Highly purified cytochrome P-448 and P-450 from rat liver microsomes

Ryan, Dene E.; Lu, Anthony Y. H.; Kawalek, Joseph C.; West, Susan; Levin, Wayne

doi:10.1016/0006-291x(75)90812-8

Cited by 185 publications

(21 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It can be deduced from the specific activity, molecular weight and stoichiometry that each band represents at least one form of cytochrome P-450. It should be noted that in the preparation [20] only one polypeptide band was found. However, immature rats were used and their product was analysed by a different electrophoretic system.…”

Section: Resultsmentioning

confidence: 81%

See 1 more Smart Citation

The biosynthesis of cytochrome P‐450 in vitro

1978

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 81%

“…Cytochrome P-450 was purified from microsomes prepared from the livers of phenobarbital-treated rats as in [20] . The preparation had spec.…”

Section: Resultsmentioning

confidence: 99%

The biosynthesis of cytochrome P‐450 in vitro

1978

View full text Add to dashboard Cite

“…Mammalian cytochrome P-450 is a membrane-bound mixed function oxidase which mediates the hydroxylation of a wide variety of substrates including steroids, aromatic compounds, hydrocarbons, and barbiturates (1). Because it has only recently been obtained in electrophoretically homogeneous form (2)(3)(4)(5), much of the information concerning P-450 has come from studies on the soluble, hence easily purified, bacterial oxidase, P-450cam (6). All cytochromes P-450 contain iron protoporphyrin IX as the prosthetic group and have a common reaction cycle with four wellcharacterized states (Fig.…”

mentioning

confidence: 99%

Magnetic circular dichroism of ferrous carbonyl adducts of cytochromes P-450 and P-420 and their synthetic models: further evidence for mercaptide as the fifth ligand to iron.

Collman¹,

Sorrell²,

Dawson³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Absorption and magnetic circular dichroism (MCD) spectra have been obtained for the ferrous carbonyl adducts of cytochromes P450 and P420 as well as synthetic model systems. Ferrous porphyrins with sodium methyl mercaptide and CO in benzene give MCD and absorption spectra which are almost identical to those of the natural enzyme, indicating that in P450 a mercaptide serves as the fifth ligand in the ferrous carbonyl adduct. MCD spectra of models with either propyl mercaptan or N-methylimidazole as the axial ligand are identical with that of P420. Thus, no unambiguous assignment of the axial ligand can be made in this case. The infrared stretching frequencies of ferrous porphyrin carbonyl complexes and the absorption spectrum of the CO adduct of Na[Fe'(meso-tetraphenylporphyrin dianion)] are consistent with the concept that in P450 considerable electron density is transferred to the iron by the mercaptide ligand. Mammalian cytochrome P-450 is a membrane-bound mixed function oxidase which mediates the hydroxylation of a wide variety of substrates including steroids, aromatic compounds, hydrocarbons, and barbiturates (1). Because it has only recently been obtained in electrophoretically homogeneous form (2-5), much of the information concerning P-450 has come from studies on the soluble, hence easily purified, bacterial oxidase, P-450cam (6). All cytochromes P-450 contain iron protoporphyrin IX as the prosthetic group and have a common reaction cycle with four wellcharacterized states (Fig. 1) beginning with a low-spin ferric resting form, 1, which is converted upon substrate binding to a high-spin ferric state, 2. Reduction gives a high-spin ferrous complex, 3, capable of binding dioxygen (for the subsequent hydroxylation step), 4, or carbon monoxide, 5.Cytochrome P-450 is unique among hemoproteins for two reasons. First, its ferrous carbonyl adduct absorbs light at the unusually long wavelength of approximately 450 nm. Additional abnormal spectral properties associated with this complex include the virtual absence of a distinct a-band in the 570 nm region and the appearance of an additional band at about 370 nm (vide infra). This very unusual but characteristic absorption spectrum has been the subject of much speculation; for instance Hill et al. (7) have implied that the state of the iron could be quite different from that found in other hemoproteins such as myoglobin. Second, only one other Abbreviations: P-450 and P-420, cytochromes P-450 and P-420; THF, tetrahydrofuran; TpivPP, meso-tetrapivalamidophenylporphyrin dianion; TPP, meso-tetraphenylporphyrin dianion; PPIXDEE, protoporphyrin diethyl ester dianion; SCH3, sodium methyl mercaptide-crown ether complex; MCD, magnetic circular dichroism; EPR, electron paramagnetic resonance.

show abstract

“…The latter was less than 3 of the cytochrome-P-450-dependent activity when the highest value determined for cytochrome P-448 was compared to the lowest value obtained in the cytochrome P-450 system. The low activity observed in the cytochrome P-448 monooxygenase system was not due to insufficient enzymatic function of cytochrome P-448 since this system supported the hydroxylation of benzo [alpyrene, a reaction known to be mediated preferentially by cytochrome P-448 [20,21].…”

Section: Discussionmentioning

confidence: 97%

“…Metabolic activity of cytochrome P-448 seems to be restricted to substrates of planar structure like benzo[a]pyrene [20,21], cthoxyresorufin [22], 2-acetaminofluorene [23] and 7-ethoxycoumarin [24]. Possibly, it is the nonplanar and bulky structure of aldrin which limits its metabobilism by cytochrome P-448 to such a low extent.…”

Section: Discussionmentioning

confidence: 99%

Aldrin Epoxidation Catalyzed by Purified Rat‐Liver Cytochromes P‐450 and P‐448 High Selectivity for Cytochrome P‐450

Wolff

Greim

Huang

et al. 1980

European Journal of Biochemistry

View full text Add to dashboard Cite

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent K , for aldrin in the complete system was 7 f 2 pM. SKF 525-A, at a concentration of 250 pM, inhibited aldrin epoxidation by 65 %, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 pM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5 % ethanol.When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3 % of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent K, for aldrin was 27 k 7 yM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The Is0 was 0.05, 0.2 and 800 mM, respectively.These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent K , and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.A variety of compounds of widely different chemical structure are oxidized by the monooxygenase system in the endoplasmic reticulum which contains cytochrome P-450 as the terminal oxidase. The broad substrate specificity of this enzyme system has been partially attributed to the existence of multiple cytochrome P-450 species of different but overlapping substrate specificities [l -51. This concept of the multiplicity of cytochrome P-450 was first supported by findings suggesting the occurrence of two major groups of microsomal hemoproteins typified by their response to inducing agents like phenobarbital and 3-methylcholanthrene, by spectral properties and by differences in substrate specificity [6 -81. Compelling

show abstract

Highly purified cytochrome P-448 and P-450 from rat liver microsomes

Cited by 185 publications

References 22 publications

The biosynthesis of cytochrome P‐450 in vitro

The biosynthesis of cytochrome P‐450 in vitro

Magnetic circular dichroism of ferrous carbonyl adducts of cytochromes P-450 and P-420 and their synthetic models: further evidence for mercaptide as the fifth ligand to iron.

Aldrin Epoxidation Catalyzed by Purified Rat‐Liver Cytochromes P‐450 and P‐448 High Selectivity for Cytochrome P‐450

Contact Info

Product

Resources

About