cDNA cloning and expression of biologically active platelet activating factor-acetylhydrolase (PAF-AH) from bovine mammary gland.
Platelet activating factor (PAF)-acetylhydrolase (PAF-AH) is an enzyme that hydrolyzes the acetyl ester at the sn-2 position of PAF, and converts it to the inactive metabolite, lyso PAF. This enzyme is distributed widely in the intracellular as well as the extracellular matrix and is believed to be a defense mechanism that protects the host against the toxic effects of PAF and other biologically active oxidized phospholipids. Purification and expression of cDNA cloning of the intracellular and extracellular types of PAF-AH from several sources from different species have been reported. In this study, the cDNA for PAF-AH was cloned by reverse transcription (RT)-PCR from total RNA of bovine mammary gland. The complete amino acid sequences from the cDNA contains 444 amino acids and was identical to that of the PAF-AH isolated from the bovine spleen cDNA library except for two mismatches of amino acid residues (Thr-247 to Met and Ile-431 to Thr). Recombinant PAF-AH was expressed in HEK 293 cells, which exhibited enzyme activity in the in vitro assay system. Furthermore, recombinant bovine PAF-AH was identified by western blot using human plasma PAF-AH antibody as a monomeric polypeptide with a molecular weight of approximately 43 kDa. This protein can be applied to in vivo models to test its protective role against the deleterious PAF actions.
Lee
Eunkyung
E
College of Pharmacy, Yeungnam University, Gyonsan, Korea.
Lee
Su Jeong
SJ
Lee
Tae Yoon
TY
Chang
Hyeun Wook
HW
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008969:Molecular Sequence Data
D018076:DNA, Complementary; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase