Phenazine Ethosulfate Research Articles

Site-Directed Mutagenesis of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus: Characterization of a Y114 → F Mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

Reactions of dimethylsulfoxide reductase in the presence of dimethyl sulfide and the structure of the dimethyl sulfide-modified enzyme.

The bis-molybdopterin enzyme dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus catalyzes the conversion of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), reversibly, in the presence of suitable e(-)-donors or e(-)-acceptors. The catalytically significant intermediate formed by reaction of DMSOR with DMS ('the DMS species') and a damaged enzyme form derived by reaction of the latter with O(2) (DMS-modified enzyme, DMSOR(mod)D) have been investigated. Evidence is presented that Mo in the DMS species is not, as widely assumed, Mo(IV). Formation of the DMS species is reversed on removing DMS or by addition of an excess of DMSO. Equilibrium constants for the competing reactions of DMS and DMSO with the oxidized enzyme (K(d) = 0.07 +/- 0.01 and 21 +/- 5 mM, respectively) that control these processes indicate formation of the DMS species occurs at a redox potential that is 80 mV higher than that required, according to the literature, for reduction of Mo(VI) to Mo(IV) in the free enzyme. Specificity studies show that with dimethyl selenide, DMSOR yields a species analogous to the DMS species but with the 550 nm peak blue-shifted by 27 nm. It is concluded from published redox potential data that this band is due to metal-to-ligand charge transfer from Mo(V) to the chalcogenide. Since the DMS species gives no EPR signal in the normal or parallel mode, a free radical is presumed to be in close proximity to the metal, most likely on the S. The species is thus formulated as Mo(V)-O-S(*)Me(2). Existing X-ray crystallographic and Raman data are consistent with this structure. Furthermore, 1e(-) oxidation of the DMS species with phenazine ethosulfate yields a Mo(V) form without an -OH ligand, since its EPR signal shows no proton splittings. This form presumably arises via dissociation of DMSO. The structure of DMSOR(mod)D has been determined by X-ray crystallography. All four thiolate ligands and Ogamma of serine-147 remain coordinated to Mo, but there are no terminal oxygen ligands and Mo is Mo(VI). Thus, it is a dead-end species, neither oxo group acceptance nor e(-)-donation being possible. O(2)-dependent formation of DMSOR(mod)D represents noncatalytic breakdown of the DMS species by a pathway alternative to that in turnover, with oxidation to Mo(VI) presumably preceding product release. Steps in the forward and backward catalytic cycles are discussed in relation to earlier stopped-flow data. The finding that in the back-assay the Mo(IV) state may at least in part be by-passed via two successive 1e(-) reactions of the DMS species with the e(-)-acceptor, may have implications in relation to the existence of separate molybdopterin enzymes catalyzing DMSO reduction and DMS oxidation, respectively.

Meiotic induction in cumulus cell-enclosed mouse oocytes: involvement of the pentose phosphate pathway.

In this study we tested the hypothesis that the pentose phosphate pathway (PPP) participates in the meiotic induction of mouse oocytes. The electron acceptors methylene blue, phenazine ethosulfate (PES), and pyrroline-5-carboxylate (P5C) oxidize NADPH to NADP and activate the NADP-dependent enzymes of the PPP. Each of these compounds triggered a dose-dependent increase in meiotic maturation in hypoxanthine-arrested cumulus cell-enclosed oocytes during 17- to 18-h cultures. More than 96% of the oocytes underwent germinal vesicle breakdown (GVB) at the highest concentrations of P5C and PES tested (250 and 1 microM, respectively) as compared to only 45-52% of control oocytes. P5C was also stimulatory to denuded oocytes. Analysis of energy substrates in microdrop cultures revealed a 3.6-fold increase in glucose consumption by PES-treated oocyte-cumulus cell complexes that was associated with stimulation of GVB. On the other hand, 2-deoxyglucose, which interferes with glucose utilization, prevented the induction of maturation brought about by P5C. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase, prevented meiotic maturation in the presence or absence of FSH. Gonadotropin-induced maturation was also prevented by 6-aminonicotinamide (6-AN) and dehydroepiandrosterone (DHEA), inhibitors of the two NADP-dependent enzymes of the PPP, and this was accompanied by suppression of glucose consumption. Phosphoribosyl-pyrophosphate (PRPP) is an important compound required in purine metabolism and can be formed from the end product of the oxidative arm of the PPP, ribose-5-phosphate. Ribose, which can be metabolized to PRPP, increased PRPP synthesis in complexes and induced meiotic maturation when added to hypoxanthine-arrested cumulus cell-enclosed oocytes in glucose-free medium in both the presence and absence of FSH. PRPP levels within complexes were also increased by glucose and FSH, but were reduced by hypoxanthine, 6-AN, and DHEA. In addition, exogenous PRPP stimulated maturation in hypoxanthine-arrested oocytes. These results support the proposition that glucose metabolism through the PPP is important in the meiotic induction mechanism and may involve the generation of PRPP that acts, at least in part, through the purine metabolizing pathways.

NAD+ Kinase Activities in Euglena Gracilis and Phaseolus Vulgaris

After an electrophoretic separation of proteins from Euglena gracilis and dry seeds of Phaseolus vulgaris in native conditions in polyacrylamide gels, gels were incubated in mixtures containing NAD+, Mg-ATP2-, glucose 6-phosphate, G6P dehydrogenase, and either phenazine ethosulfate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (PES/MTT) or phenazine methosulfate and nitro blue tetrazolium (PMS/NBT) as coupled redox system for NAD+ kinase activity detection. In the presence of PES/MTT, 4 bands were revealed for E. gracilis, among which two corresponded to NAD+ kinase activity, the other corresponding to a NAD+ reductase activity due to alcohol dehydrogenase (ADH). In the presence of PMS/NBT, only the bands of NAD+ kinase activity were revealed. With Phaseolus vulgaris, 3 bands of ADH were always revealed in both mixtures, and only the use of PMS/NBT allowed the detection of NAD+ kinase as a fourth band. With both materials, NAD+ reductase staining in gels was intensifed in the presence of GTP or ATP and even further with ADP or GDP. The results demonstrate that: 1) the NAD+ kinase and NAD+ reductase are two distinct enzymes; 2) the NAD+ reductase corresponds to ADH.

Induction of the SOS response and mutations by reactive oxygen-generating compounds in various Escherichia coli mutants defective in the mutM, mutY or soxRS loci.

Derivatives of E. coli WP2s (uvrA trpE) defective in 7,8-dihydro-8-oxoguanine (8-OG) DNA glycosylase activity (mutM), MutY glycosylase activity on an A:8-OG mispair (mutY), and/or an adaptive response to oxidative stress by superoxide (soxRS) were constructed to compare the mutability to various reactive oxygen-generating compounds. Induction of Trp+ reversion was assayed both in the presence and absence of plasmid pKM101. Phenazine methosulfate and phenazine ethosulfate showed mutagenic activity at a relatively low dose in soxRS mutants. In comparison to the parent strain WP2s, however, the introduction of mutM, mutY, or soxRS mutations, in any combination, did not make the strain hypersensitive in terms of mutability (i.e. mutation induction at relatively low doses) to hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or phenylhydrazine. Mutagenicity of formaldehyde was detected only in the pKM101-carrying strains. On the other hand, bleomycin, menadione, plumbagin, paraquat, and diquat were not mutagenic to any strain, with or without pKM101. The SOS-response inducing activity was measured by monitoring the expression of a umu'-'lacZ fusion gene, carried on the plasmid pSK1002. The induction of the SOS response by hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, formaldehyde, phenylhydrazine, and bleomycin was of almost the same magnitude between the parent strain and a mutM or soxRS mutant. Phenazine methosulfate and phenazine ethosulfate induced the SOS response only in the soxRS derivatives. No induction was detected by treatment with redox-cycling compounds, such as menadione, plumbagin, paraquat, or diquat.

A new kinetic model for the steady-state reactions of the quinoprotein methanol dehydrogenase from Paracoccus denitrificans

The reactions of methanol dehydrogenase from Paracoccus denitrificans with artificial electron acceptors, ammonia, cyanide, and substrates have been characterized by steady-state kinetic analysis. Phenazine ethosulfate, a commonly used electron acceptor for this enzyme, was shown to exhibit pronounced substrate inhibition with a K(i) value approximately 20-fold lower than its Km. Wurster's Blue exhibited only relatively mild substrate inhibition and was deemed a more appropriate electron acceptor. Ammonia was an obligatory activator of the enzyme at low concentrations and inhibited a high concentrations. The K(i) value for this inhibition correlated closely with the Kd calculated from a titration of perturbations of the absorption spectrum of methanol dehydrogenase which were caused by the addition of ammonia. Cyanide, which suppressed the peculiar endogenous reaction of methanol dehydrogenase, was also both an activator of substrate-dependent activity and a competitive inhibitor with respect to methanol. Kinetic analysis indicated that the latter two activities corresponded to two distinct binding sites for cyanide. The Ka for cyanide activation correlated closely with the concentration required to inhibit 50% of the endogenous reaction, suggesting that a single binding event is responsible for both of these effects. A model is presented to describe the effects of ammonia and cyanide in the reaction cycle of methanol dehydrogenase, and the physiological relevance of the activation and inhibition by these compounds in vitro is discussed.

Apparent oxygen-dependent inhibition by superoxide dismutase of the quinoprotein methanol dehydrogenase.

Methanol dehydrogenase activity, when assayed with phenazine ethosulfate (PES) as an electron acceptor, was inhibited by superoxide dismutase (SOD) and by Mn2+ only under aerobic conditions. Catalase, formate, and other divalent cations did not inhibit the enzyme. The enzyme also exhibited significantly higher levels of activity when assayed with PES under anaerobic conditions relative to aerobic conditions. The oxygen- and superoxide-dependent effects on methanol dehydrogenase were not observed when either Wurster's Blue or cytochrome c-55li was used as an electron acceptor. Another quinoprotein, methylamine dehydrogenase, which possesses tryptophan tryptophylquinone (TTQ) rather than pyrroloquinoline quinone (PQQ) as a prosthetic group, was not inhibited by SOD or Mn2+ when assayed with PES as an electron acceptor. Spectroscopic analysis of methanol dehydrogenase provided no evidence for any oxygen- or superoxide-dependent changes in the redox state of the enzyme-bound PQQ cofactor of methanol dehydrogenase. To explain these data, a model is presented in which this cofactor reacts reversibly with oxygen and superoxide, and in which oxygen is able to compete with PES as an electron acceptor for the reduced species.

Methyl viologen as a preferred electron acceptor in metabolic experiments

Widely used artificial electron acceptors, including dichlorophenol-indophenol, methylene blue, and phenazine ethosulfate, were shown to induce the nonenzymatic decarboxylation of pyruvate. This characteristic rendered these electron acceptors unsuitable for use in metabolic experiments. In contrast, methyl viologen stimulated the pentose phosphate pathway in mouse preimplantation embryos without decarboxylating pyruvate in the process. Furthermore, methyl viologen had no effect on the Embden-Myerhoff pathway and was not overtly toxic to mouse embryos. These features made methyl viologen a preferred artificial electron acceptor for metabolic studies.

L-pipecolic acid oxidation in the rabbit and cynomolgus monkey: Evidence for differing organellar locations and cofactor requirements in each species

L-Pipecolic acid oxidation was studied in the rabbit and cynomolgus monkey. Tissue homogenates from both species incubated with L-[2,3,4,5,6-3H]pipecolic acid produced a single radioactive product identified as alpha-aminoadipic acid. In the rabbit, L-pipecolic acid oxidation was greatest in kidney cortex with progressively lesser specific activities in liver, heart, and brain. When rabbit kidney cortex was fractionated by differential centrifugation or on Percoll gradients, activity paralleled that of the mitochondrial marker, glutamate dehydrogenase. In sonicated mitochondria, 92% of the activity was in the soluble fraction. Activity was inhibited by both rotenone and antimycin A and was maximal when FAD, phenazine ethosulfate, and glycerol were included in the assay; Km,app was 0.74 +/- 0.16 mM. Nipecotic acid, piperidine, and cis-2,4-piperidine dicarboxylic acid did not inhibit L-pipecolic acid oxidation, while L-proline had a Ki greater than or equal to 10 mM. D-Alanine and kojic acid, substrate and inhibitor of D-amino acid oxidase, respectively, were also not inhibitory. When monkey kidney cortex was fractionated on Percoll gradients, L-pipecolic acid oxidation activity paralleled that of the peroxisomal marker, catalase. After organellar subfractionation, the activity was membrane-associated and maximal at pH 8.5; Km,app was 4.22 +/- 0.30 mM. L-Pipecolic acid oxidation produced hydrogen peroxide, suggesting involvement of an oxidase in alpha-aminoadipic acid formation. Antimycin A did not inhibit the reaction. No specific cofactor requirements were identified and phenazine ethosulfate inhibited the reaction. D-Pipecolic acid, L-proline, and the other compounds cited above did not significantly inhibit the activity.

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1.

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.

Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans.

Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. The enzyme was able to catalyze the oxidation of a wide variety of primary aliphatic amines and diamines, but it did not react with secondary, tertiary, or aromatic amines. The enzyme exhibited optimal activity at pH 7.5, with Km values of 12.5 microM for methylamine and 156 microM for phenazine ethosulfate and a Vmax of 16.9 mumol/min per mg of protein. No loss of enzyme activity was observed after incubation for 48 h at pH values ranging from 3.0 to 10.5, and the enzyme was very stable to thermal denaturation. Enzyme activity and immunological detection of each subunit were only observed with cells which had been grown on methylamine as a carbon source.

A photosynthetic photoelectrochemical cell using phenazine methosulfate and phenazine ethosulfate as electron acceptors

Several recent studies have demonstrated that photosystem I (PSI), one of the two light-active complexes of photosynthesis, can be used as a light transducer in a biological photoelectrochemical cell. This paper examines the results of using phenazine methosulfate (PMS) and phenazine ethosulfate (PES) as an electron acceptor in such a cell. The PMS and PES have relatively high formal potentials compared to flavin mononucleotide (FMN) and other acceptors used in the past, yet the PMS and PES resulted in power outputs and conversion efficiencies second only to the use of FMN as an acceptor. The mechanism of action has been interpreted in terms of electroactive products of parent compounds formed during the normal function of the cell under illuminated conditions. For example, photolysis and cyclic voltammetry data demonstrate that pyocyanin (Py) [formal potential = -0.37 vs saturated calomel electrode (SCE) at pH 8.5], the photoproduct of PMS, is the electroactive species in cells containing PMS (formal potential = -0.19 vs SCE at pH 8.5). Similar phenomena were observed for PES and FMN. The power output of the cell results from about equal contributions from a cyclic photosynthetic component and a component caused by the direct photoreduction of the acceptor and reoxidation by sacrificial donors in the buffer. Future research directions are discussed in terms of designing cells that function purely in the photosynthetic or photochemical modes.

Xanthine oxidase from human liver: Purification and characterization

Xanthine oxidase [EC 1.2.3.2] was purified 2000-fold from human liver. The last step of the procedure involved affinity chromatography. The resulting preparation showed two closely migrating bands of enzyme activity after gel electrophoresis under nondenaturing conditions. No other proteins were detected on these gels. The average particle mass of the enzyme was 300 kDa as determined by size-exclusion chromatography. This together with results of gel electrophoresis under denaturing conditions suggested that the native enzyme was composed of two subunits of approximately 150 kDa each. The electrophoretic patterns also indicated that a portion of these subunits had undergone partial proteolysis. The substrate specificity of the purified human enzyme was studied using an assay in which phenazine ethosulfate coupled the transfer of electrons from the reduced enzyme to cytochrome c. Hypoxanthine, 2-hydroxypurine, xanthine, 2-aminopurine, and adenine were among the most efficient purine substrates studied. Most purine nucleosides tested were oxidized at detectable rates, but with relatively high K m values. The 2′-deoxyribonucleosides were more efficient substrates than were the corresponding ribonucleosides or arabinonucleosides. In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates. However, considerable differences between the bovine and human enzymes were observed with nucleoside substrates. With xanthine as the substrate for the human enzyme, 20% of the total electron flow was univalently transferred to oxygen to produce superoxide radicals.

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.

Quantitative determination of the pentose phosphate pathway in preimplantation mouse embryos.

A quantitative calculation was made of the pentose phosphate pathway (PPP) activity in preimplantation mouse embryos from the 2-cell through the late blastocyst stage. This activity varied with development and showed repeated high and low values. Peak activities occurred at both the 2-cell (15.8%) and compacted morula (13.6%) stages, with lowest activity at the development of the late blastocyst (3.2%). The metabolic effectors dimitrophenol (DNP) and phenazine ethosulfate (PES) had opposite effects on PPP activity. Dinitrophenol, although stimulating total CO2 production, virtually eliminated PPP activity while PES stimulated the pentose cycle activity 6-fold. These results indicated that the PPP was under metabolic control and that the embryos had a potential for much larger PPP activities. There was no correlation between the C-1/C-6 ratio obtained from the metabolism of [1-14C] and [6-14C] glucose and calculated PPP activities. A metabolic incubation chamber was devised for these experiments that exhibited certain unique features, including continuous collection of 14CO2 and 3H2O. Single embryos were placed in the chamber and sampled momentarily for metabolic activity. Subsequently, such embryos were successfully transferred to pseudopregnant recipients.

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (pI= 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.

Purification of membrane-bound polyvinyl alcohol oxidase inPseudomonassp. VM15C

Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (pI = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.

Kinetics of yeast alcohol dehydrogenase and its coenzyme coimmobilized in a tubular flow reactor

Yeast alcohol dehydrogenase and nicotinamide adenine dinucleotide (NAD) were coimmobilized, with covalent attachment, to the interior surface of a nylon tube. The NAD was attahed at the N(6) group of the adenine moiety; an NAD derivative was prepared and attached to free carboxyl groups at a partially hydrolyzed nylon surface. The enzyme was attached, through glutaraldehyde residues, to free amino groups on the surface. Kinetic studies were carried out in which the reduced NAD was recycled by means of phenazine ethosulfate and 2,6-dichlorophenol indophenol. The reaction was studied over a range of flow rates and ethanol concentrations. The variation of rate with flow rate suggested that there was little diffusion control with respect to ethanol and that there was no observable inhibition by the reaction products. These conclusions were confirmed by evidence based on dimensionless parameters for the reaction and by direct inhibition experiments. The apparent Michaelis constant was lower than when only the enzyme was immobilized, suggesting that the immobilized enzyme-coenzyme system is of high efficiency. Overall rates of reaction were lower than when there was saturation with NAD. The tube showed no measurable loss of catalytic activity over a period of one month.

Mixed function oxidase in the mammary gland and liver microsomes of lactating rats: Effects of 3-methylcholanthrene and β-naphthofla vone

Mammary gland and liver microsomes of lactating rats were examined for the components of mixed function oxidase and related enzyme activities. Cytochrome b 5, NADH- and NADPH-dependent cytochrome c reductase activities were 15-, 6- and 10-fold lower, respectively, in the mammary gland than in the liver microsomes. The determination of cytochrome P-450 (P-448) in the mammary gland microsomes required elimination of the spectral interferences by hemoglobin and cytochrome aa 3. The presence of the latter in this fraction was also shown by cytochrome c oxidase activity. Cytochrome aa 3 was reduced by anaerobic incubation of mammary gland microsomes, in the presence of antimycin A, with sodium succinate, phenazine ethosulfate, and sodium ascorbate for 30 min at room temperature. Spectral resolution of the dithionite-reduced cytochrome P-450 (P-448) carbon monoxide complex occurred 30 min after gassing. The basal level of cytochrome P-450 was about 500-fold greater in the liver than in the mammary gland microsomes. Pretreatment of lactating rats with the inducers of hepatic cytochrome P-448, 3-methylcholanthrene and β-naphthoflavone, increased the cytochrome content 3- to 10-fold and 2-fold, in the mammary gland and liver microsomes, respectively. The induction of cytochrome P-448 in microsomes of both tissues was also shown by type I binding spectra obtained with N-2-fluorenylacetamide. Using hydroxylation of benzo[ a]pyrene and N-2-fluorenylacetamide as a measure of mixed function oxidase activity, we found that the basal activities, which were 4- to 8-fold greater in the liver microsomes, were increased in both tissues after treatment of rats with the inducers. The induced activities were inhibited by 0.1 mM α-napthoflavone in vitro, indicating a dependence on cytochrome P-448. The data suggest that the mammary gland, an extrahepatic target for carcinogens, is capable of their metabolism.

Phenazine ethosulfate as a preferred electron acceptor to phenazine methosulfate in dye-linked enzyme assays

Rapid nonenzymatic reduction of 2,6-dichlorophenolindophenol by N-methyl phenazonium methosulfate has been observed in aqueous solution and has been found to increase with increasing pH and ionic strength. The instability of N-methyl phenazonium methosulfate in aqueous solution has been explored in terms of change of absorption spectrum and formation of free radicals as evidenced by EPR spectroscopy. N-Ethyl phenazonium ethosulfate has been found to be much more stable than the methyl analog and did not reduce dichlorophenolindophenol nonenzymatically. The implications of these findings with respect to use of these dyes as artificial electron acceptors are discussed and the recommendation made that, wherever possible, use of N-methyl phenazonium methosulfate be discontinued in favor of use of the N-ethyl analog.