Charles B. Kasper

Preferential induction of aryl hydroxylase activity in rat liver nuclear envelope by 3-methylcholanthrene

Abstract The specific activity of aryl hydroxylase associated with the nuclear envelope from rat hepatocytes is increased 17-fold by the intraperitoneal administration of 3-methylcholanthrene. This is in marked contrast to the failure of phenobarbital to induce nuclear hydroxylase activity. Since both 3-methylcholanthrene and phenobarbital stimulate the induction of aryl hydroxlase in the endoplasmic reticulum, it appears that the cellular controls regulating the expression of the nuclear envelope enzyme differ from those operative for the control and regulation of the microsomal hydroxylase.

Conformational changes of NADPH-cytochrome P450 oxidoreductase are essential for catalysis and cofactor binding

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase☆

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development☆

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.

Mechanistic Studies on the Reductive Half-reaction of NADPH-Cytochrome P450 Oxidoreductase

Site-directed mutagenesis has been employed to study the mechanism of hydride transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specifically, Ser457, Asp675, and Cys630 have been selected because of their proximity to the isoalloxazine ring of FAD. Substitution of Asp675 with asparagine or valine decreased cytochromec reductase activities 17- and 677-fold, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytochromec reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A., and Kasper, C. B. (1996) Biochemistry 35, 9451–9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly multiplicative decreases in cytochrome c reductase activity (774- and 22000-fold, respectively) with corresponding decreases in the rates of flavin reduction. For each mutation, increases were observed in the magnitudes of the primary deuterium isotope effects with NADPD, consistent with decreased rates of hydride transfer from NADPH to FAD and an increase in the relative rate limitation of hydride transfer. Asp675substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shifted the pK a of an ionizable group previously identified as necessary for catalysis (Sem, D. S., and Kasper, C. B. (1993)Biochemistry 32, 11539–11547) from 6.9 to 7.8. These results are consistent with a model in which Ser457, Asp675, and Cys630 stabilize the transition state for hydride transfer. Ser457 and Asp675interact to stabilize both the transition state and the FAD semiquinone, while Cys630 interacts with the nicotinamide ring and the fully reduced FAD, functioning as a proton donor/acceptor to FAD.

Nuclear Receptor Involvement in the Regulation of Rat Cytochrome P450 3A23 Expression

Many genes of the cytochrome P450 3A (CYP3A) subfamily, including several human and rat isoforms, are inducible by glucocorticoids. In the ratCYP3A23 gene, a 110-base pair segment of the proximal 5′-flanking region mediates dexamethasone activation. Three binding sites (DexRE-1, DexRE-2, and Site A), identified by DNase I footprinting analysis, were characterized for their relative contribution to both basal activity and dexamethasone inducibility. Site-directed mutagenesis of DexRE-1 (−144 to −169) and DexRE-2 (−118 to −136) demonstrated that each contained a core imperfect AGGTCA direct repeat, which comprised a consensus nuclear receptor binding site, and was essential for dexamethasone responsiveness but was not required for basal activity. Competition gel shift and supershift analyses revealed that both sites can bind the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor. Site A (−85 to −110) was shown to be important for both basal activity and dexamethasone responsiveness. Point mutants displayed a reduced (2–3-fold) induction response, compared with 15-fold for wild-type, which was accompanied by a 40–60% drop in basal activity. Site A was shown to bind the liver-enriched nuclear receptor hepatocyte nuclear factor 4. Our studies demonstrate that the mechanism mediating glucocorticoid-inducible transcriptional activity ofCYP3A23 involves multiple binding sites for members of the nuclear receptor superfamily.

Hepatic cytochrome P-448 and epoxide hydrase: enzymes of nuclear origin are immunochemically identical with those of microsomal origin.

Abstract Nuclear and microsomal sources of hepatic cytochrome P -448 and epoxide hydrase were compared using antibodies made against the pure antigens isolated from rat liver microsomes. Both antigens were easily detected in detergent-solubilized nuclei and microsomes from rats using the Ouchterlony double-diffusion technique. Epoxide hydrase from either whole nuclei or nuclear envelope was immunochemically identical with the enzyme isolated from microsomes. Similarly, in rats pretreated with 3-methylcholanthrene, the cytochrome P -448 of nuclear origin was immunochemically indistinguishable from the enzyme derived from microsomes. These results establish the immunochemical identity of these hepatic nuclear and microsomal enzymes and provide a firm basis for applying the knowledge gained with the microsomal system of metabolism to the nuclear system.

Rapid method for preparation of crystalline human ceruloplasmin from Cohn Fraction IV-1☆

Abstract Crystalline ceruloplasmin has been prepared in high yield by a relatively rapid method from Cohn Fraction IV-1 of human plasma. The material contains near eight molecules of copper per 160,000 g. protein. It shows a single component on electrophoretic and ultracentrifugal assay.

Differential Contributions of NADPH-Cytochrome P450 Oxidoreductase FAD Binding Site Residues to Flavin Binding and Catalysis

Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg454, Tyr456, Cys472, Gly488, Thr491, and Trp677) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411–8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr456, which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4′-hydroxyl, or Arg454, which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome creductase activity. In contrast, substitution of Thr491, which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456SK m cyt  c and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp677 is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp677 and the carboxyl-terminal Ser678decreased catalytic activity 50-fold without affecting FAD content.

Invitro translation of epoxide hydratase messenger RNA

Abstract Rat liver epoxide hydratase mRNA, found exclusively associated with polysomes tightly bound to the endoplasmic reticulum, was translated using a rabbit reticulocyte protein synthesizing system. Epoxide hydratase synthesized in vitro was found to be identical to the native enzyme in terms of molecular weight and partial proteolytic peptide patterns, indicating the absence of a cleavable signal sequence. Translatable levels of epoxide hydratase mRNA increased 3-fold at 4 hours after phenobarbital administration. This increase was inhibited by cordycepin, an RNA synthesis chain terminator. These results demonstrate that epoxide hydratase is induced by phenobarbital through transcriptional and/or post-transcriptional nuclear events.