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The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.

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Stimulation of soluble guanylyl cyclase in rat fetal lung fibroblasts (RFL-6 cells) was used as a sensitive assay for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) formation. Intact N1E-115 cells released an EDRF/NO-like material that enhanced cyclic GMP levels in RFL-6 cells. The synthesis of this substance could be stimulated with the receptor agonist neurotensin (10 microM) or by addition of the EDRF/NO substrate L-arginine (100 microM). In Ca2(+)-free Locke's solution, stimulation of EDRF/NO production by both neurotensin and L-arginine was abolished. The EDRF/NO-synthesizing activity was localized in the cytosol of N1E-115 cells. The activity was lost after boiling and it was highly sensitive to Ca2+ with the major increase in activity occurring between 100 and 500 nM Ca2+. L-Arginine and NADPH were required for maximal synthesis of EDRF/NO by the enzyme(s). The synthesis of EDRF/NO was inhibited by the following antagonists of calmodulin-regulated functions (with the approximate IC50 values given in parentheses): calmidazolium (7 microM), trifluoperazine (10 microM), fendiline (80 microM), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide) (120 microM), and compound 48/80 (3 micrograms/ml). The EDRF/NO-synthesizing activity was partially purified from N1E-115 cytosol by DE 52 anion exchange chromatography. The activity was eluted with 0.1 M KCl. The enzyme(s) showed very little activity in the presence of L-arginine (100 microM) and NADPH (100 microM), but the activity could be fully restored by addition of exogenous calmodulin (EC50, approximately 2 units/ml). At 0.3 M KCl, a fraction eluted from the DE 52 column that was also able to fully restore the EDRF/NO-synthesizing activity. Thus, this fraction is likely to contain the endogenous Ca2(+)-binding protein. It is concluded that the activity of the EDRF/NO-synthesizing enzyme(s) in N1E-115 neuroblastoma cells is regulated by Ca2+ and calmodulin.

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Stimulation of soluble guanylyl cyclase and increase in cyclic GMP in rat fetal lung fibroblasts (RFL-6 cells) was used as a bioassay to detect EDRF/NO formation. The cytosolic fraction of whole rat brain synthesized an EDRF/NO-like material in a process dependent on L-arginine and NADPH. The enzymatic activity was destroyed by boiling and inhibited by N omega-nitro-L-arginine. Hemoglobin and methylene blue blocked the effect of EDRF/NO. When different brain regions were analyzed in the presence of L-arginine and NADPH, the cytosolic fraction from cerebellum showed the highest EDRF/NO-forming activity (2-3 times higher than whole brain). Activity similar to whole brain was found in hypothalamus and midbrain. Enzymatic activities in striatum, hippocampus and cerebral cortex were about two thirds of whole brain. The lowest activity (less than half of whole brain) was found in the medulla oblongata.

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Accumulation of cyclic GMP in cultured rat lung fibroblasts was used to test the hypothesis that N1E-115 neuroblastoma cells produce an endothelium-derived relaxing factor (EDRF)-like activity. By using this assay, the production of an EDRF-like activity in homogenates and cytosolic fractions of N1E-115 neuroblastoma cells was observed. Detection of the activity required the presence of superoxide dismutase and was inhibited by hemoglobin. Production of the EDRF-like factor was dependent on L-arginine and NADPH. The apparent Km for L-arginine was 1.25 microM and the apparent Km for NADPH was 1.67 microM. The production of the EDRF-like activity was inhibited by the L-arginine analogs, NG-monomethyl-L-arginine and NG-nitro-L-arginine, with apparent Ki values of 1.0 and 0.3 microM, respectively.

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The cytosolic fraction of N1E-115 neuroblastoma cells catalysed the L-arginine- and NADPH-dependent formation of a substance that relaxed endothelium-denuded strips of rabbit aorta. Relaxations in response to this substance were enhanced in the presence of superoxide dismutase. N omega-Nitro-L-arginine and NG-monomethyl-L-arginine, two inhibitors of EDRF synthesis, markedly attenuated the relaxations. Hemoglobin, a scavenger of EDRF, and methylene blue, an inhibitor of soluble guanylate cyclase, completely abolished the relaxation to N1E-115 cytosol. In contrast, the cyclo-oxygenase inhibitor indomethacin did not alter the relaxations. These data demonstrate that the cytosol of a neuronally-derived cell line is able to synthesize a substance with pharmacological properties similar to EDRF.

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Hepatocytes were isolated from the livers of ethanol-pretreated rats, and the relationship between the generation of CO2 and the loss of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) from the incubation mixtures was examined. The evolution of CO2 by hepatocytes isolated from untreated, control rats was compared with the evolution of CO2 by hepatocytes isolated from rats treated with 10% EtOH in their drinking water. The CO2 generated from either NDMA or NDEA represented only a fraction of the parent compound that was metabolized during the incubation period. Therefore, the measurement of CO2 evolution as an indication of the metabolism of these simple dialkylnitrosamines is inadequate, and the actual loss of the parent compound must be measured directly when utilizing isolated hepatocytes as a model system to study the metabolism of nitrosamines. The liver microsomal metabolism of NDMA and NDEA was also examined. Pretreatment of the rats with ethanol resulted in a marked increase in the microsomal metabolism of NDMA but had a relatively small effect on NDEA metabolism. Phenobarbital pretreatment did not result in any increase in NDMA metabolism whereas there was a very significant (6-fold) increase in NDEA metabolism. These results suggest that different isozymes of cytochrome P-450 may be primarily responsible for the metabolism of the two nitrosamines. The inhibition patterns observed when an antibody inhibitory to cytochrome P-450j was added to microsomes derived from control and ethanol- and phenobarbital-pretreated rats conclusively demonstrate that NDMA and NDEA are preferentially metabolized by distinct isozymes of cytochrome P-450.

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The metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to reactive intermediates which covalently bind to cellular proteins was investigated. Isolated hepatocytes were used to determine whether protein alkylation is random in nature or whether it results in the alkylation of specific proteins. Isolated hepatocytes from rats treated with either ethanol (EtOH) or phenobarbital were incubated with the 14C-labeled nitrosamines for 1-3 h, after which the cells were separated from the incubation medium in order to distinguish secreted proteins from intracellular proteins. SDS-PAGE of the proteins in the medium followed by fluorographic analysis of the gels revealed that a heavily labeled protein was secreted into the medium which represents the predominantly labeled protein. Intracellularly, the major portion of the covalently bound label was found in the region of the gel where the cytochromes P-450 migrate. Pretreatment of the hepatocytes with diethyl maleate and buthionine sulfoximine to decrease the intracellular levels of glutathione had no effect on the labeling, indicating that glutathione does not protect cellular proteins from labeling by these carcinogens. Pretreatment of the cells with D-(+)-galactosamine to inhibit UDP-glucuronyltransferases resulted in a significant decrease in protein labeling by NDEA, suggesting that a glucuronide intermediate may be involved in the activation of NDEA to an alkylating species. The heavily labeled protein secreted into the incubation medium was identified as albumin on the basis of its apparent molecular weight of 66K, as determined by SDS-PAGE, and its cross-reactivity with anti-rat albumin IgG.

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There is significant evidence to suggest that protein kinase C and DNA topoisomerases are functionally linked in signal transduction pathways. Much of this is based on the observation that phosphorylation of topoisomerase II by protein kinase C may lead to its activation in vitro and that inhibitors of topoisomerase II block phorbol diester-induced differentiation in HL-60 cells. In the present study, the activities of the DNA topoisomerases I and II have been quantitated to examine their regulation in phorbol diester-treated HL-60 cells undergoing differentiation. The activity of topoisomerase I increased rapidly after treatment with phorbol myristate acetate (PMA); it increased maximally (150% of control activity) at 3 hr post-treatment and remained elevated for at least 24 hr. Conversely, from the onset of exposure to PMA through 12 hr, there was no measurable alteration in topoisomerase II activity in PMA-treated cells. Moreover, there was a measurable decrease in topoisomerase II activity at the later time points, a result that occurred concomitantly with the loss of proliferative potential in differentiating HL-60 cells. Similar results were obtained when the activities of both enzymes were measured in nuclear extracts. The apparent increase in topoisomerase I activity was not due to an increase in the mass of the enzyme after PMA treatment, as measured by both western blotting and by the formation of camptothecin-dependent, topoisomerase I-DNA complexes. Taken together, these data suggest that the activities of the topoisomerases I and II may have been regulated independently in PMA-treated HL-60 cells, that the activity of topoisomerase II was not increased under conditions in which protein kinase C was activated in vivo, and that an increase in the activity of topoisomerase I may have had a role in the mechanism through which HL-60 cells underwent monocytic maturation in response to phorbol diesters.

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The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.

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Evidence is presented that the method of reconstitution of the cytochrome P-450-containing liver microsomal enzyme system with cytochrome b5 (b5), including the order of addition of the components, the concentration of the b5, and the length of incubation prior to initiation of the reaction by NADPH, governs the steady state catalytic activity obtained. For example, the addition to cytochrome P-450 isozyme 2, NADPH-cytochrome P-450 reductase, and phosphatidylcholine of concentrated b5 (0.4 microM) results in extensive inhibition of benzphetamine demethylation and NADPH oxidation, whereas the addition of dilute b5 (0.02 microM) to the other components results in extensive stimulation of the demethylation reaction. The inhibition is partly relieved by prolonged incubation. The effects of pH and buffer concentration were determined, and the optimal molar ratio of b5 to cytochrome P-450 isozyme 2 was shown to be about 2.0 for stimulation of benzphetamine demethylation, dimethylaniline demethylation, and cyclohexanol oxidation to cyclohexanone. Cytochrome P-450 isozyme 4-catalyzed aminopyrine demethylation and aniline p-hydroxylation are not stimulated by b5, as predicted from a model based on stopped flow kinetic measurements [Pompon and Coon: J. Biol. Chem. 259, 15377 (1984)]. End-point stoichiometry measurements were carried out with cytochrome P-450 isozyme 2 in the absence of b5 or in the presence of b5 under optimal conditions. The results indicate that when b5 is reconstituted with the cytochrome P-450 isozyme 2 enzyme system under optimal conditions, substrate monooxygenation is enhanced, NADPH oxidation is unaffected, and hydrogen peroxide formation is decreased.

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The mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (P-450 PB-B2) by chloramphenicol has been investigated. Preparations of the enzyme from animals treated in vivo with chloramphenicol (CAP PB-B2) have been isolated, and their catalytic, spectral, and physical properties have been compared with those of the native PB-B2. The CAP PB-B2 exhibited: 1) a 60-70% loss in the rate of NADPH-supported monooxygenase activity with the substrates benzphetamine, 7-ethoxycoumarin, and p-nitroanisole; 2) a 60% decrease in the extent of enzymatic P-450 reduction catalyzed by NADPH-cytochrome P-450 reductase under both aerobic and anaerobic conditions; 3) a 60% decrease in the steady-state level of the ferrous dioxygen complex in the presence of substrates; 4) a 60% decrease in the magnitude of the type I spectral change induced by benzphetamine; and 5) a shift in the wavelength maximum for the chemically reduced ferrous-carbonyl complex from 450 to 451.5 nm. On the other hand, the ability of the CAP PB-B2 to catalyze the iodosobenzene-supported metabolism of 7-ethoxycoumarin and p-nitroanisole was unaltered. The results are consistent with a scheme whereby the binding of metabolites of chloramphenicol to amino acid residues in the PB-B2 close to the heme moiety blocks electron transport from NADPH-cytochrome P-450 reductase, thereby leading to a loss of monooxygenase activity.

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The possible role of free hydroxyl radicals in the oxidation of cyclohexanol to cyclohexanone and of benzene to phenol was examined in a reconstituted system containing rabbit phenobarbital-inducible P-450LM2. From steady state kinetic studies, a KM for cyclohexanol of 8.7 mM and a Vmax of 5.7 nmol of cyclohexanone formed/min/nmol of P-450 were determined. Similarly, a KM for benzene of 105 mM and a Vmax of 22 nmol of phenol formed/min/nmol of P-450 were obtained. With intact microsomes from phenobarbital-treated rabbits, a KM for benzene of 18 mM and a Vmax of 1.7 nmol of phenol formed/min/nmol of P-450 were determined. With the use of substrate concentrations in the range of the respective KM values, superoxide dismutase, desferrioxamine, and dimethyl sulfoxide were found to have no significant effect on the P-450-catalyzed reactions. When the oxidation of benzene or cyclohexanol was examined in a model hydroxyl radical-generating system containing xanthine, xanthine oxidase, and Fe-EDTA, no dependence of the rate of oxidation on the substrate concentrations used was observed. Since the rate of hydroxyl radical generation by the model system was adjusted to be greater than the rate of product formation in the P-450 system, the lack of dependence on substrate concentration suggests that free hydroxyl radicals are not involved in the P-450-catalyzed reactions studied. Taken together, these findings indicate that the free hydroxyl radical-mediated pathway observed by other investigators does not contribute significantly to product formation when these substrates are present at concentrations within the range of their respective KM values.

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This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.

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The formation and autoxidative decomposition of the oxygenated forms of two isozymes of rabbit liver microsomal cytochrome P-450 were studied: 5,6-benzoflavone- or isosafrole-inducible P-450LM4, isolated in the high spin state, and P-450LM3b, isolated in the low spin state. When an anaerobic solution of photochemically reduced isozyme 4 was mixed with aerobic buffer in a stopped flow spectrophotometer, the dioxygen complex with absorption maxima at 555 and 418 nm was rapidly formed. The monophasic reaction had a pseudo-first order rate constant of about 58 s-1. Autoxidation of the complex, which was complete in about 20 s, exhibited biphasic first order kinetics at 580 nm with rate constants of about 0.92 and 0.22 s-1. The results obtained with isozyme 3b were similar, except that the decomposition of the ferrous oxy intermediate appeared to be triphasic. Superoxide could not be detected as a product of the autoxidative decomposition of the oxy form of P-450 isozyme 4. Hydrogen peroxide was produced in about 70% yield when oxygen was in excess, whereas in a titration in which small increments of oxygen were added to an excess of the ferrous cytochrome the reduction apparently led to the formation of water. The biphasic kinetics of some of the reactions involving purified mammalian cytochrome P-450 has in some instances been attributed to the formation of aggregates by these hydrophobic proteins. This was ruled out as an explanation of the kinetics observed for the autoxidative decay of ferrous dioxygen P-450LM4, since two phases were also observed with a preparation converted from the usual aggregated state to the monomeric state by exposure to a zwitterionic detergent.

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Since there exists some controversy in the literature as to whether paraquat augments microsomal lipid peroxidation via superoxide anion (O2-), the role of paraquat and active oxygen species in NADPH-dependent lung microsomal lipid peroxidation was investigated. Incubation of buffered aerobic mixture of bovine lung microsome and NADPH, in the presence or absence of exogenously added iron, resulted in a progressive formation of lipid peroxides whose accumulation could be followed at 535 nm as malondialdehyde. Paraquat strongly inhibited this lipid peroxidation. Thus, malondialdehyde formation was 50% inhibited by 4 X 10(-5) M paraquat in the reaction mixture. The malondialdehyde color development by lipid peroxides was not affected by this concentration of paraquat. Lipid peroxidation was also strongly inhibited by singlet oxygen scavengers, e.g. dimethylfuran and diphenylfuran, and by catalase. Hydroxyl radical scavengers, e.g. mannitol, benzoate, and ethanol, had little effect in malondialdehyde production. Superoxide dismutase, which removes O2- efficiently, did not inhibit malondialdehyde production by lung microsomes and rather enhanced its formation. A scheme in which paraquat and active O2 species may be involved with microsomal lipid peroxidation is presented.

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