A cDNA designated hIIB1, representing the entire coding sequence of a P450 in the IIB gene family, was isolated from a human liver lambda gt11 library by using the rat IIB1 cDNA as a probe. The hIIB1 protein, deduced from the cDNA sequence, contained 491 amino acids, had a calculated molecular weight of 56,286, and displayed 76% amino acid similarity with the rat IIB1 protein. Expression of this cDNA, using the vaccinia virus system, yielded a P450 that had a reduced CO-binding spectrum with an absorption maximum of 452 nm. The expressed human enzyme was able to catalyze the deethylation of 7-ethoxy-coumarin. Total RNA from 13 livers was probed for levels of hIIB mRNA. Two livers had high levels, four contained moderate levels, and eight contained very low, or no detectable, mRNA. These data suggest either that defective hIIB1 genes exist in humans or that the hIIB1 gene is regulated and variably induced in our liver specimens. To search for mutant mRNA transcripts, libraries were constructed from livers expressing low levels of hIIB1 mRNA. A cDNA, designated hIIB2, was isolated that was identical with the hIIB1 cDNA except for the presence of an unusual alteration of the DNA near its 5' end corresponding to the putative exon 4. This alteration was caused by a deletion of 29 bp and an insertion of 44 bp of nonhomologous DNA. This sequence replacement occurs at the junction of the third and fourth exons as predicted from the structure of the rat IIB1 gene, suggesting that a faulty splice might have given rise to the variant hIIb2 transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

A cDNA coding for a P450, designated IIF1, was isolated from a human lung lambda gt11 library by screening with a human IIC9 cDNA probe. The cDNA-encoded IIF1 protein had 491 amino acids and a calculated molecular weight of 55,507. IIF1 cDNA, expressed by using a vaccinia virus vector, produced a cytochrome with a lambda max of 454 nm when reduced and complexed with carbon monoxide. This enzyme was able to dealkylate ethoxycoumarin, propoxycoumarin, and pentoxyresorufin but possessed no activity toward ethoxyresorufin and only trace dearylation activity toward benzyloxyresorufin. A variant cDNA, designated IIF1v, was isolated that was identical with IIF1 except for the loss of two segments of 161 and 388 bp within the cDNA coding region. Two mRNAs, consistent with the predicted size of the IIF1 and IIF1v transcripts, were found at very low abundance in lung specimens by Northern blot analysis. A 2-kb transcript, hybridizing with the human IIF1, was also detected as an abundant mRNA in rat lung. The CYP2F gene subfamily was localized to human chromosome 19 and mouse chromosome 7. On the basis of Southern blotting analysis with multiple restriction enzymes, we conclude that the CYP2F1 gene is flanked by a second highly similar gene.

Studies were conducted to address glucuronidation of 3‐trifluoromethyl‐4‐nitrophenol (TFM) in sea lampreys Petromyzon marinus, channel catfish Ictalurus punctatus, rainbow trout Oncorhynchus mykiss, and bluegills Lepomis macrochirus. The ability of these species to biotransform TFM was investigated by determining the kinetics of UDP‐glucuronyltransferase (UDPGT; also known as glucuronosyltransferase) in vitro from hepatic microsomal preparations. Maximal velocity (Vmax, nmol/min·mg) for UDPGT activity toward TFM was significantly greater (P < 0.05) in bluegills (1.52), rainbow trout (1.82), and channel catfish (1.46) than in sea lampreys (0.68). Binding affinities (Km) of UDPGT for TFM varied significantly among species in the following order: Bluegill (58 μM) > rainbow trout (97 μM) > channel catfish (172 μM) > sea lamprey (261 μM). Analysis of Vmax/Km ratios, a measure of enzyme efficiency (nmol/min‐mguM TFM), indicated that the efficiency of UDPGT activities in all species examined was influenced more by binding affinity (Km) than by the Vmax of the reaction. These calculated ratios were progressively lower for species that were previously reported to be more sensitive to aqueous TFM (i.e., to have lower LC50s, TFM concentrations lethal to half the test fish). Sea lampreys appear to have relatively low UDPGT activity and binding affinity for phenolic substrates. This, in part, may account for the sensitivity of the sea lamprey to aqueous TFM.

Our prior work, and the work of others, demonstrated that chronic administration of ethanol to cells in culture or to mice resulted in decreased responsiveness of adenylyl cyclase (EC4.6.1.1) to a number of stimulatory agents. In this study, we substantiated the ethanol-induced changes in cerebral cortical adenylyl cyclase activity in alcohol-tolerant and alcohol-dependent mice, and we examined whether chronic ethanol treatment of mice altered the quantity of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) in cerebral cortex and other mouse brain areas. Amounts of various G protein subunits--including the alpha subunits of GS (GS alpha), Gi alpha 1-3, G(o) alpha, and beta subunits--were examined by Western blot analysis. There was no change in quantity of these G protein subunits in cerebral cortex, hippocampus, or cerebellum of ethanol-fed mice, compared with controls. In striatum of ethanol-fed mice, small increases in Gi alpha 1 and G(o) alpha were observed, but these changes could not explain the ethanol-induced desensitization of adenylyl cyclase in brain areas such as the cerebral cortex. Forskolin activation of cerebral cortical adenylyl cyclase activity showed two components of activation, with high and low "affinity" for forskolin. Ethanol treatment caused a decrease in the efficacy of forskolin for both components, whereas the EC50 of forskolin for each component did not change. Adenylyl cyclase activity measured in the presence of manganese was also diminished in cortical membranes of ethanol-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)