Hepatic microsomal NADPH-cytochrome P-450 reductase from little skate, Raja erinacea. Comparison of thermolability and other molecular properties with a mammalian enzyme.

Components of little skate (an elasmobranch) and rabbit hepatic microsomal cytochrome P-450 dependent monooxygenase systems were examined for differences which might explain the decreasing xenobiotic-metabolizing activity of little skate microsomes assayed at temperatures above 30 degrees C. The proportion of saturated fatty acids in microsomal lipids and the habitat temperature are both lower in skate as compared to rabbit, which is consistent with the known adaptive pattern. The more thermolabile enzyme of the skate system in microsomal preparations is NADPH-cytochrome P-450 reductase. The optimal assay temperature for purified skate reductase (30 degrees C) is 10 degrees C lower than that for the purified rabbit reductase. The purified skate reductase differs from rabbit reductase in monomeric molecular weight, in peptides produced by partial proteolysis, in immunochemical properties, but not in flavin content.

Covalent binding of metabolites of 4-ipomeanol to rabbit pulmonary and hepatic microsomal proteins and to the enzymes of the pulmonary cytochrome P-450-dependent monooxygenase system.

The covalent binding of metabolites of 4-ipomeanol, a potent lung toxin, to proteins in rabbit pulmonary and hepatic microsomal preparations and in purified monooxygenase systems was investigated. The rate of binding was 12-fold greater in pulmonary preparations than in hepatic preparations. Covalent binding in pulmonary microsomal fractions was inhibited 39 to 49% by antibodies to rabbit pulmonary cytochrome P-450II or P-450I and 90% by antibodies to cytochrome P-450 reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scintillation autoradiography of pulmonary microsomal proteins revealed the presence of heavily labeled bands throughout the molecular weight range (Mr) examined. Two of these bands corresponded in mobility to pulmonary cytochrome P-450I (Mr 52,000) and P-450II (Mr 58,000). In addition, there was a great deal of binding associated with very high molecular weight proteins, probably in the form of cross-linked aggregates which were unable to penetrate the gel matrix. In the absence of cofactor, no binding was observed. Binding was decreased by the addition of the following: antireductase greater than glutathione = NADH (without NADPH) greater than anti-II greater than anti-I. The electrophoretic patterns of the proteins from incubation of [3H]-4-ipomeanol with purified pulmonary P-450-dependent monooxygenase enzymes were also examined. In the complete system, the majority of the binding was associated with high molecular weight species located at the origin and with low molecular weight species that migrated with the tracking dye. In the absence of cofactor, some binding to proteins that corresponded with cytochrome P-450 and P-450 reductase was observed. Protease digestion of incubation mixtures resulted in the migration of all bound material at the dye front.

Identification of cytochrome P-450 isozymes in nonciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated from rabbit lung.

Two forms of cytochrome P-450 (P-450I and P-450II) have been shown by several techniques to be present in both nonciliated bronchiolar cells (Clara) and alveolar type II cells isolated from rabbit lung. In contrast, the alveolar macrophage contains little or none of these cytochromes. Cross-reactivity between antibodies to cytochrome P-450I or P-450II and detergent-digested microsomes prepared from 80% type II or 70% Clara cell fractions was shown by Ouchterlony double immunodiffusion. The presence of both cytochromes was also demonstrated by histochemical immunofluorescence in smears of type II cells stained by a modified Papanicolaou procedure and Clara cells stained with nitroblue tetrazolium. However, this same fluorescent antibody technique used for localization of rabbit pulmonary cytochromes P-450I and P-450II in tissue sections showed most of the immunofluorescence in the Clara cells of the bronchiolar epithelium. SDS-polyacrylamide gel electrophoresis of microsomes from either the type II or Clara cell fractions produced bands which corresponded to cytochrome P-450I (52,000 daltons) and cytochrome P-450II (58,000 daltons).

The rabbit pulmonary monooxygenase system. partial structural characterization of the cytochrome P-450 components and comparison to the hepatic cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450, P-450I and P-450II, are distinguished by unique peptides observed upon electrophoresis of the products of limited proteolysis (with papain or chymotrypsin) in the absence or presence of sodium dodecyl sulfate. In contrast, the peptides from the proteolytic digestions of pulmonary cytochrome P-450I are indistinguishable from those of the major form of hepatic cytochrome P-450 induced by phenobarbital (P-450PB). Electrophoresis of the fragments of P-450, and P-450II produced by treatment with cyanogen bromide also shows different peptides, whereas the peptides produced from P-450I and P-450PB are the same. The amino acid composition and P-450II is different from that of P-450I or P-450PB. P-450II is distinguished further from the other two cytochromes on the basis of amino acid composition and subunit molecular weight (either as calculated from the amino acid composition or as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis). The sequence of the first five residues at the NH2-terminal segment of P-450I and P-450PB are identical, while that of P-450II is different.

Purification and structural comparison of pulmonary and hepatic cytochrome P-450 from rabbits.

A procedure is described for the purification of a major form of cytochrome P-450 from the livrs of rabbits treated with phenobarbital and a major form of the cytochrome from the lungs of untreated rabbits. Preparations in good yield (13--17%) and of high purity (up to 21 nmol of cytochrome per mg of protein) that were free of lipid and contained minimal amounts of non-ionic detergent were obtained from either tissue. The two cytochromes cannot be distinguished from each other on the basis of absorption spectra, extinction coefficients, apparent molecular weights (52 000), amino acid compositions, or peptide fragments produced by treatment of the proteins with cyanogen bromide. These data are consistent with a major indigenous form of rabbit pulmonary cytochrome P-450 being the same as the major form of hepatic cytochrome induced by phenobarbital.

Membrane-bound redox proteins of the murine Friend virus-induced erythroleukemia cell.

We have obtained and studied a 105,000-g pellet from T-3-Cl-2 cells, a cloned line of Friend virus-induced erythroleukemia cells. By difference spectrophotometry, the pellet was shown to contain cytochrome b5 and cytochrome P-450, hemeproteins that have been shown to participate in electron-transport reactions of endoplasmic reticulum and other membranous fractions of various tissues. The pellet also possesses NADH-cytochrome c reductase activity which is inhibited by anti-cytochrome b5 gamma-globulin, indicating the presence of cytochrome b5 reductase. This is the first demonstration of membrane-bound forms of these redox proteins in erythroid cells. Dimethyl sulfoxide-treated T-3-Cl-2 cells were also shown to possess membrane-bound cytochrome b5 and NADH-cytochrome c reductase activity. We failed to detect soluble cytochrome b5 in the 105,000-g supernatant fraction from homogenates of untreated or dimethyl sulfoxide-treated T-3-Cl-2 cells. In contrast, erythrocytes obtained from mouse blood were shown to possess soluble cytochrome b5 but no membrane-bound form of this protein. These findings are supportive of our hypothesis that soluble cytochrome b5 of erythrocytes is derived from endoplasmic reticulum or some other membrane structure of immature erythroid cells during cell maturation.