IHARA Hayato National Cardiovascular Center Research Institute, Department of Pharmacology, R, 薬理部, 室員 (00223298)
MIYATA Atsuro National Cardiovascular Center Research Institute, Department of Pharmacology, R, 薬理部, 室員 (60183969)
YOKOYAMA Chikeo National Cardiovascular Center Research Institute, Department of Pharmacology, R, 薬理部, 室員 (90200914)
Biosynthetic mechanisms of long-chain fatty acids and the derivatives, prostanoids, were studied in the present project, and the following results were obtained.
1. Biosynthsis of long-chain fatty acid is regulated by modulating the activity of acetyl-CoA carboxylase (ACC). To study relationships between the enzyme structure and its function, we cloned the Escherichia coli gene coding for a subunit of ACC, biotin carboxylase. The gene was found 13 bp '3-down stream of the gene encoding another subunit, biotin carboxyl carrier protein. E. coli biotin carboxylase gene encodes 449 amino acids with a molecular weight of 49, 320, which agrees with the molecular weight of 51, 000 determined by SDS-polyacrylamide gel electrophoresis. The deduced primary structure of the enzyme was very similar to that of E. coli carbamyl phosphate synthetase. With use of the determined primary structure of E. coli biotin carboxylase and the reported primary structures of other biotin enzymes, a phylogenetic tr
ee is under construction. Furthermore, the regulation of fatty acid synthesis in rat brain has been studied by measuring ACC mRNA levels during the development.
2. Arachidonate, a polyunsaturated fatty acid is converted to bioactive compounds by cyclooxygenase, and lipoxygenases such as 12-lipoxygenase. The biosynthesis of thromboxane from arachidonate was catalyzed by two enzymes, cyclooxygenase and thromboxane synthase, gene expression of which have been investigated by RNA blot analysis. It was found in MC3T3-El cells that cAMP-producing compounds such as epinephrine and PGE2, elevated cyclooxygenase mRNA levels. Furthermore, cDNA coding for cyclooxygenase was cloned by a combined method of PCR and cDNA cloning techniques. The sequence of the cDNA completely coincided with the sequence predicted from the nucleotide sequence of human cyclooxygenase gene. Next, human platelet cDNA coding for thromboxane synthase was isolated according to the partial aminoacid sequences by a combined method of PCR and cDNA cloning techniques. The open reading frame of thromboxane synthase mRNA encodes 533 amino acids with a molecular weight of 60, 487. The deduced primary structure of the enzyme exhibited a 34-36 % homology to the amino acid sequences of cytochrome P450s classified in the P450 Ill gene family. The results show that thromboxane synthase constitutes a new gene familiy of P450. On the other hand, arachidonate in platelet is metabolized to 12-hydroxyeicosatetraenoic acid by 12-lipoxygenase. The cDNA for human platelet 12-lipoxygenase was cloned from a cDNA library of human erythroleukemia cells. The cDNA has an open reading frame encoding 663 amino acids with a molecular weight of 75, 513. Less