Emi Kusunose

Expression and molecular cloning of human liver leukotriene B4 omega-hydroxylase (CYP4F2) gene.

Human liver leukotriene B4 (LTB4) omega-hydroxylase (CYP4F2) plays an important role in the metabolic inactivation and degradation of LTB4, a potent mediator of inflammation. The regulatory mechanism for the transcription of CYP4F2 has not yet been clarified. Here, we report that CYP4F2 is constitutively expressed in a human hepatoma cell line, HepG2, and is not induced by clofibrate. We isolated the gene encoding CYP4F2 and determined its genomic organization and the functional activity of its promoters. The CYP4F2 gene contains at least 13 exons with its open reading frame being encoded from exon II to exon XIII. Exon I includes 49 bp of a 5 untranslated sequence. The structure of this gene is very similar to that of the CYP4F3 gene earlier reported by Kikuta et al. (DNA Cell Biol 1998;17:221-230). The 5 flanking sequence downstream from -165 of the CYP4F2 gene has 75% similarity to the corresponding region of the CYP4F3 gene. However, common putative regulating elements in the two human CYP4F genes were not detected except for the TATA box. The elements recognized by nuclear receptors were not observed within its 5 flanking region. Deletion of the 5 flanking regions containing putative regulating elements recognized by HNF-3beta, CDP CR, and p300 caused alterations in the transcriptional activity. The region from -83 to -67 was necessary for transcription, but the TATA sequence was not. Our results indicate that the human two CYP4F genes evolved by duplication and alterations of the transcription regulation region and the site of exon III.

PROPERTIES AND ORIGIN OF DPNH DIAPHORASES FROM MYCOBACTERIUM TUBERCULOSIS.

Abstract A series of DPNH diaphorases has been isolated from cell-free extracts of the H37Ra strain of Mycobacterium tuberculosis . These diaphorases are either soluble or can be solubilized by treatment of the DPNH-oxidase particles with either digitonin or with alkaline buffers following lyophilization. Each diaphorase preparation can use more than one electron acceptor (nitro-BT, 5 DCPP, ferricyanide, K 3 , or cytochrome c). This multiplicity of electron acceptors is a reflection of the multiplicity of diapliorases in each preparation and is not due to lack of electron acceptor specificity. The soluble and particulate diaphorases are related. The DPNH-diaphorase activities solubilized from the washed DPNH-oxidizing particles are identical with the soluble DPNH diaphorases found in the original cell-free extract. We conclude that the soluble DPNH diaphorases found in the original cell-free extract are derived from the comminution of the DPNH-oxidase particles during preparation of the cell-free extract. The DPNH-oxidase particles are probably derived from the cytoplasmic membrane as opposed to pre-existing intracellular particles. Evidence is presented suggesting the production of more than one diaphorase with the same electron acceptor specificity, the same enzymatic constants but different physical characteristics.

ω- AND (ω-1)-HYDROXYLATION OF FATTY ACIDS IN RABBIT INTESTINAL MUCOSA MICROSOMES

Publisher Summary This chapter discusses the ω– and (ω-l)–hydroxylation of fatty acids in rabbit intestinal mucosa microsomes. It has been found that fatty acids are rapidly converted to ω– and (ω-l)–hydroxy fatty acids in the microsomes from rabbit intestinal mucosa that had been washed with a sucrose solution containing phenylmethylsulfonyl fluoride as a trypsin inhibitor. Small intestine is the site of fat absorption with subsequent activation and esterification of fatty acids, thus, studies on fatty acid hydroxylation help to elucidate its physiological significance in the fatty acid metabolism of intestinal mucosa. The chapter describes some properties of the fatty acid ω– and (ω-1)–hydroxylation system of rabbit intestinal mucosa microsomes. The microsomes from rabbit intestinal mucosa catalyze the hydroxylation of fatty acids in the presence of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) and molecular oxygen. Myristic and palmitic acids are converted to the corresponding ω–and (ω-l)–hydroxy fatty acids whereas lauric acid is converted to 12–hydroxy–lauric acid and capric acid,to 9– and 10–hydroxycapric acids together with an unknown polar acid. Among these fatty acids, myristic and lauric acids appear to be the most efficient substrates.