Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--II. Evidence for a co-operative interaction between NADPH and NADH.

There was a significant reduction in apparent K_m (NADPH) values for both aminopyrine and ethylmorphine demethylases when the kinetic constants for NADPH were determined in the presence of constant NADH concentrations. NADH was also shown to significantly stimulate apparent V for NADPH cytochrome P450-reductase (in the presence of aminopyrine) without changing the apparent K_m (NADPH) value. Further, NADH stimulated the initial rapid phase of the biphasic reduction kinetics of NADPH cytochrome P450-reductase in the presence of aminopyrine. These findings suggest that in the presence of both pyridine nucleotides, there has been a change in the rate limiting step which, with NADPH alone, is generally accepted to be the reduction of the cytochrome P450-substrate complex. It has been necessary to make certain modifications to a previously proposed mechanism to explain the results obtained in the present study.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--III. The influence of the 1,4,5,6-tetrahydronicotinamide analogue of NADP on the NADPH kinetics of aminopyrine-N-demethylation.

1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (NADH₃), a structural analogue of NADH, was unable to support the demethylation of aminopyrine or the reduction of the cytochrome P450-aminopyrine complex. However, the combination of NADH₃ with NADPH stimulated the NADPH dependent reduction of the cytochrome P450-aminopyrine complex. There was no significant alteration in the apparent K_m (NADPH) value, but there was an 80 per cent increase in apparent V of NADPH for NADPH-cytochrome P450-reductase (plus aminopyrine) when the kinetic constants were determined in the presence of 100 μM NADH₃. The inclusion of NADH₃ in the medium for aminopyrine demethylation also resulted in a significant increase in apparent V compared to the value obtained in the absence of NADH₃. The results suggest that the structure of NADH, as well as its capacity to donate the electron, is responsible for the NADH mediated increase in aminopyrine metabolism.     

Mestranol-induced hypertension: characterization of cytochrome P-450 dependent catechol estrogen formation in brain microsomes

An animal model of estrogen-induced hypertension was used to study the effects of chronic administration of the synthetic estrogen mestranol on cytochrome P-450 content and catechol estrogen formation in brain microsomes. Cytochrome P-450 content of brain microsomes from untreated female rats in estrus was 0.034 nmole/mg protein and the dithionite-reduced carbon monoxide absorbance peak (γ_max) was 452 nm. Catechol estrogen formation in brain microsomes was optimal in the presence of both NADPH and NADH cofactors with an apparent K_m value of 71 μM for 17β-estradiol substrate. Brain microsomes from animals in estrus and diestrus were compared, and no significant differences were observed in cytochrome P-450 content, or in the apparent K_m and V_max values of catechol estrogen formation. Administration of mestranol, 15 μg biweekly, for 3–4 weeks resulted in a significant increase in systolic blood pressure in unanesthetized female rats. Mestranol treatment was not associated with a change in microsomal cytochrome P-450 content or the spectral γ_max. At 10 μM substrate concentration, catechol estrogen formation in microsomes from mestranol-treated animals was increased 2- to 3-fold, with enzyme activity being expressed either per mg protein or per nmole cytochrome P-450. In contrast, no difference was observed between groups when enzyme activity was measured at 100 μM substrate concentration. These data suggest that chronic administration of a synthetic estrogen can regulate the enzyme system involved in formation of brain catechol estrogen metabolites, a mechanism which may alter the biological impact of the parent steroid.     

Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc.

Rat liver microsomes were incubated in the presence of zinc and the rate of NADPH oxidation and related metabolism of aniline and ethylmorphine by appropriate oxidases were studied. A competitive mechanism of the inhibition of NADPH oxidation by zinc was found, with V_max = 10.3 nmoles NADP/min/mg of protein and K_i amounting to 7.22 μM zinc. In microsomes dialyzed against EDTA, addition of Mn²⁺ but not of Mg²⁺ enhanced the rate of NADPH oxidation. A complex relation of Zn²⁺ and Mn²⁺ in liver microsomes was found, the data not obeying the rigorous treatment for enzyme kinetics. The activity of aniline hydroxylase and ethylmorphine-N-demethylase was inhibited by zinc; 50 per cent inhibition was reached at 60 and 55 μM Zn²⁺ respectively. Another microsomal enzyme, glucose 6-phosphatase, independent of NADPH, was not affected by zinc. The content and spectral characteristics of cytochrome P-450 were not affected by zinc. It is concluded that Zn²⁺ inhibits oxidation of NADPH and prevents this pyridine nucleotide from functioning in the microsomal electron transport system. The possibility that Zn²⁺ may interfere with other ions or enzymes involved in microsomal electron transport cannot be excluded.     

Kinetic studies with 5-azacytidine-5'-triphosphate and DNA-dependent RNA polymerase.

In order to further understand the biochemical mode of action of 5-azacytidine, a potent antileukemic agent, kinetic studies were performed with 5-azacytidine-5'-triphosphate (5-aza-CTP) and purified DNA-dependent RNA polymerase from Escherichia coli and calf thymus. RNA polymerase could catalyze the incorporation of the fradulent nucleotide, 5-aza-CTP, into RNA. The apparent K_m value for 5-aza-CTP was estimated to be 350 and 390 for the E. coli and calf thymus enzymes respectively. The K_m value for 5-aza-CTP was about 18-fold greater than the K_m value for CTP (20 μM). The apparent V_max value for CTP was about 2-fold greater than the V_max value for 5-aza-CTP. 5-Aza-CTP was a weak competitive inhibitor with respect to CTP; the apparent K_i value for 5-aza-CTP was estimated to be 680 and 810 μM for the E. coli and calf thymus enzymes respectively. On the other hand, CTP was a potent competitive inhibitor with respect to 5-aza-CTP; the apparent K_i value of CTP was estimated to be 16 μM. 5-Aza-CTP did not appear to inhibit the incorporation of UTP into RNA in the reaction catalyzed by RNA polymerase. These data suggest that the inhibition of RNA synthesis in cells by 5-aza-cytidine is not produced by the inhibition of RNA polymerase by 5-aza-CTP.     

Monoamine oxidase in human platelets: Kinetics and methodological aspects

The kinetic properties of human platelet monoamine oxidase (MAO) were examined in 20 apparently healthy controls. The mean value (±S.D.) of the maximum velocity (V) was found to be 5.36 ± 1.97 pmoles of product formed/10° platelets/min and the Michaelis-Menten constants were for phenethylamine (K^PEA_m) 14.6 ± 8.20 μM and for oxygen (K_m^O₂)254 ± 125 μM, when assayed in 0.1 M phosphate buffer, pH 7.4. The relation between the value of the corresponding apparent constants was studied. Inhibition of the enzyme activity was seen at 20 μM of PEA and 180 μM of oxygen. The enzyme kinetics were also studied at different pH. Two pK values were found, pK₁ = 6.65 and pK₂ = 6.95. The influence of homogenization on the MAO activity was compared with the activity in the undisrupted platelet. At PEA concentrations below 10 μM higher MAO activities were found in the intact cell. A 15 per cent loss of activity was detected in platelet samples after storing at ?20° for three and a half years.     

Activation of tryptophan hydroxylase from central serotonergic neurons by calcium and depolarization.

The activity of tryptophan hydroxylase in a low speed supernatant preparation from rat thalamus, midbrain and medulla pons. assayed in the presence of subsaturating concentrations of 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6MPH₄), was enhanced 100 per cent by addition of 10 mM CaCl₂ to the reaction medium. A similar increase in activity was also observed if CaCl₂ (10 or 100 mM final concn) was added directly to the enzyme and the unbound CaCl₂ then removed by gel filtration on Sephadex G-25. This increase in activity could not be reversed by the addition of ethylene glycol bis-(β-aminoethyl ether)-N, N′-tetraacetic acid (EGTA). It was associated with a modest increase in V_max (16 per cent) and an approximate doubling of the affinity of the enzyme for substrate and cofactor (apparent K_m for tryptophan decreased from 79 to 46 μM; apparent K_m for 6MPH₄ decreased from 148 to 101 μM). An increase in activity was also observed when tryptophan hydroxylase was prepared from slices of the brain preparation which had been (1) depolarized with a medium containing 66 mM KCl; (2) incubated overnight in an Na⁺-free medium or (3) treated with ouabain (0.05 or 0.1 mM), procedures believed to increase free intraneuronal levels of Ca²⁺. The increase in activity was, however, not observed when the slices were incubated in a Ca²⁺-free, K-rich medium to which 100 μM EGTA had been added. A kinetic analysis of the enzyme prepared from slices depolarized in K⁺ revealed an increase in V_max and a decrease in the apparent K_max for substrate (74–45 μM) and artificial cofactor (177–118 μM). The activation observed after depolarization could not be reversed with EGTA.     

The effect of albumin on the metabolism of ethoxyresorufin through O-deethylation and sulphate-conjugation using isolated rat hepatocytes

Ethoxyresorufin was metabolised by suspensions of isolated rat hepatocytes through O-deethylation to resorufin, followed by sulphate-conjugation of the resorufin. The deethylation but not the conjugation was greatly induced by 3-methylcholanthrene pretreatment in vivo. Induction altered the apparent deethylation V_max but not the apparent K_m value. With control hepatocytes there was a 3-fold difference between the apparent V_max values for deethylation (0.07) and conjugation (0.22 nmole/min/10⁶ hepatocytes) but none between the apparent K_m values (1.3 μM, deethylation and 1.0 μM, conjugation). After induction the deethylation V_max (10 nmole/min/10⁶ hepatocytes) was almost 13-fold higher than the conjugation V_max (0.81 nmole/min/10⁶ hepatocytes), but again there was no difference in the K_m values for the two reactions (2 μM). A significant proportion of the deethylation product, resorufin, passed out from the hepatocytes and then re-entered them in order to undergo conjugation. Extracellular bovine serum albumin inhibited the conjugation by binding resorufin that had left the hepatocytes. Albumin greatly increased the total resorufin formed from ethoxyresorufin, despite inhibiting very slightly the initial rate of deethylation.     

Differential Induction of Peroxygenase-Dependent Microsomal Aniline Hydroxylase by Chronic Ethanol Ingestion

ABSTRACT

The liver microsomal-mediated hydroxylation of aniline, which is selectively induced by chronic (EtOH) ingestion, has been studied as a function of NADPH plus dioxygen (O₂)- or hydroperoxide-dependent reactions. Consistent with the well-documented induction of aniline hydroxylase following chronic ethanol -ingestion, the results showed selectivity towards aniline hydroxylase by the NADPH plus 0₂- and tert-butyl hydroper-xide (t-BuOOH)-dependent reactions with microsomes from EtOH-fed rats. On the other hand, the cumene hydroperoxide (CumOOH)-dependent aniline hydroxylase activity was not discriminated between microsomes from EtOH-and pair-fed rats. In parallel experiments with positive controls, CumOOH did show selectivity for phenobarbital (PB)- induced microsomal aniline hydroxylase compared to chow-fed rats. The K_cat/K_m values, which indicate the efficiency of enzyme catalysis, for NADPH plus 0₂-, t-BuOOH, and CumOOH-dependent aniline hydroxylase from EtOH-fed rats were 102, 37, and 5 and from pair-fed rats were 68, 4, and 4 (nmol p-aminophenol/min/nmol cytochrome P-450)/mM aniline, respectively. The relative K_cat/K_m ratio for EtOH-fed to that of pair-fed microsomal aniline hydroxylase from NADPH plus 0₂-, t-BuOOH-, and CumOOH-dependent reactions were 1.5, 7.4. and 1.2, respectively. The present preliminary studies indicate that the catalytic efficiency of EtOH-induced aniline hydroxylase is significantly greater for the t-BuOOH-dependent reaction.     

Biotransformation of nitrosobenzene,phenylhydroxylamine, and aniline in the isolated perfused rat liver

1. Haemoglobin-free single-pass perfusion of isolated rat liver with [¹⁴C]aniline, [¹⁴C]phenylhydroxylamine, and [¹⁴C]nitrosobenzene was carried out.

2. Perfusion with aniline revealed apparent enzyme kinetics for 4-aminophenol formation with K_m = 144μM, V_max = 51 nmol/min per g liver wet; for 2-aminophenol K_m = 144μM, V_max = 16nmol/min per g; for acetanilide K_m = 33μM, V_max = 25 nmol/min per g. Formation of phenylhydroxylamine and nitrosobenzene was observed at a rate of 1.5 nmol/min per g provided that these metabolites had been trapped within red cells.

3. Perfusion with phenylhydroxylamine displayed a metabolic pattern similar to aniline with apparent phenylhydroxylamine reduction kinetics of K_m = 260μM and V_max = 600 nmol/min per g. In addition an acid-labile phenylhydroxylamine glucuronide was formed.

4. Perfusion with nitrosobenzene showed very rapid reduction to phenylhydroxylamine and to the metabolites observed with phenylhydroxylamine. In postmicrosomal supernatant, enzymic reduction of nitrobenzene by NADH and NADPH showed K_m = 12μM nitrosobenzene and V_max = 5000nmol/min per g.

5. Three per cent of nitrosobenzene was irreversibly bound to liver proteins. After 20min perfusion with nitrosobenzene, 0.95 μmol of liver glutathione was lost per 10μmol nitrosobenzene infused; 0.16μmol of glutathione was released with effusate and bile, 0.46μmol of glutathionesulphinanilide was produced, the rest, 0.33μmol, may have formed mixed disulphides.     

Kinetics, activation, and induction of aortic mono-oxygenases--biotransformation of benzo[a]pyrene.

Aortic aryl hydrocarbon hydroxylase (EC 1.14.14.2), a cytochrome P-450-dependent mono-oxygenase complex potentially important in the etiology of atherosclerosis, was detected previously in aortic homogenates of humans, monkeys and rabbits [M. R. Juchau, J. A. Bond and E. P. Benditt, Proc. natn. Acad. Sci. U.S.A.73, 3723 (1976)]. The present study more fully characterizes the mono-oxygenase activity in aortas of treated and untreated New Zealand White rabbits. A 2-fold activation was obtained with NADH (7 × 10^?4 M) at saturating concentrations of NADPH. Addition of heme (9 μm) increased the enzymatic activity 2- to 4-fold during a 15-min incubation and over 25-fold during a 2-hr incubation. The results suggest the presence of relatively high concentrations of apoprotein in the aortic tissues. Kinetic studies in the presence of heme yielded an apparent K_m of 1 × 10^?4 M and V_max of 15.24 pmoles/mg of protein/min with respect to NADPH. A sigmoidal curve was obtained with varying benzo[a]pyrene concentrations (0.5 to 80 μM), suggesting the possibility of allosterism. Aroclor 1254,3-methylcholanthrene and 5,6-benzoflavone acted to induce the cytochrome P-450-dependent mono-oxygenase, while pheno-barbital and pregnenolone 16α-carbonitrile demonstrated little, if any, induction capacity. Analyses of metabolites formed in induced aortas with high-pressure liquid chromatography revealed the formation (in each case) of primarily the phenolic metabolites of benzo[a]pyrene.     

Adenosine 3',5'-monophosphate phosphodiesterase activity in experimental animal tumours which are either sensitive or resistant to bifunctional alkylating agents.

The adenosine 3′,5′-monophosphate phosphodiesterase of Walker rat carcinoma 256, ADJ/ PC6 plasma cell tumour, NK lymphoma. Sarcoma 180 and TLX5 lymphoma behaves kinetically as if two separate activities exist, one with a low affinity for the substrate and the other with a high affinity. The high K_m values are 82·5, 566, 590, 1975 and 1075 μM for the enzyme from each tumour respectively, and the low K_m values are 1·1, 17·7, 5·75, 7·1 and 4·4 μM. In the Walker carcinoma and PC6 plasma cytoma, tumours which are sensitive to alkylating agents, the apparent V_max of the low K_m forms are respectively 38 and 25 per cent of the total activity. In those tumours which are naturally resistant to the growth inhibitory properties of the alkylating agents, the apparent V_max of the low K_m form is less than 10 per cent of the total activity. Walker carcinoma showing a 20-fold resistance to chlorambucil[p-(di-2-chloroethylamino) phenylbutyric acid] has the V_max of the high affinity form decreased to 15 per cent of the total, while a 70-fold resistant line has the V_max of this form of the enzyme decreased to 9 per cent of the total. This decrease in the activity of the high affinity form of the enzyme in the resistant tumours does not appear to be due to the presence of an endogenous inhibitor. While the high K_m form of the enzyme in the Walker carcinoma is mainly confined to the cytosol, about half of the activity of the low K_m form appears to be associated with particulate fractions. This subcellular distribution does not differ appreciably in the sensitive and resistant tumours. The possible reasons for such an isoenzyme shift are discussed.     

Metabolism of tetraorganolead compounds by rat-liver microsomal mono-oxygenase: II. Enzymic dealkylation of tetraethyl lead

1. The biological degradation of tetraethyl lead to the triethyl lead cation by rat-liver microsomes from untreated, phenobarbital-pretreated and methylcholanthrene-pretreated rats has been studied; NADPH and oxygen are essential.

2. The reaction is inhibited by CO and can be reactivated in the presence of O₂ by irradiation with u.v. light with a max. at 450nm.

3. Substrate binding to cytochrome P-450 is of type 1.

4. Apparent K_m values for triethyl lead formation in microsomes were determined. The highest activities (i.e. about 2 nmol triethyl lead per nmol cytochrome P-450 per min) and the lowest apparent K_m values (i.e. 7 × 10^-6 M) are found in microsomes from methylcholanthrene-pretreated rats.

5. In microsomes from control and phenobarbital-pretreated rats K_s values from substrate-binding studies (about 2 × 10^-6M) are one order of magnitude lower than the apparent K_m values (3 × 10^-5 M).     

Studies on the mechanism of cadmium-induced inhibition of the hepatic microsomal monooxygenase of the male rat.

Cadmium is a potent inhibitor of hepatic drug metabolism but little is known about the biochemical mechanisms involved. Male rats receiving a single dose of cadmium acetate (2.0 mg Cd²⁺/kg, ip) exhibited significant decreases in hepatic microsomal metabolism of hexobarbital (79%), ethylmorphine (71%), and aniline (47%), as well as decreased amounts of both microsomal cytochrome P-450 (39%) and cytochrome b₅ (30%) 72 hr after administration of the metal. In addition, the magnitudes of microsomal hexobarbital, ethylmorphine, and aniline binding spectra were significantly reduced. NADPH-cytochrome c reductase activity was not altered. In vitro addition of Cd²⁺ (10^?6 to 10^?3m) to liver microsomes produced a concentration-dependent inhibition of the metabolism of the substrates tested, a reduction in the level of cytochrome P-450 which was accompanied by an increase in the level of the inactive form of the hemo-protein, P-420, and decreases in the microsomal binding spectra of the three substrates. In kinetic studies, apparent V_max values were significantly lowered for ethylmorphine N-demethylase (75%) and aniline hydroxylase (57%) by cadmium treatment. The apparent K_m value for aniline hydroxylase was not altered, but that for ethylmorphine N-demethylase was significantly decreased following cadmium treatment. After in vitro cadmium addition, concentration-dependent decreases were observed in the apparent V_max and K_m values for both microsomal reactions.     

Effects of disulfiram on the oxidation of benzaldehyde and acetaldehyde in rat liver

The in vitro oxidation of benzaldehyde and acetaldehyde was studied in liver samples from disulfiram-treated and control rats. With 25 μM substrate, both cytosol and mitochondria appeared to make a nearly equal contribution to the oxidation of benzaldehyde, whereas ca. 90% of acetaldhyde oxydation occurred in mitochondria. When the K_m values for benzaldehyde with aldehyde dehydrogenase (ALDH) were determined, two K_m values (3 and 120 μM) were obtained with mitochondria, but only a single K_m value (25 μM) was obtained with the cytosolic fraction. The relatively high K_m (2.9 mM) found with microsomes makes it unlikely that microsomes are important in the oxidation of benzaldehyde. In intact mitochondria, with 200 μM acetaldehyde or benzaldehyde the matrix space enzyme accounted for 77 and 62%, respectively, of the total ALDH activity. When the activity was determined in a mixture containing both substrates, the activity was found not to be additive, indicating that both substrates are oxidized by the same matrix space enzyme. With subcellular fractions, from livers of disulfiram-treated and control rats, a greater degree of inhibition of ALDH was obtained when acetaldehyde was a substrate compared to that with benzaldehyde in cytosol and mitochondria. Microsomal ALDH was not inhibited by disulfiram. In liver slices from rats given disulfiram, a statistically significant inhibition was found when either 25 or 250 μM acetaldehyde was used (46 and 33%). With benzaldehyde, a significant inhibition (24%) was observed only with the lower substrate concentration. Finding that both mitochondrial fractions and slices were less inhibited at the higher substrate concentration implies that the high K_m enzyme is not inhibited. It can be concluded that, in rat, disulfiram inhibiting liver ALDH not only affects oxidation of acetaldehyde, but also that of benzaldehyde.     

Compensatory increase in synaptosomal aldehyde reductase activity in rat brain after chronic barbital treatment

The changes in brain aldehyde reductase (AIR) activities during long-term treatment of rats with barbital were studied. NADH-linked AIR activity in the synaptosomal fraction increased rapidly, and supernatant AIR activity rose later during barbital treatment. With respect to NADPH-linked AIR, two distinct K_m values were observed for p-nitrobenzaldehyde. The low-K_m enzyme had a higher K_i value than the high-K_m enzyme. Although the elevation of NADPH-linked AIR activity under the routine assay conditions was less remarkable, V_max values of the low-K_m enzyme were greatly increased in both the synaptosomal and the supernatant fractions by chronic barbital treatment. In addition, the K_i value of low-K_m AIR in synaptosomes was greater in barbital-treated animals than in control animals. K_m values were unchanged in either fraction by chronic barbital treatment. These data suggest that chronic barbital treatment resulted in a compensatory increase in the activity of AIR with low K_m, which is less sensitive to barbital and utilizes either NADH or NADPH, in synaptosomes.     

N-demethylation of aminopyrine in vivo and in the isolated hepatocyte of the rat.

The N-demethylation of [dimethylamine-¹⁴C]aminopyrine was monitored in vivo and in isolated hepatocytes of the rat by collection of ¹⁴CO₂, The isolated hepatocyte is capable of aminopyrine N-demethylation rates similar to those occurring in vivo. The kinetics of N-demethylation in the isolated cells indicate the presence of two separate reactions with K_m values of 32 and 710 μM. The kinetics of N-demethylationin vivo, however, indicates the presence of one reaction with an intermediate K_m value of 260 μM. It is thought that the two reactions observed in the hepatocytes also occur in vivo but that it is not possible to differentiate between them.     

Effect of cyclic nucleotides on activity of cyclic 3',5'-adenosine monophosphate phosphodiesterase

A series of cyclic 2′,3′-nucleotides, cyclic 3′,5′-nucleotides and derivatives of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with a substituent at the C-8 position were investigated as inhibitors of partially purified cyclic AMP phosphodiesterases (PDE) of cat heart and rat brain. The assays were carried out at a substrate concentration (0.06 μM) where the contribution to the total enzyme activity by phosphodiesterases with K_m values for cyclic AMP above 100 μM was insignificant; consequently the activity measured was that of low K_m enzymes.Cyclic 3′,5′-guanosine monophosphate (cyclic GMP) and cyclic 3′,5′-inosine monophosphate (cyclic IMP) were shown to be the most potent inhibitors of PDE of cat heart (I₅₀ = 1 and 2 μM, respectively). Of the cyclic AMP derivatives tested that have a substituent at the C-8 position, 8-bromo cyclic AMP was the most potent inhibitor; next most potent were the derivatives with a sulfur atom, whereas derivatives with oxygen- or nitrogen-containing substituents were the least potent inhibitors of PDE of cat heart or rat brain. Most of the cyclic nucleotides that were tested were more potent inhibitors of the PDE of cat heart than that of PDE of rat brain.The kinetic properties of PDE of cat heart were also investigated in the presence of cyclic GMP, cyclic IMP and 8-bromocyclic AMP. All three compounds were found to be competitive inhibitors, with apparent K_i values of 0.51, 2.3 and 20 μM respectively. The possible pharmacologic role of cyclic nucleotides is discussed.     

Saturation of an α,β-unsaturated ketone: A novel xenobiotic biotransformation in mammals

1. Reduction of an α,β-unsaturated ketone to the corresponding saturated ketone in a 2-benzylidene-3-ketocoumaran derivative has been investigated.

2. Reductase activity resides in the cytosolic fraction of liver, lung and kidney. Rat and human blood also contain significant reductase activity.

3. Hepatic reductase activity was high in guinea-pigs followed by hamsters, rabbits, rats and mice. The substrate had an apparent K_m and V_max of 5·6 μM and 1·3 nmol/min per mg protein, respectively. The reduction was dependent upon NADPH having an apparent K_m of 14·8 μM and a V_max of 1·0 nmol/min per mg protein.

4. Only the A side hydrogen of NADPH was incorporated into the reduced product.

5. The reaction was inhibited by cyanide, and sulphydryl reagents. Phenobarbital did not induce the activity in rats.     

Nitrite formation from 2-nitropropane by microsomal monooxygenases.

Rat liver microsomes in the presence of NADPH and dioxygen catalyze the oxidative denitrification of 2-nitropropane to actone and nitrite. Pretreatment of the animals with phenobarbital and 3-methylcholanthrene increases the specific activity. Inhibitors of microsomal monooxygenases inhibit the formation of nitrite and the photochemical action spectrum of the CO-inhibited reaction indicates the involvement of cytochrome P450. The K_m value and the spectral dissociation constant, K_s, of the substrate binding spectrum are both around 10^?2M. 1-nitropropane also undergoes oxidative denitrification. It is suggested that aliphatic nitro compounds may exhibit mutagenic or carcinogenic effects as recently demonstrated for 2-nitropropane in rats.