Alteration of liver microsomal monooxygenases and substrate competition with aniline hydroxylase from rats chronically fed low-fat and high-fat-containing alcohol diets

Male Sprague-Dawley rats fed ethanol (EtOH) 36% of total calories for four weeks in a liquid diet containing either 34% (HF) or 12% (LF) of calories as fat were studied with respect to induction of microsomal monooxygenases (MFO) and substrate competition with EtOH-inducible aniline hydroxylase. The specific activity and turnover of aniline hydroxylase were induced to similar extents by HF-EtOH and LF-EtOH diets. Whereas, both LF-EtOH and HF-EtOH caused a decrease in the turnover of arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and aldrin epoxidase compared to pair-fed (PF) controls, LF-EtOH but not HF-EtOH increased the turnover of ethoxycoumarin and ethoxyresorufin O-deethylase (ECOD and EROD). The increase in ECOD and EROD and the decrease in AHH by EtOH is contrary to the parallel induction of these activities by J-methylcholanthrene (3-MC) and Aroclor 1254 (Aroclor). Benzo(a)pyrene (BaP) stimulated aniline hydroxylase in the HF-EtOH and PF systems, whereas with LF diet, stimulation was seen only in the EtOH group. Ethoxycoumarin (EC) inhibited aniline hydroxylase by microsomes from EtOH- and pyrazole-treated rats, whereas it stimulated aniline hydroxylase by control microsomes, suggesting that the EC effects were associated with EtOH-inducible cytochrome P-450. Ethoxyresorufin (ER) inhibited aniline hydroxylase in EtOH and PF groups, thus the differential effects of EC were not nonspecific O-deethylase effects. The effects of EtOH feeding on ECOD, EROD, and AHH (ie, substrates for 3-MC-inducible cytochrome P-450) displayed a greater differential between the experimental and control group with the LF- than with the HF-containing diet. The findings suggest that the alteration of certain MFO activities by chronic EtOH ingestion can be modified by the content of dietary fat. Moreover, the competition dynamics of MFO substrates toward EtOH-inducible aniline hydroxylase are altered by EtOH feeding and, in turn, modified by dietary fat.     

The locus of inhibition of NADH oxidation by benzothiadiazoles in beef heart submitochondrial particles

6-Chloro-1,2,3-benzothiadiazole (6-Cl-BTD) is an effective inhibitor of NADH oxidase (site I) but not of succinate oxidase in beef heart submitochondrial particles. For NADH oxidase activity maximal inhibition (80-85%) was achieved at 0.75mM 6-Cl-BTD. A similar level of inhibition was also observed (half maximal inhibitory concentration 0.5mM) towards NADH-duroquinone reductase; NADH-juglone reductase was slightly inhibited (23%) at 0.5mM 6-Cl-BTD while NADH-ferricyanide reductase was unaffected. The data suggest that 6-Cl-BTD interacts with an electron transport site on the oxygen side of NADH dehydrogenase and inhibitory studies with 6-Cl-BTD and rotenone indicate that it might correspond with one of the two sites affected by rotenone. The substituted 1,2,3-benzothiadiazoles (BTDs) are perhaps best known for their activity as inhibitors of cytochrome P-450-mediated mixed-function oxidation (MFO). In vitro, the BTDs are potent inhibitors of MFO activities in microsomes from mammalian liver and insect tissues and they have been demonstrated to inhibit aminopyrine metabolism in perfused rat liver. In vivo, they reportedly prolong hexobarbital sleeping time in mice, inhibit the irreversible binding of labeled trichloro-ethylene to microsomal protein and effectively enhance the toxicity (synergize) of pyrethrin, organophosphorus-containing and carbamate insecticides to insects.     

Kepone induction of hepatic mixed function oxidases in the male rat.

Effect of preexposure to kepone on hepatic drug metabolizing enzymes and several parameters of mixed function oxidase (MFO) system was investigated. Male rats were exposed to 0, 50, 100 and 150 ppm kepone in the daily ration for 16 days. Gain in body weight was declined after kepone to 86, 62 and 33% of controls for 50, 100 and 150 ppm kepone respectively. Liver weight was unaltered at all three levels of kepone. While hydroxylation of both aniline and pentobarbital was enhanced, the increased in aliphatic hydroxylation (i.e., pentobarbital) were greater at all levels of exposure to kepone. Aminopyrine demethylase was enhanced roughly 3 fold at 50 ppm kepone and was not further increased at higher levels of kepone. Cytochrome P₄₅₀, NADPH-cytochrome c reductase, and aniline binding were all increased suggesting that there were general increases with intermediate steps of the electron transfer and drug oxidation system. Cytochrome b₅ and NADPH dehydrogenase were unaltered after kepone treatment. These results suggest that kepone is an efficient inducer of hepatic MFO system.     

An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus.

Four chromogenic substrates for characterizing serine protease of Vibrio alginolyticus were evaluated. The protease activity of bacterial extracellular products, or the fractions of 33 kD protease purified by the AKTA purifier system with various columns, was completely inhibited by ethylenediamine tetra-acetic acid, ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), antipain and phenylmethylsulphonyl fluoride (PMSF) using water-soluble substrates (azoalbumin and azocasein). It was only completely inhibited by antipain and PMSF using water-insoluble substrates (azocoll and hide powder azure). The protease activity was not, or only partially, inhibited by 1,10-phenanthroline and sodium dodecyl sulphate (SDS) using all four substrates. Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor. Chelating agents may be still applicable as inhibitors using water-insoluble substrates and 1,10-phenanthroline is highly recommended in the characterization for metalloprotease to avoid confusion. In the present study, the 33 kD protease was further confirmed as an SDS-resistant serine protease and not a metalloprotease.     

Effect of Plasmodium berghei infection and chloroquine on the hepatic drug metabolizing system of mice

The hepatic microsomal mixed-function oxidase (MFO) system was markedly impaired during Plasmodium berghei infection in mice. Cytochrome P-450 and other mono-oxygenases, viz. aniline hydroxylase, aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase, were significantly decreased while microsomal heme showed a four-fold increase at peak parasitemia (greater than 50%). Oral treatment with chloroquine (16 mg kg-1 body wt for 4 days) of P. berghei-infected mice cleared the parasitemia within 72 h and almost normalized the altered levels of MFO indices, a week after cessation of treatment. The findings were further supported by the isoenzymic profile and drug-binding properties of terminal mono-oxygenase, cytochrome P-450.     

Purification of the alliin lyase of garlic, Allium sativum L

1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at -10 degrees , particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6.5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10mum) and cysteine (0.5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The K(m) values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the K(m) was 6mm and the V(max.) was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The K(i) decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the K(i) was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg(2+), Mn(2+), Co(2+) or Fe(2+) stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg(2+).     

Studies on the substrate specificity and inducibility of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system in Bacillus megaterium A.T.C.C. 13368 was investigated with regard to its appearance in the cell with respect to the growth curve of the organism, with regard to its inducibility by a number of agents (among them some of the classical inducers of the mammalian liver microsomal cytochrome P-450 system) and with regard to its capacity to convert non-steroidal substances into oxygenated compounds. The enzyme was found to reach a maximum concentration in the cell during the stationary phase of the growth curve. Of all the agents tested as inducers, none showed any capacity to induce cytochrome P-450meg. Finally, of the substances tested as substrates only aniline (p-hydroxylation) was metabolized by the microbial enzyme system. This conversion might be related to the general oxygenase activity of haemoproteins. It is concluded that the substrate specificity of the B. megaterium hydroxylase system is narrow.     

Purification and properties of thiaminephosphate pyrophosphorylase of Escherichia coli

Thiaminephosphate pyrophosphorylase (EC 2.5.1.3) in Escherichia coli has been purified 175-fold by conventional methods of enzyme purification. General properties of the partially purified enzyme were similar to those of the yeast enzyme except for a small molecular weight of 17,000. The E. coli enzyme was inhibited by a variety of high-energy phosphate compounds. Acetyl phosphate was the most potent inhibitor and resulted in 50% inhibition at 0.5 mm concentration. ATP and acetyl phosphate were both uncompetitive inhibitors with respect to both substrates. Low-energy phosphate compounds and pyridine nucleotides were not able to inhibit the activity. These results, together with the other results obtained, indicate that these high-energy phosphate compounds did not inhibit the enzyme activity after conversion to a common compound. The physiological significance of this type of inhibition was discussed from the point of cellular energy charge.     

Feminization of the hepatic monooxygenases by growth hormone is mimicked by puromycin and correlates with a decrease in male-type cytochrome P450

The hepatic monooxygenase system (MFO) was studied in hypophysectomized male rats treated with growth hormone (GH), puromycin, or both. GH significantly decreased the amount of cytochrome P450 and the activity of ethylmorphine demethylase but did not affect aniline hydroxylase or NADPH cytochrome c reductase. Puromycin significantly increased the activity of the reductase but otherwise had effects identical to GH. The agent's effects were additive. By labelling the P450 with [3H]-heme we found that GH decreased the amount of male-type (slow turnover) P450 by 56% but lowered the female-type (fast turnover) by only 10%. The hormone increased the half-life of both types by 56 and 100% respectively. We conclude that GH feminizes the MFO by decreasing the synthesis of male-type cytochrome P450.     

ATP and ADP hydrolysis in fish, chicken and rat synaptosomes

Ecto-enzymes capable of hydrolyzing ATP and ADP (NTPDase) are present in the central nervous system of various species. In the present investigation we studied the synaptosomal NTPDase (ATP diphosphohydrolase, apyrase, E.C. 3.6.1.5) from fish, chicken and rats under different conditions and in the presence of several classical inhibitors. The cation concentration required for maximal activity was 0.5 mM for fish, 1.0 mM for chickens and 1.5 mM for rats with both substrates. The results showed that the pH optimum for all animal preparations was close to 8.0. The temperature used was 25–27°C for fish and 35–37°C for chicken and rat preparations. The inhibitors azide and fluoride only inhibited the preparation at high concentrations (10 mM). Lanthanum (0.1–0.4 mM), N-ethylmaleimide (0.4–3.0 mM) and ouabain (0.5–3.0 mM) had no effect on NTPDase activity from fish, chickens or rats. Orthovanadate (0.1–0.3 mM) only inhibited fish synaptosomal NTPDase. Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. Suramin was the most potent compound in causing inhibition, presenting inhibition at 30 μM. Our results demonstrate that the synaptosomal NTPDase response to several factors is similar in fish, chickens and rats, and that the enzyme presents functional homology.     

Adenosine Triphosphate and Other Requirements for the Utilization of Glucose by Agents of the Psittacosis-Trachoma Group.

Weiss, Emilio (Naval Medical Research Institute, Bethesda, Md.). Adenosine triphosphate and other requirements for the utilization of glucose by agents of the psittacosis-trachoma group. J. Bacteriol. 90:243-253. 1965.-The agent of meningopneumonitis cultivated in the allantoic cavity of chick embryos and purified by differential centrifugations was employed for most of the studies of the requirements for glucose utilization. The evolution of C(14)O(2) from glucose-1-C(14) was used as the criterion of metabolic activity in most experiments. The rate of glucose utilization increased somewhat during the first hour of incubation at 34.4 C and became approximately constant during the second hour. Changes in glucose concentration from 1 to 5 mm did not appreciably affect metabolic activity. More vigorous CO(2) production was obtained when the ratio of K(+)-Na(+) was >1 and, under certain conditions, when the concentration of inorganic phosphate was relatively high (0.05 m). Glucose utilization was entirely dependent on added adenosine triphosphate (ATP) and Mg(++). The effect of ATP was greatly reduced when the microorganisms were partially disrupted with sonic energy. Adenosine diphosphate (ADP) could be substituted for ATP, but the activity was reduced to less than 20%. ATP was not required when glucose-6-phosphate was substituted for glucose. With ADP and glucose, glucose-6-phosphate was an effective competitor of glucose utilization. Nicotinamide adenine dinucleotide phosphate (NADP) enhanced CO(2) production from carbon 1, but not from other carbons, with glucose and, especially, glucose-6-phosphate as substrates. ATP and NADP produced the above-described effects only when their concentrations were comparable to those of the substrates. These concentrations always exceeded the amount of CO(2) produced (0.05 to 0.5 mumole/mg of agent protein). The concentration of NADP could be reduced when oxidized glutathione was added. Diphosphothiamine had no effect on CO(2) production. Qualitatively similar results were obtained with the agent of trachoma purified from yolk sac. These experiments furnish evidence that agents of the psittacosistrachoma group, despite their enzymatic capabilities, require an exogenous source of energy.     

The effects of hydropsychid colonization on algal response to nutrient enrichment in a small Michigan stream, U.S.A.

1. We tested the hypothesis that the indirect effects of colonization by Hydropsyche spp. (Trichoptera: Hydropsychidae) may be greater than direct effects of nutrients on the benthic algal community growth. Two sets of nutrient-releasing substrates (a total of twenty-four) were deployed into a small pristine stream in northern Michigan. Each set was composed of four treatments replicated three times: (i) no nutrient enrichment (C), (ii) 0.5 M phosphate-P enrichment (P), (iii) 0.5 M nitrate-N enrichment (N) and (iv) 0.5 M phosphate-P plus 0.5 M nitrate-N enrichment (P + N). All hydropsychids colonizing on the substrate in one set (twelve substrates) were removed regularly and the other set (twelve substrates) with undisturbed hydropsychids served as the controls. 2. Algal biomass and gross primary productivity were estimated as chlorophyll a (chl a) concentration, algal biovolume, and carbon fixation rate, respectively. There was a significant interactive effect of hydropsychid colonization and P enrichment on algal biomass measured as chl a concentration. With removal of hydropsychids, chl a concentration increased 11-fold in the P enrichment treatments relative to the controls. The effects of P on chl a was, however, not significant in the presence of hydropsychids. Such interactive effects were not observed when algal responses were measured as biovolume and carbon fixation rate (GPP). 3. It is recommended that algal responses to nutrient enrichment should be measured as biovolume or carbon fixation rate in small streams where hydropsychids are commonly present.     

On the inhibition of microsomal drug metabolism by polychlorinated biphenyls (PCB) and related phenolic compounds.

The in vitro metabolism of p-nitroanisole, aminopyrine, and aniline by rat liver microsomal monoxygenases were studied in the presence of different polychlorinated biphenyl (PCB) mixtures and some related hydroxybiphenyls. The tested PCB mixtures contained preferably dichloro- (di-CB), tetrachloro- (tetra-CB), or hexachlorobiphenyls (hexa-CB). All PCB were competitive inhibitors of only aminopyrine demethylation by normal microsomes (Ki 22-39 micron). In microsomes of PCB-pretreated rats the aminopyrine demethylation was inhibited noncompetitively by di-CB and hexa-CB whereas tetra-CB remained a competitive inhibitor (Ki 12 micron). Moreover, after PCB pretreatment all PCB were competitive inhibitors of p-nitroanisole demethylation. 2-OH-biphenyl and 4-OH-biphenyl caused competitive inhibition of aminopyrine demethylation and aniline hydroxylation but failed to inhibit p-nitroanisole metabolism by normal microsomes. Chlorinated 4-hydroxybiphenyls inhibited competitively the metabolism of both type I and type II substrates. However, after PCB pretreatment all phenolic compounds caused uncompetitive inhibition of aniline hydroxylation.     

Effects of excess and deficiency of dietary methionine on mixed-function oxidase system of hepatic microsomes in male broilers.

1. Three experiments were conducted to determine the effect of dietary methionine levels on the hepatic microsomal mixed-function oxidase (MFO) system in male broiler chicks. 2. The level of dietary methionine for the highest growth coincided with that for the MFO activity. 3. Dietary methionine deficiency reduced MFO activity, but the reduction effect was not constant. 4. Dietary methionine excess also reduced MFO activity, even though growth rate was held at a maximum.     

Effect of lead on the reaction of O-demethylase in p-nitro anisole in the cladoceran Moina macrocopa

The presence of the Mixed-Function Oxydase (MFO) system, in Moina macrocopa was detected through the transformation of p-nitroanisole to para-nitrophenol. The presence of this enzymatic system suggested that this cladoceran participates in the biotransformation of xenobiotics in aquatic ecosystems. This capacity, in conjunction with the aquatic flea's high tolerance to environmental stress, suggested that M. macrocopa could be used in bioremediation efforts to increase ecosystem health. The effects of lead on the MFO system in M. macrocopa, were also studied. Lead acted as an inhibitor of the enzymatic complex. Therefore, induction of MFO-activity as an early warning may not work in waterbodies affected by both inducers of MFO and inhibitors like lead.     

黄绿绿僵菌Mf82悬乳剂对褐飞虱作用的时间-剂量-死亡率模型分析

为了寻找褐飞虱Nilaparvata lugens生物防治的新途径,用新分离出的黄绿绿僵菌Metarhiziumflavoviride(Mf82)菌株与实验室保存的黄绿绿僵菌、金龟子绿僵菌和球孢白僵菌3种菌种9个菌株作对比,测定了它们对褐飞虱成虫的毒力。结果表明:Mf82菌株对褐飞虱成虫的毒力最高,以1.0×108个孢子/mL的孢子液喷雾接种到褐飞虱成虫体表上,累积死亡率高达81.7%,LT50为4.6d,致病效果显著高于其他受测菌株。在此基础上研制了黄绿绿僵菌悬乳剂,并研究了其对褐飞虱的致病力。结果表明:随着黄绿绿僵菌浓度的增加,褐飞虱的累计死亡率增加,在浓度为1,048个孢子/mm2时,累计死亡率达到85.0%。利用时间-剂量-死亡率模型对数据进行处理,所建模型均顺利通过Hosmer-Lemeshow拟合异质性检验,表明模型拟合良好,并由模型估计出了该剂型对褐飞虱的致死剂量与致死时间。在接种后第7天和第9天,LC50值分别为2.1×103、9.9×102个孢子/mm2,LC90分别为7.8×104、3.7×104个孢子/mm2。黄绿绿僵菌悬乳剂对褐飞虱的致死时间与对数剂量相关,供试菌剂LT50值随着对数剂量的增加而递减,对数剂量由7.0增加到8.0时,LT50由8.9d降为5.7d。可见该黄绿绿僵菌悬乳剂对褐飞虱具有较强的毒力,在褐飞虱生物防治中具有广阔的应用前景。     

Characterization of full length and truncated type I human methionine aminopeptidases expressed from Escherichia coli

Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the initiator methionine and is recognized as a potential target for developing antibacterial, antifungal, and anticancer agents. We have established an Escherichia coli expression system for human type I MetAP (HsMetAP1) and characterized the full length HsMetAP1 and its N-terminal-truncated mutants HsMetAP1(Delta1-66) and HsMetAP1(Delta1-135) for hydrolysis of several thiopeptolide and peptide substrates and inhibition by a series of nonpeptidic inhibitors. Although the N-terminal extension with zinc finger motifs in HsMetAP1 is not required for enzyme activity, it has a significant impact on the interaction of the enzyme with substrates and inhibitors. In hydrolysis of the thiopeptolide substrates, a relaxation of stringent specificity for the terminal methionine was observed in the truncated mutants. However, this relaxation of specificity was not detectable in hydrolysis of tripeptide or tetrapeptide substrates. Several nonpeptidic inhibitors showed potent inhibition of the mutant HsMetAP1(Delta1-66) but exhibited only weak or no inhibition of the full length enzyme. With the recombinant HsMetAP1 available, we have identified several MetAP inhibitors with submicromolar inhibitory potencies against E. coli MetAP (EcMetAP1) that do not affect HsMetAP1. These results have demonstrated the possibility of developing MetAP inhibitors as antibacterial agents with minimum human toxicity. In addition, micromolar inhibitors of HsMetAP1 identified in this study can serve as tools for investigating the functions of HsMetAP1 in physiological and pathological processes.     

Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in Human P-glycoprotein. Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 micromol x min x mg(-1) with a K(m) for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K(m) for ATP or 8-azidoATP and K(i) for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [alpha-(32)P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. The K(m) for [alpha-(32)P]8-azidoATP (20 microm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [alpha-(32)P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, P(i) exhibits very low affinity for Pgp (K(i) approximately 30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [alpha-(32)P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.     

Modulation of the cytotoxicity and mutagenicity of benzo[a]pyrene and benzo[a]pyrene 7,8-diol by glutathione and glutathione S-transferases in mammalian cells (CHO/HGPRT assay)

Biologically reactive metabolites of benzo[a]pyrene (BP) and benzo[a]-pyrene 7,8-diol (BP-diol), formed by the mixed-function oxidase (MFO) system, are substrates for conjugation and detoxication by glutathione (GSH) when catalyzed by glutathione S-transferases (GSHT). We have investigated the detoxication of BP- and BP-diol-induced cytotoxicity and mutagenicity with GSH by supplementing the S9 mix used in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay with GSH (6.5 mM) or GSH plus GSHT. The addition of GSH to the S9 mix resulted in a reduction of BP- and BP-diol induced cytotoxicity. GSH plus GSHT eliminated BP-induced cytotoxicity and reduced the mutagenicity of BP. GSH inhibited the mutagenicity at low (essentially non-lethal) concentrations of BP-diol, but did not do so at toxic concentrations. GSH plus GSHT inhibited the cytotoxicity and mutagenicity of BP-diol at concentrations not affected by GSH alone. These studies indicate that biochemical mechanisms of detoxication can affect the biological activity of a carcinogen, such as BP or BP-diol as profoundly as bioactivation by the MFO system.     

Nitrogenase. VII. Effect of component ratio, ATP and H2 on the distribution of electrons to alternative substrates.

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL