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Biomedical subjects

D J Reed

Publications and source records attributed to D J Reed.

At least 91 records · Page 5Linked to original sources
PubMed1986

Defense mechanisms of normal and tumor cells.

This paper reviews the protective systems of normal and tumor cells against chemical and radiation injury. The glutathione redox cycle is an important cell defense system that can be compromised by various chemical modifiers. Acute cell injury can involve the glutathione pools of both the cytosol and the mitochondria. Intracellular calcium may have a role in cell death following acute cell injury but extracellular calcium does not seem to initiate the events leading to cell death. Changes in the glutathione redox status affects the distribution of intracellular calcium and the protein thiol-disulfide redox status. Formation of glutathione protein-mixed disulfides is discussed in terms of a possible protective mechanism against oxidative injury.

Animals↗
PubMed1985

Interactions of S-(2-haloethyl)-mercapturic acid analogs with plasmid DNA.

A series of related S-(2-haloethyl)-L-cysteine analogs were synthesized and their interaction with DNA was studied with plasmid pBR322. Both S-(2-chloroethyl)-L-cysteine (CEC) and S-(2-bromoethyl)-L-cysteine (BrEC) rapidly induced relaxation of the supercoiled plasmid as determined by agarose gel electrophoresis and electron microscopy, whereas S-(2-fluoroethyl)-L-cysteine did not interact with DNA. The relaxation was most probably due to strand scission at alkylated labile sites in the DNA. When 35S-labeled CEC or BrEC was used as the substrate, covalent binding of 35S to DNA was obtained; CEF displayed a somewhat higher binding than BrEC. No binding of 35S was obtained with (2-hydroxyethyl)-L-[35S]cysteine, [35S]cysteine, or [35S]cystine, substrates which did not induce relaxation of the DNA. Esterification of the carboxyl group resulted in a somewhat lower rate of DNA strand scission, whereas N-acetylation prevented the cysteine analogs from inducing DNA strand breaks. S-(2-Chloroethyl)-glutathione (GSH) did not interact with DNA as determined by lack of effect on the superhelicity of DNA, a finding which is in agreement with the hypothesis that the primary amine groups of CEC or BrEC may participate in the formation of reactive intermediates which can interact with DNA. S-(2-Hydroxyethyl)-GSH and S-(2-hydroxyethyl)-L-cysteine were unable to induce DNA strand breaks. Neutral denaturation of supercoiled pBR322 treated with the analogs revealed that compounds which were able to induce DNA strand breaks also interfered with denaturation of double-stranded circular DNA. No such interference was observed when double-stranded linear DNA (obtained by BamH1 restriction digestion) was treated with the analogs prior to denaturation. These data indicate that a marked difference exists between S-(2-chloroethyl)-L-cysteine and S-(2-chloroethyl)-glutathione in their reaction with supercoiled plasmid DNA. Either a major difference exists in the reactivity of the corresponding episulfonium ions of these conjugates or a separate mechanism of alkylation based on a free alpha-amino of the cysteine conjugate is participating in DNA strand breakage and possible crosslinking. In vivo toxic effects of these S-(2-chloroethyl) conjugates are predicted to be distinctly different.

Alkylation↗
PubMed1985

Glutathione changes occurring after S-adenosylhomocysteine hydrolase inhibition.

Freshly isolated rat hepatocytes, which metabolize methionine through the cystathionine pathway, and cultured L5178Y cells, which do not, were compared for their response to the inhibition of S-adenosylhomocysteine (SAH) hydrolase (EC 3.3.1.1). When cells were incubated in Fischer's medium lacking cystine but containing 0.67 mM methionine and 10% serum, the addition of periodate-oxidized adenosine (POA), an inhibitor of SAH hydrolase, increased the level of SAH approximately 4-fold in L5178Y cells (5 mM POA) and 30-fold in hepatocytes (1 mM POA). POA treatment also decreased the amount of intracellular glutathione (GSH) in hepatocytes by 6-fold, and in L5178Y cells by 3-fold. Incubation of hepatocytes with adenosine plus homocysteine, 2-chloroadenosine, or 2',3'-acyclic adenosine increased intracellular SAH and also lowered GSH levels. Neither GSH oxidation nor efflux of GSH or GSH conjugates appeared to account for the GSH loss. Intracellular GSH, covalently bound to proteins as mixed disulfides, increased when hepatocytes were incubated with POA, but the increase was insufficient to account for the total GSH loss. In hepatocytes with prelabeled [35S]GSH, POA caused the cellular GSH content to decrease while the specific activity of [35S]GSH remained constant, suggesting that inhibitor treatments that caused elevated SAH levels may have increased the degradation of GSH while GSH synthesis was inhibited.

Adenosine↗
PubMed1985

Mechanism of chemical-induced toxicity. I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes.

A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death. This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells. In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis. Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds. As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable. The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced. The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS. These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K+ and glutathione concentrations. Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment. These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult.

Animals↗
PubMed1985

Mechanism of chemical-induced toxicity. II. Role of extracellular calcium.

Previous studies disagree as to if chemical-induced cell death is caused by the influx and accumulation of extracellular Ca2+. To determine the role of extracellular Ca2+ in toxic cell death, the viability (leakage of intracellular K+ and lactate dehydrogenase) and total Ca2+ content of isolated hepatocytes incubated in the presence or absence of extracellular Ca2+ were determined during a toxic insult with bromobenzene, ethyl methanesulfonate (EMS), Ca2+ ionophore A23187, and adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The present study utilized the dibutyl phthalate separation technique which enabled the analysis of only viable hepatocytes for changes in intracellular Ca2+ and K+ content during toxic cell injury. The three chemical treatments, bromobenzene, EMS, and ADR-BCNU, each caused an accelerated loss of viability in hepatocytes incubated without extracellular Ca2+ as compared to cells incubated with Ca2+. Furthermore, the total Ca2+ content of viable hepatocytes incubated in the presence of extracellular Ca2+ did not increase during chemically induced cell injury as compared to control cells. In fact, a significant decline in total cellular Ca2+ was observed in viable hepatocytes incubated in Ca2+-free medium during toxic cell injury. Treatment with Ca2+ ionophore A23187 was also toxic to hepatocytes incubated in the presence or absence of extracellular Ca2+. At high concentrations of ionophore (20 microM or 4 micrograms/10(6) cells), cell death was accelerated in hepatocytes incubated with Ca2+ as compared to cells incubated in Ca2+-free medium. In contrast, after treatment with lower concentrations of ionophore (10 microM or 2 micrograms/10(6) cells), the rate of cell death was reversed with hepatocytes incubated without extracellular Ca2+ dying first. Thus, depending on the concentration of A23187 and the time of exposure, the presence of extracellular Ca2+ can be shown either to accelerate or protect against cell death. Surprisingly, reversible and irreversible cell injury were not observed in hepatocytes incubated with extracellular Ca2+ and 2 microM A23187 though this treatment resulted in an 800% increase in total intracellular Ca2+ content. We conclude that chemical-induced hepatic cell death is not caused by an increase in total cellular Ca2+ resulting from the influx of extracellular Ca2+.

Animals↗
PubMed1985

Buthionine sulfoximine inhibition of cystine uptake and glutathione biosynthesis in human lung carcinoma cells.

Intracellular glutathione (GSH) content of human lung carcinoma cells, A549, in log phase was 25 +/- 5 nmol/10(6) cells, which is considerably higher than that reported in other tumor cells. After partial depletion of GSH with diethyl maleate (DEM), addition of cystine to the medium allowed full resynthesis of GSH in 4 hr, cysteine in the same time period led to less resynthesis, and methionine provided minimal resynthesis. Using cystine as the sole sulfur source and with buthionine sulfoximine (BSO, 5 mM) included in the medium after cells were depleted with DEM, inhibition of both cystine uptake and resynthesis of GSH occurred. BSO inhibited [35S]cystine uptake (as early as 10 min) in a concentration-dependent process, ranging from a 28% decrease for 1 microM BSO to an 85% decrease for 100 microM BSO compared to the control cells after 240 min of incubation. In addition, GSH resynthesis from [35S]cystine for 240 min was inhibited in a parallel dose-dependent manner, in that 1 microM BSO caused a 27% decrease and 100 microM BSO provided a 75% decrease from control values. BSO did not inhibit the uptake of [35S]methionine, but inhibited the low amount of resynthesis of GSH when methionine was the sole sulfur source. BSO did not inhibit the uptake of arginine, phenylalanine, and leucine. DL-, L-, and methyl ester-BSO each inhibited [35S]cystine uptake and incorporation into GSH to a similar extent. The half-life of GSH was 3.5 +/- 0.4 hr in A549 cells that were grown in complete medium with GSH synthesis occurring.

Buthionine Sulfoximine↗
PubMed1985

Vitamin E reversal of the effect of extracellular calcium on chemically induced toxicity in hepatocytes.

Isolated rat hepatocytes were incubated in the presence or absence of extracellular calcium and alpha-tocopherol succinate with three different toxic chemicals; namely, adriamycin in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea, ethyl methanesulfonate, and the calcium ionophore A23187. In the absence of extracellular calcium these three compounds were far more toxic to the cells than in its presence. The addition of vitamin E to calcium-free medium, however, protected hepatocytes against toxic injury, whereas cells incubated in medium containing calcium were not protected. Hepatocyte viability during each toxic insult correlated well with the cellular alpha-tocopherol content but not with the presence or absence of extracellular calcium. These results suggest that cellular alpha-tocopherol maintains the viability of the cell during a toxic insult and that the presence or absence of vitamin E in the incubation medium probably explains the conflicting reports on the role of extracellular calcium in toxic cell death.

Animals↗
PubMed1985

Characterization of the growth inhibition induced by tumor-promoting phorbol esters and of their receptor binding in A549 human lung carcinoma cells.

Exposure of A549 human lung carcinoma cells to 10(-8) M 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a change in cell morphology and caused the arrest of cell growth. After 4-5 days of exposure to TPA the cells started to proliferate again. However, on removal of the cells from the culture flask and reseeding, the cells had regained their sensitivity towards TPA. Cells which were subcultured in the presence of 10(-8) M TPA for 9 weeks were permanently refractory to the growth-inhibitory properties of TPA. Incubation of A549 cells with [3H]phorbol-12,13-dibutyrate ([ 3H]PDB) showed that the cells possess specific phorbol ester receptors. Exposure of the cells to 10(-7) M PDB preceding the receptor binding assay led rapidly to a decline in the binding of 6 nM [3H]PDB, in case of preincubation for 24 h to 38% of the binding in cells not pre-exposed to PDB. The receptor binding capacity after pretreatment with PDB was only weakly decreased in the cells which were desensitized towards the TPA-induced growth inhibition. Thus the decrease in receptor binding on exposure to phorbol esters does not appear to cause the refractoriness of the cells towards the effect of TPA. It seems more likely that this decrease in binding capacity is part of the events by which phorbol esters cause inhibition of cell growth.

Caenorhabditis elegans Proteins↗
PubMed1985

Oxidative properties of 12-O-tetradecanoylphorbol-13-acetate-stimulated human blood monomorphonuclear leukocytes and their toxicity against a human lung carcinoma cell line.

Human monomorphonuclear leukocytes (MMNs) stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) were found to be toxic towards human A549 lung carcinoma cells which have been desensitized against the direct growth-inhibitory effect of TPA. This toxicity was dependent on the TPA concentration and the ratio of MMNs to A549 cells. Using a TPA concentration of 10(-7) M and an effector:target cell ratio of 30:1, experiments were performed to give clues as to the mechanisms by which TPA-stimulated MMNs cause toxicity. Levels of the endogenous thiol glutathione were reduced by 37% in MMNs exposed to TPA for 24 h, but the glutathione levels in the A549 target cells were not markedly affected by TPA-stimulated MMNs. The supernatant of incubations of MMNs with TPA contained a species which was capable of oxidizing the thiol agent 5-thio-2-nitrobenzoic acid. Within 2 h, 9 nmol of this oxidant were produced by 10(7) MMNs. The oxidant exhibited a half-life of 20 h, and its formation was abolished by adding catalase (150 units/ml), azide (1 mM), or cyanide (1 mM) to the incubations of MMNs with TPA. The addition of superoxide dismutase (100 units/ml) enhanced oxidant formation. These results indicate that its generation was dependent on the myeloperoxidase:H2O2:halide system. Large amounts of an oxidizing species with properties identical to those described here have been characterized recently in polymorphonuclear leukocytes [S. J. Weiss, M. B. Lampert, and S. T. Test. Science (Wash. DC), 222: 625-627, 1983]. The toxicity exerted by TPA-stimulated MMNs was partially inhibited by superoxide dismutase and by retinoic acid (30 microM) but not at all by catalase, azide, or cyanide. Therefore, the 5-thio-2-nitrobenzoic acid oxidant does not appear to be involved in the process which led to cytotoxicity by TPA-stimulated MMNs in A549 cells.

Cell Line↗
PubMed1984

Extracellular calcium protects isolated rat hepatocytes from injury.

The incubation of isolated rat hepatocytes in calcium-free medium resulted in a pronounced increase in lipid peroxidation, mitochondrial and cytoplasmic glutathione depletion, glutathione disulfide formation and efflux of reduced glutathione as compared with hepatocytes incubated in calcium containing medium. These data suggest that extracellular calcium ions serve a protective role in isolated rat hepatocytes against cell injury.

Animals↗
PubMed1984

Sizes, laminar and topographic origins of cortical projections to the major divisions of the red nucleus in the monkey.

The retrograde transport of horseradish peroxidase was used to study the topographic and laminar origins of the cortical projections to the parvocellular and the magnocellular divisions of the red nucleus in Macaca mulatta and Macaca fascicularis. Approximately 90% of the corticorubral projection is directed to the parvocellular division of the nucleus. Corticoparvocellular (CRp) neurons are pyramidally shaped, are smaller in size than corticospinal neurons, and are more numerous. They are found principally in sublamina Va of cytoarchitectonic areas 4 and 6, and in moderate quantities in sublamina Vb of posterior area 8 and area 5. In areas 4 and 6, the cells are grouped in clusters of three to 15 neurons each and are arranged in cellular bands of varying rostrocaudal thickness which course mediolaterally. With respect to functionally defined zones, CRp neurons are found throughout the supplementary motor area and the precentral motor cortex. In addition, they are found in parts of areas 5, 6, and 24 that project to these cortical motor areas, and that are thought to have "premotor" or movement-programming functions. The corticomagnocellular (CRm) projection arises principally from cells in sublamina Vb of the precentral arm and leg areas (area 4), and from adjacent parts of posterior area 6, CRm cells are pyramidally shaped, and their size distribution is bimodal, with peaks that correspond, respectively, to the modal diameters of CRp and of corticospinal neurons. These results and those of previous studies suggest that CRm neurons are involved principally in the control of hand and foot movements, with little effect on more proximal musculature. The massive CRp projection, however, is clearly part of a large cerebrocerebellar communication system, with motor and/or movement programming functions that have yet to be clearly defined.

Animals↗
PubMed1984

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes.

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an alpha-hydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.

Animals↗
PubMed1984

A sequential pulse generator for producing true biphasic stimuli.

The ability to generate biphasic pulses during electrical stimulation of nervous tissue has important advantages over monophasic or capacitively coupled stimulation. A comparatively simple circuit is described which, when used with standard electrophysiological laboratory equipment, can economically implement biphasic stimulation. The resultant system is quite flexible, yet easy to operate.

Electric Stimulation↗
PubMed1984

Measurement of glutathione-protein mixed disulfides.

We have undertaken the development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Treatment with 25 mM dithiothreitol (DTT) in 50 mM N-morpholinopropane sulfonic acid buffer, pH 8.0, reduces disulfides on the protein, and free thiols resulting from this procedure are analyzed by HPLC. We have found up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in our chromatography system with a retention volume similar to cysteine. Future experiments concerning this unidentified thiol are in progress.

Animals↗
PubMed1983

The hydrolysis and alkylation activities of S-(2-haloethyl)-L-cysteine analogs--evidence for extended half-life.

A series of S-(2-haloethyl)-L-cysteine derivatives, which are analogs of the proposed glutathione half-mustard metabolites of dihaloethanes, were synthesized and studied with respect to their hydrolysis and alkylation rates in aqueous solution. The trend of relative hydrolysis rates, Br greater than Cl much greater than F, paralleled their respective leaving group abilities; however, a dramatic rate increase was seen at pH 8 versus pH's 6 or 4. Hydrolysis of S-(2-chloroethyl)-L-cysteine analogs, where the ionizable groups were blocked (carboxyl esterified and/or N-acetylated), revealed that the amine moiety was responsible for the increased hydrolysis of mustard gas (beta, beta'-dichlorodiethyl sulfide) gave similar results with S-(2-chloroethyl)-L-cysteine, a finding which is consistent with the reaction intermediate being a highly charged species. The alkylation rates with 4-(p-nitrobenzyl)-pyridine were not affected by blocking the ionizable groups. A mechanism of internal cyclization is proposed to explain the accelerated alkaline hydrolysis rates noted with S-(2-haloethyl)-L-cysteines but not with the N-acetylated analogs (mercapturic acids). This scheme proposes the formation of 3-(thiomorpholine)-carboxylic acid as an alternative pathway to the generally accepted hydrolysis reaction. This compound and not S-(2-hydroxyethyl)-L-cysteine was the identified product following pH 10 hydrolysis. Increased hydrolysis half-time of amine-blocked cysteine analogs versus parent cysteine analogs may exist with S-(2-haloethyl)-glutathione derivatives which may explain the substantial nucleic acid alkylation seen with S-(2-haloethyl) derivatives of glutathione.

Alkylation↗
PubMed1983

Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links.

Plasmid [3H]pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS). Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands. All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion. Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA. Electron microscopy of this band showed that it consisted of relaxed circles. Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites. A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA. The level of alkylation was quantitated by incubating [3H]pBR 322 with [2-chloroethyl-U-14C]chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration. Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA. Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.

Alkylation↗
PubMed1983

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase.

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.

Anaerobiosis↗