The dissociation of flavin coenzymes from trypsin-solubilized NADPH/Cytochrome c (P-450) reductase of pig-liver microsomes.
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The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein. (+info)
NADPH-cytochrome P-450 reductase. Circular dichroism and physical studies.
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NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis after mineral or organic acid hydrolysis. The free mobility of cytochrome P-450 reductase in sodium dodecyl sulfate was identical to that of several soluble proteins used as standards (i.e. ovalbumin, bovin serum albumin, erythrocuprein, beta-galactosidase). Molecular weight estimates from sedimentation equilibrium studies in the presence of guanidine hydrochloride (76,500) are consistent with those determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at various per cent gel concentrations (79,000 to 80,000). Computer analysis of circular dichroism spectra of cytochrome P-450 reductase in the far ultraviolet region indicated the presence of 34 per cent alpha helical and 16 per cent beta structure. The amount of random structure was calculated to be 50 per cent. (+info)
Properties of the scrapie agent-endomembrane complex from hamster brain.
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Subcellular fractionation of scrapie-infected hamster brain indicated the association of the scrapie agent with a component of the endomembrane system. Characterization by equilibrium density gradient centrifugation, electron microscopy, and marker enzymes suggested a primary association with rough and smooth endoplasmic reticulum and a possible incorporation into the plasma membrane. DNA polymerase activity demonstrated a direct correlation with regions of scrapie activity from the gradient fractions. A scrapie-related product was detected after (3H)TMP incorporation and analysis on 2.2% polyacrylamide gels. Analysis of nucleic acid species extracted from subcellular fractions resulted in a greater quantity from healthy brain; however, no qualitative distinctions were detected. (+info)
Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes.
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In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function. (+info)
Localization on mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase.
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Four cytoplasmic mutants of Saccharomyces cerevisiae showing loss of mitochondrial rutamycin-sensitive ATPase activity but having significant cytochrome oxidase and NADH-cytochrome c reductase have been isolated. Genetic studies indicate the mutations to be closely linked to each other and have been assigned to a new locus, PHO1. The mutations show a low frequency of recombination with the OL12 locus, suggesting a linkage to this marker. They are not, however, linked to the OLI1 locus. Linkage of the ATPase mutations to the OLI2 locus is also indicated by restoration of wild-type diploids by sigma- clones that retain the segment of mitochondrial DNA carrying OLI2. Based on the recombinants issued from crosses of the mutants with a triple drug-resistant strain and an analysis of the resistance markers present in sigma- clones that are effective in restoring a wild-type phenotype, the PHO1 locus has been placed in the segment of DNA located between PAR1 and OLI2. (+info)
Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein.
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An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome. (+info)
Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochromes P-450.
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The interaction of trans-cinnamic acid with the cytochrome P-450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction. Evidence is presented that the trans-cinnamic acid interacts with the cytochrome P-450, and that this interaction is analogous to "type 1" interactions of other cytochrome P-450 systems. This evidence includes the formation of a "type 1" substrate binding spectrum, an increased rate of reduction of cytochrome P-450 by NADPH in the presence of trans-cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans-cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P-450 system and enzymological parameters of the trans-cinnamic acid 4-hydroxulation reaction. The investigation has been extended to cytochrome P-450 systems of other tissues and it has been found that the trans-cinnamic acid 4-hydroxylation reaction cannot account for the presence of most of th cytochrome P-450 in several tissues. This suggests that other functions of higher plant cytochrome P-450 chains exist, and that the substrate specificityof the hemoprotein may vary in different plant tissues. (+info)
Characterization of the role of lysine 110 of NADH-cytochrome b5 reductase in the binding and oxidation of NADH by site-directed mutagenesis.
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An expression vector for bovine NADH-cytochrome b5 reductase was used for site-directed mutagenesis of lysine 110, the residue previously implicated in NADH interactions with this flavoprotein. Replacement of this basic residue with an uncharged glutamine resulted in an increase of 3 orders of magnitude in the Km for NADH and a decrease in kcat of an order of magnitude, strongly implicating lysine 110 in both binding of NADH to the reductase and the orientation of the reduced nicotinamide group for rapid hydride ion transfer to the flavin. Substitution of lysine 110 by histidine, to provide a pH-sensitive positive charge at this position in the neutral pH range, exhibited only a moderate 25-fold increase in Km and a normal kcat at pH 6.0, whereas at pH 8.5 the Km for NADH rose to 238 microM with a decrease of 45% over unmodified enzyme in the kcat. A similar pH sensitivity in the inhibition constant for adenosine diphosphate ribose, lacking only the nicotinamide moiety of NADH, emphasizes the crucial role of the positive charge at this locus and is consistent with charge-pairing of lysine 110 with the pyrophosphate group of NADH or adenosine diphosphate ribose. (+info)