Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CYTOCHROMES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 181 records · Page 10Linked to original sources
PubMed1987

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions.

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.

Animals↗
PubMed2004

Expression, purification, and characterization of the CuA-cytochrome c domain from subunit II of the Bacillus subtilis cytochrome caa3 complex in Escherichia coli.

Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.

Bacillus subtilis↗
PubMed1999

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver.

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.

Animals↗
PubMed1987

Cytochrome P-450 related to P-4504 from phenobarbital-treated rabbit liver: molecular cloning of cDNA and characterization of cytochrome P-450 obtained by its expression in yeast cells.

cDNA complementary to mRNA coding for a minor form of cytochrome P-450 from phenobarbital-treated rabbit liver (pHP3) was isolated using cDNA for the major phenobarbital-inducible cytochrome P-450 of rat liver as a probe in the first screening of a cDNA library. The nucleotide sequence of pHP3 was determined and contained a continuous reading frame encoding 490 amino acids. The deduced amino acid sequence of pHP3 protein exhibited about 50% homology with the major cytochrome P-450 from phenobarbital-treated rabbit liver, while the homology was as high as 80% between two minor cytochrome forms, pHP2 and pHP3. Two expression plasmids, pAHF3 and pAH delta N3, were constructed by insertion of pHP3 fragment between yeast alcohol dehydrogenase 1 (ADH1) promoter and terminator regions. pAHF3 contained the entire coding sequence of pHP3, but nucleotide sequences for the N-terminal region of pHP3 protein (from the 2nd to the 3rd amino acid) were deleted in pAH delta N3. On introduction of the constructed plasmids into Saccharomyces cerevisiae AH22 cells, the absorption spectrum of cytochrome P-450 was detected in the microsomal fraction from the transformed cells carrying pAHF3. On the other hand, cytochrome P-450 could not be detected spectrophotometrically in any subcellular fractions from the yeast cells carrying pAH delta N3, although the transcript of pHP3 insert was detected in RNA blot analysis. These results suggest that the N-terminal region of pHP3 protein plays an important role in accumulation of the newly synthesized pHP3 protein in yeast cells. Cytochrome P-450 (pHP3) was solubilized from microsomal membranes of the transformed yeast cells and purified partially on an aminooctyl Sepharose column (specific content, about 6 nmol per mg of protein). In the oxidized state the cytochrome preparation exhibited an absorption spectrum characteristic of a low-spin ferric cytochrome P-450. The reduced CO complex of the cytochrome showed a Soret absorption maximum at 450 nm. The monooxygenase activity of cytochrome P-450 (pHP3) was examined in a reconstituted system containing the cytochrome preparation and NADPH-cytochrome P-450 reductase. Cytochrome P-450 (pHP3) catalyzed N-demethylation of benzphetamine and aminopyrine and denitrification of 1-nitropropane. Addition of cytochrome b5 to the reconstituted system resulted in stimulation of the N-demethylation activities but inhibition of the denitrification activity. Neither 7-ethoxycoumarin O-deethylation activity nor acetanilide p-hydroxylation activity was detected, either in the presence or absence of cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Membrane-bound cytochrome c is an alternative electron donor for cytochrome aa3 in Nitrobacter winogradskyi.

We purified membrane-bound cytochrome c-550 [cytochrome c-550(m)] to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The cytochrome showed peaks at 409 and 525 nm in the oxidized form and peaks at 416, 521, and 550 nm in the reduced form. The molecular weight of the cytochrome was estimated to be 18,400 on the basis of protein and heme c contents and 18,600 by gel filtration. The N-terminal amino acid sequence of cytochrome c-550(m) was determined to be A-P-T-S-A-A-D-A-E-S-F-N-K-A-L-A-S-A-?-A-E-?-G-A-?-L-V-K-P. We previously purified soluble cytochrome c-550 cytochrome c-550(s)] from N. winogradskyi and determined its complete amino acid sequence (Y. Tanaka, Y. Fukumori, and T. Y. Yamanaka, Biochim. Biophys. Acta 707:14-20, 1982). Although the sequence of cytochrome c-550(m) was completely different from that of cytochrome c-550(s), ferrocytochrome c-550(m) was rapidly oxidized by the cytochrome c oxidase of the bacterium. Furthermore, the liposomes into which nitrite cytochrome c oxidoreductase, cytochrome c oxidase, and nitrite were incorporated showed nitrite oxidase activity in the presence of cytochrome c-550(m). These results suggest that cytochrome c-550(m) may be an alternative electron mediator between nitrite cytochrome c oxidoreductase and cytochrome c oxidase.

Amino Acid Sequence↗
PubMed1986

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5.

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.

Animals↗
PubMed1986

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: isozyme specificity and role of cytochrome b5.

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process.

Animals↗
PubMed1981

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides.

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.

Bacteriochlorophylls↗
PubMed1980

NADH- and NADPH-dependent reconstituted p-nitroanisole O-demethylation system containing cytochrome P-450 with high affinity for cytochrome b5.

A reconstituted system containing a form of cytochrome P-450, cytochrome b5, NADPH-cytochrome P-450 reductase, and NADH-cytochrome b5 reductase, all purified from rabbit liver microsomes, could catalyze O-demethylation of p-nitroanisole in the presence of both NADPH and NADH. Omission of either cytochrome P-450 or cytochrome b5 from the system led to complete loss of the activity. The reconstituted activity was sensitive to carbon monoxide, metyrapone, phenyl isocyanide, and cyanide, indicating that the cytochrome P-450 used is cyanide-sensitive and is involved in the catalytic process. The maximal demethylase activity was attained when the system contained cytochrome P-450 and cytochrome b5 at a 1 : 1 molar ratio. Trypsin digestion of cytochrome b5 abolished the capacity of this cytochrome to reconstitute the demethylase activity. These results suggest that O-demethylation of p-nitroanisole by this particular form of cytochrome P-450 absolutely requires the intact form of cytochrome b5 and that the second electron needed for the demethylation may be donated to the cytochrome P-450 only by way of cytochrome b5.

Animals↗