ABSTRACT
A cytochrome P450 cDNA, encoding a new form of CYP3A protein, was isolated from a liver cDNA library of a male rat using anti-P450(6)beta-1 and anti-P450(6)beta-2 antibodies and the CYP3A2 cDNA. The cDNA (CYP3A18 cDNA) consisted of 1987 nucleotides, in which were contained an open reading frame of 1491 bp (corresponding to 497 amino acids), 5'-(59 bp) and 3'-noncoding regions (437 bp). The deduced amino acid sequence of CYP3A18 cDNA was completely identical in the first 27 N-terminal residues of P450(6)beta-2 previously isolated by us (Nagata et al. J Biochem 1990: 107, 718-725) from livers of rats treated with dexamethasone, and also shared higher extents of similarity with hamster CYP3A10 (78.5%) than with rat CYP3As previously sequenced (66.3-69.3%). Northern blot analyses indicated a male-dominant expression of this new CYP3A mRNA and enhanced expression in dexamethasone-or pregnenolone-16 alpha-carbonitrile (PCN)-treated, but not phenobarbital-or 3-methylcholanthrene-treated rats. Expressed CYP3A18 protein in COS-1 cells migrated at a position identical to that of purified P450(6)beta-2 on sodium dodecyl sulfate-acrylamide gel electrophoresis and catalyzed 16 beta- and 6 alpha-hydroxylations of testosterone. In contrast to CYP3A1 and CYP3A2, cytochrome b5 was not essential for maximal catalytic activities of recombinant CYP3A18 protein. These results, together with ontogenic profiles of CYP3A18 mRNA and P450(6)beta-2 protein, indicate that the newly isolated CYP3A18 cDNA encodes P450(6)beta-2 in rat liver.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , Mixed Function Oxygenases/genetics , Testosterone/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Catalysis , Cell Line , Cloning, Molecular , Cricetinae , Cytochrome P-450 CYP2E1 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Cytochromes b5/metabolism , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Female , Hydroxylation , Male , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Steroid Hydroxylases/metabolism , TransfectionABSTRACT
This study described the relationship between the concentration of the branched-chain fatty acid (BCFA) in rat skin surface lipid and the serum level of the branched-chain amino acid (BCAA) and branched-chain alpha-keto acid (BCKA). The concentrations of the BCFAs in the monoester fraction of the skin surface lipid, and BCAAs and BCKAs in the serum were analyzed in rats fed varying amounts of protein (0 to 40%) and different types of BCAAs (3%) for 2 weeks. The serum concentrations of BCAA were proportional to the protein level by day 10 of the feeding period. This dose response was not sustained by day 14 at the end of the feeding. Protein level dependence was not so evident in the concentration of BCKA. The concentrations of BCFA, even carbon number iso-acid in particular, increased in linear proportion to protein intake in the skin surface lipid. Supplementation of valine and isoleucine to the diet at a 3% level specifically raised the concentration of the respective BCAA and corresponding BCKA in serum, and related BCFA in the skin surface lipid. Addition of leucine, however, did not affect the related BCFA concentration in spite of elevated concentration of leucine and its alpha-keto acid. A good linear correlation between the average concentration of the substrates in the circulation and the concentrations of the product BCFA on the skin surface was thus obtained for valine, isoleucine and their respective alpha-keto acid. This relationship did not appear to hold up for leucine and its alpha-keto acid.
Subject(s)
Amino Acids, Branched-Chain/blood , Fatty Acids/analysis , Keto Acids/blood , Skin/chemistry , Animals , Dietary Proteins/metabolism , Male , Rats , Rats, Inbred StrainsABSTRACT
1. Age-related change of the branched-chain fatty acid distribution in rat skin surface lipid was studied for 24 months. 2. The proportion of even carbon number iso-acid increased from infancy to month 5 and thereafter decreased with advancing age toward senescence. 3. Concentration of odd carbon number iso-acid depicted a similar shape of time course, but with a lesser magnitude and a peak value at month 1. 4. Anteiso- fatty acid reached the plateau level at month 5 and remained roughly constant through maturity to senescence.
Subject(s)
Aging/metabolism , Fatty Acids/metabolism , Skin/metabolism , Aging/blood , Amino Acids, Branched-Chain/blood , Animals , Chromatography, Thin Layer , Fatty Acids/blood , Fatty Acids/chemistry , Kinetics , Lipid Metabolism , Male , Rats , Rats, Inbred StrainsABSTRACT
1. Fatty acid and lipid compositions of cultured rat keratinocytes were compared with those of intact epidermis prepared from newborn and adult rats. 2. The uniqueness of the lipid profile of cultured cell manifested itself in the accumulation of a novel lipid which co-migrated with monoalkyldiacylglyceride on thin-layer chromatography. 3. Concentration of the branched-chain fatty acid was specifically high in the cholesterol ester fraction of the intact cell, and was decreased by cultivation under submerged conditions.