fischer-344 strains as a model of low and high metabolizers, respectively. A simple Mendelian genetic analysis for the frequency distribution of acetohexamide reductase activity in liver microsomes of male rats led us to conclude that the phenotype is genetically regulated by an autosomal co-dominant fashion. Female rats, unlike male rats, did not exhibit microsomal enzyme activity in parental, first filial (F1) and second filial (F2) generations, indicating that the inheritance of the microsomal enzyme activity is sex-limited. On the other hand, the frequency distribution of acetohexamide reductase activities in liver cytosol of male and female rats was unimodal in all generations and there was no significant difference among these cytosolic enzyme activities.

The Safety Evaluation of Fluvastatin, an HMG–CoA Reductase Inhibitor, in Beagle Dogs and Rhesus Monkeys

Fluvastatin is a potent synthetic competitive inhibitor of β-hydroxy-β-methyl-glutaryl-coenzyme A (HMG–CoA) reductase, the rate-limiting enzyme in the biosynthetic pathway for hepatic cholesterol synthesis. The therapeutic indication is reduction of elevated total and low-density lipoprotein cholesterol levels. Results from four toxicity studies in beagle dogs and one study in rhesus monkeys following oral administration of fluvastatin are reported. In two 26-week dog studies, doses were 0, 1, 8, or 48 mg/kg/day (reduced to 36 mg/kg/day in Week 7) and 0, 6, 24, or 36 mg/kg/day (reduced to 30 mg/kg/day in Week 2). In a 2-year dog study, doses were 0, 1, 8, or 16 mg/kg/day. Dose levels in the 26-week monkey study were 0, 0.6, 12, and 48 mg/kg/day (raised to 84 mg/kg/day in Week 17 and to 108 mg/kg/day in Week 22). In these studies, evaluations included clinical and physical examinations, body weight and food consumption, electrocardiography, ophthalmoscopy, hematology and clinical chemistries, urinalysis, blood drug concentration, and macroscopic and microscopic examinations of observed lesions and representative tissues. In the 26- and 52-week dog studies and the monkey study, lenticular biochemistry, the HMG–CoA reductase activity of liver microsomes, and serum lipid concentrations were investigated. The fourth dog study was a single-dose toxicokinetic study in which 48 mg/kg [3H]fluvastatin was monitored for up to 2 weeks. Sampling was limited to ocular tissues for enzyme analysis. Doses of ≥24 mg/kg/day were lethal in dogs. At lethal doses, ataxia, convulsions, fecal blood, multifocal congestion and hemorrhage, isolated foci of malacia in the medulla oblongata, and liver necrosis were observed. Reduced weight gain, emesis, cataracts, elevated liver enzymes, reduced cholesterol, and gallbladder inflammation with mucosal hyperplasia occurred at ≥8 mg/kg/day. In contrast to other HMG–CoA reductase inhibitors, fluvastatin did not cause significant central nervous system hemorrhage or testicular changes in dogs. Monkeys tolerated exposure to fluvastatin well with only mild gallbladder changes observed. Reduced serum cholesterol and slight hyperplasia of the gallbladder mucosa occurred in the 12 and 48/84/108 mg/kg/day groups.

Strain- and sex-related differences of acetohexamide reductase activities in liver microsomes and cytosol of rats: The Wistar-Imamichi strain lacks the male-specific microsomal enzyme activity

A marked strain-related difference was observed among acetohexamide reductase activities in liver microsomes of male rats. The microsomal enzyme activities in the Fischer-344 (Fischer), Sprague-Dawley (SD) and Wistar strains were 2.58±0.50, 1.60 ± 0.44 and 0.79±0.41 nmol/min/mg protein, respectively. The microsomal enzyme activities in these rat strains were much higher in males than in females, indicating that the microsomal enzyme is a male-specific enzyme. The Wistar-Imamichi (Wistar-IM) strain was found to lack the male-specific microsomal enzyme activity. In Fischer, SD and Wistar strains of testectomized male rats, the microsomal enzyme activities were significantly increased by the treatment with testosterone. However, testosterone treatment was ineffective on the microsomal enzyme activity in the Wistar-IM strain. These results suggest that Wistar-IM rats has a genetic deficiency of the microsomal enzyme. There was no strain-related difference among the cytosolic enzyme activities in male rats. The cytosolic enzyme activities in Fischer and Wistar rats were higher in females than in males.

Extrahepatic lipogenesis contributes to hyperlipidemia in the analbuminemic rat

Hepatic lipid and apolipoprotein synthesis is increased in the nephrotic syndrome. Catabolism of triglyceride-rich lipoproteins is impaired in nephrotic syndrome but not in rats with hereditary analbuminemia (NA), suggesting that lipid synthesis should be increased by analbuminemia in the absence of proteinuria. In this study the rate of cholesterol and fatty acid synthesis in liver and extrahepatic tissue was measured in female NA and control Sprague-Dawley (SD) rats to determine whether lipid synthesis was indeed increased in isolated analbuminemia and to identify the site(s) of increased lipogenesis. We also measured the concentrations of apolipoproteins (apo) AI, B, and E in plasma, as well as the levels of the respective mRNAs in liver. Plasma cholesterol, triglycerides, and apo AI, B, and E were all increased severalfold in the NA rat (P < 0.001). Although liver apolipoprotein mRNA content was significantly increased (P < 0.001) for apo AI (643%), B (273%), and E (299%), 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in liver microsomes and hepatic cholesterol synthesis were not significantly increased in the NA rats. Hepatic fatty acid synthesis and intestinal cholesterol synthesis were not increased in the NA rats. Surprisingly, intestinal fatty acid synthesis was elevated by 60% (P < 0.01). The NA rats demonstrated approximately fourfold increases in the incorporation of 3H2O into circulating cholesterol and fatty acids (P < 0.001). A 56% increase in the synthesis of total nonsaponifiable lipid was found in the extravisceral carcass (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Attenuation of Dysfunction in the Ischemia-Reperfused Liver by Glycyrrhizin

The present study evaluated the effect of glycyrrhizin (GR) on an injury of the liver caused by ischemia-reperfusion in rats. In the liver ischemia-reperfusion model, levels of serum GOT, GPT and LDH activities and lipid peroxides in the liver tissue were significantly increased. On the contrary, total glutathione content in the liver tissue and NADPH cytochrome P-450 reductase activity of liver microsomes were decreased. Pretreatment with GR 20 mg/kg. i.v. 10 min before induction of ischemia resulted in significant decreases in serum GOT, GPT, LDH activities and the lipid peroxide level and a higher tissue glutathione content during the period of reperfusion. Electron microscopic studies revealed various hepatocellular damages with an almost intact sinusoidal endothelium in ischemia-reperfused livers. However, the degree of damage was less severe in the livers from the rats pretreated with 20 mg/kg GR. The results indicate that GR is able to provide partial protection against ischemia-reperfused damage.

Attenuation of dysfunction in the ischemia-reperfused liver by glycyrrhizin.

The present study evaluated the effect of glycyrrhizin (GR) on an injury of the liver caused by ischemia-reperfusion in rats. In the liver ischemia-reperfusion model, levels of serum GOT, GPT and LDH activities and lipid peroxides in the liver tissue were significantly increased. On the contrary, total glutathione content in the liver tissue and NADPH cytochrome P-450 reductase activity of liver microsomes were decreased. Pretreatment with GR 20 mg/kg, i.v. 10 min before induction of ischemia resulted in significant decreases in serum GOT, GPT, LDH activities and the lipid peroxide level and a higher tissue glutathione content during the period of reperfusion. Electron microscopic studies revealed various hepatocellular damages with an almost intact sinusoidal endothelium in ischemia-reperfused livers. However, the degree of damage was less severe in the livers from the rats pretreated with 20 mg/kg GR. The results indicate that GR is able to provide partial protection against ischemia-reperfused damage.

Inhibition of carnitine palmitoyltransferase leads to induction of 3-hydroxymethylglutaryl coenzyme A reductase activity in rat liver

The relation between carnitine palmitoyltransferase (CPT) activity and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated. Rats were treated with aminocarnitine or 1-carnitine overnight. In rats, in which CPT activity was inhibited by aminocarnitine, plasma and hepatic triacylglycerol contents were increased 5- to 6-fold. The plasma cholesterol concentration was unchanged, while the hepatic cholesterol content was lowered (−16%). Hepatic cholesterol synthesis, determined by following the incorporation of 14C-acetate and 3H 2O into digitonin-precipitable sterols, in liver slices was increased 5- to 7-fold. HMG-CoA reductase activity in liver microsomes was increased to the same extent.

Warfarin resistance: Vitamin K epoxide reductase of scottish resistance genes is not irreversibly blocked by warfarin

The dithiothreitol-dependent vitamin K 2,3 epoxide (vitamin KO) reductase activity in liver microsomes of Scottish-derived warfarin-resistant Wistar rats (Tolworth Laboratory) was compared to that of susceptible Wistar rats. Under the test conditions, reductase activities in liver homogenates and in liver microsomes were comparable for both strains. The in vitro 150 of S-warfarin for microsomal reductase activity was 1 to 2 μM in both strains. The effect of in vivo S-warfarin was investigated after single doses, i.e. 0.2 and 1mg/kg for the susceptible rats, and 1 and 5mg/kg for the resistant rats. At 20 hr following the warfarin administrations in the susceptible strain, microsomal reductase was suppressed to about 30% of control. Microsomal reductase activity in the resistant strain was not reduced. Tissue and microsomal warfarin concentrations, however, were comparable in both strains. Wash experiments with microsomes which were treated in vitro with S-warfarin showed that vitamin KO reductase of the warfarin-resistant strain was not irreversibly inactivated by warfarin. The reactivation was mediated by DTT. The results suggest the following characteristic of the vitamin KO reductase of the Scottish resistance gene: contrary to the ◂normal” enzyme, the tight complex between the inhibitor and the resistant enzyme is liable to reactivation by reduction of the disulfide bridge in the active centre of the enzyme. This property explains the resistance for 4-hydroxycoumarin anticlotting activity.

Metabolism, Covalent Binding, and Mutagenicity of Aflatoxin B1 by Liver Extracts From Rats of Various Ages2

The ability of S-9 fractions isolated from the livers of 4-, 12-, and 26-month-old male inbred F344 rats to activate and metabolize the hepatocarcinogen aflatoxin B1 [(AFB1) CAS: 1162-65-8] was studied. The following observations were made: The activation of AFB1 to compounds that are mutagenic in the Ames Salmonella-microsome test and to compounds that covalently bind DNA in vitro was similar for liver S-9 from 4- and 12-month-old rats. A 30-50% decrease in the activation of AFB1 occurred in rats between 12 and 26 months of age. The in vitro metabolism of AFB1 to chloroform-soluble and water-soluble metabolites was similar for 4- and 12-month-old rats and decreased significantly in rats after 12 months of age. The proportion of most of the chloroform-soluble metabolites of AFB1 formed by liver S-9 from 4-, 12-, and 26-month-old rats was similar. However, the proportion of aflatoxicol (CAS: 29611-03-8) produced by liver S-9 increased approximately twofold in rats between 12 and 26 months of age. The cytochrome P450 content and the NADPH cytochrome c reductase activity of liver microsomes decreased 40-45% in rats between 12 and 26 months of age. However, the activities of UDPglucuronyltransferases and most forms of glutathione S-transferase did not change significantly with increasing age in liver microsomes and cytosol, respectively.

Effect of environmental temperature changes on rat liver fatty acid desaturases.

Female rats warm-adapted at 30-32 C for 20-25 days and then shifted to 13-15 C for 12, 24, 48, 72 and 120 hr showed that delta 9 desaturase and fatty acid synthetase activity decay after 24 hr of cold exposure, while delta 6 and delta 5 desaturases were increased after this period of time. These results were confirmed by an increase of arachidonic acid of heart and liver microsomes phosphatidylcholine and a decrease of oleic acid. Neither NADH-cyt b5 reductase nor NADH-cyt c reductase activity of liver microsomes were significantly affected. Male rats warm-adapted under the same conditions and then shifted to 13-15 C for 120 hr did not show significant changes in fatty acid synthetase, delta 9 and delta 6 desaturases and enzymes of the microsomal electron transport chain. Therefore, the desaturase response to environmental temperature changes could be plausibly linked to female hormones.

Genetic differences in the induction of aryl hydrocarbon hydroxylase and its components by 3-methylcholanthrene in liver and lung microsomes among four strains of guinea pigs

Four strains of guinea pigs (Hartley, No. 2, No. 13 and JY-1) were examined for the effects of intraperitoneal treatment with 3-methylcholanthrene on aryl hydrocarbon hydroxylase activity, total cytochrome P-450 content in liver and lung microsomes, and NADPH-cytochrome c reductase activity in liver microsomes. Following treatment with 3-methylcholanthrene at a dose of 50 mg/kg body weight, aryl hydrocarbon hydroxylase activity and cytochrome P-450 content in liver were both increased in all the strains used, and the activity of NADPH-cytochrome c reductase in liver was also increased in all strains except No. 13. While the cytochrome P-450 content in lung was increased in all the strains except No. 13, there was no increase in the aryl hydrocarbon hydroxylase activity in lung from any strain of guinea pig examined. When the dose of 3-methylcholanthrene was increased to 250 mg/kg body weight, an apparent induction of aryl hydrocarbon hydroxylase was detected in the lung from the Hartley strain of guinea pigs, but not in the other three strains. In summary, marked differences were seen in sensitivity to 3-methylcholanthrene between liver and lung, and apparent strain differences were observed among the guinea pigs used in this experiment.

Synthesis of cholesterol in the human fetus: 3-hydroxy-3-methylglutaryl coenzyme A reductase activity of liver microsomes.

The specific activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in microsome-enriched fractions prepared from normal human fetal liver tissue was assessed. The mean specific activity was 0.58 +/- 0.18 nmol mevalonate formed min-1 mg-1 protein. The activity of the enzyme was inhibited by preincubation of microsomes with ATP (4 mM) and was greatly reduced when microsomes were prepared from tissue homogenized in the presence of NaF (50 mM). It can be computed that the activity of HMG CoA reductase in human fetal liver microsomes is adequate to provide cholesterol to meet the requirements of the fetal adrenal for steroid precursor provided that cholesterol synthesized in the fetal liver appears in the plasma in the form of low density lipoprotein.

Reduction of N-hydroxy-2-acetylaminofluorene by liver microsomes

The reduction of N-hydroxy-2-acetylaminofluorene( N-hydroxy-AAF) to 2-acetylamino-fluorene and 2-aminofluorene by liver microsomes was studied. The reductase preferred NADPH rather than NADH as a cofactor and was strongly inhibited by carbon monoxide and oxygen. Further, the inhibitors of hepatic mixed function oxidase effectively inhibited the reductase activity. Pretreatments of rats with 3-methylcholanthrene and PCB markedly induced the reductase activity. From these results it was suggested that the reduction of N-hydroxy-AAF was catalysed by cytochrome P-450, especially by cytochrome P-448. The reductase activity was also detected in microsomes from lung, kidney and small intestinal mucosa, and the rates of the reduction were about 6, 7 and 27 per cent, respectively, of that in liver microsomes. Species difference in the reductase activity of liver microsomes was also examined. The highest activity of the reductase was found in hamsters, followed by guinea pigs and rabbits.

Reduced nicotinamide adenine dinucleotide-dependent reduction of tertiary amine N-oxide by liver microsomal cytochrome P-450

NADH-supported reduction of tertiary amine N-oxides in rat liver microsomes was investigated with imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide as substrate in the presence of NADH. The reductase activity is sensitive to carbon monoxide. NADH-cytochrome b 5 reductase or cytochrome b 5, solubilized by trypsin or subtilisin, showed no N-oxide reductase activity. The NADH-dependent reduction of tertiary amine N-oxides was markedly inhibited by antibody to NADH-eytochrome b 5 reductase and antibody to cytochrome b 5. These results confirmed that NADH-dependent N-oxide reduction was catalyzed by cytochrome P-450 and the reducing equivalent for the N-oxide reduction was transferred from NADH to cytochrome P-450 mainly via NADH-cyto-chrome b 5 reductase and cytochrome b 5 in the microsomal membranes. NADH-dependent N-oxide reductase is also sensitive to oxygen and 4 μM oxygen gave 50 per cent inhibition with imipramine N-oxide. Kinetic study shows that K m values for the reduction of imipramine N-oxide, tiaramide N-oxide and N, N-dimethylaniline N-oxide were 0.05 mM, 0.14 mM and 0.16 mM, respectively. NADH-dependent N-oxides reductase activity is affected by azo and nitroso compounds and hydrazide, although the degree of inhibition was rather weak compared with those of NADPH-dependent activity. Furthermore, NADH-dependent reduction of tertiary amine N-oxide was only slightly affected by n-octylamine, 2,4-diehloro-6-phenylphenoxyethylamine and aniline. NADH-dependent N-oxide reductase activity in liver microsomes was less sensitive to phenobarbital or 3-methyleholanthrene pretreatment than NADPH-dependent activity. Some characteristics of NADH-dependent N-oxide reductase activity were discussed and compared with those of NADPH-dependent activity.

Does cytochrome P-420 exist? Haem reductase activity in liver microsomes

Does cytochrome P-420 exist? Haem reductase activity in liver microsomes

Purification and properties of NADH-cytochrome b5 reductase solubilized by lysosomes from rat liver microsomes.

A new method for the purification of NADH-cytochrome b5 reductase [EC 1.6.2.2] from rat liver microsomes is presented. Solubilization of the reductase was achieved by incubating liver microsomes with lysosomes of the same tissue, and external addition of solubilizing agents was not needed. The method is suitable for a large scale preparation of the purified reductase starting from 1 kg or more of liver. The purification ratio of the reductase activity was 600 to 700. The overall yield from microsomes was about 40%. The properties of the purified enzyme were almost identical with those reported by previous workers for “snake venom-solubilized” enzyme. The purified enzyme preparations obtained by this method still contained two protein components with identical enzymatic activities and very similar physicochemical properties. These two components were immunologically indistinguishable. Examination of solubilization treatment suggested that these two NADH-cytochrome b5 reductase components had been derived from a single enzyme present in microsomes, and long incubation with lysosomes was responsible for the appearance of two components. NADH-ferricyanide reductase activity of liver microsomes could be mostly explained by NADH-cytochrome b5 reductase. The reduction of neotetrazolium by microsomes was, however, dependent on the interaction of the reductase with cytochrome b5 in microsomal membranes as in the case of microsomal NADH-cytochrome c reductase.