Characterization of a platelet activating factor acetylhydrolase from rat adipocyte.
Platelet activating factor-acetylhydrolases (PAF-AHs) are a family of enzymes with the common property of hydrolyzing and inactivating PAF and thus regulating its levels. In the course of studying the role of PAF in rat adipocytes and its possible implication in body weight regulation and immune response, conditions in which adipocytes are involved, we investigated the existence of PAF-AH in these cells. We detected PAF-AH activity in rat adipocytes which is mainly distributed in the cytosol. The behaviour of the enzyme during hydrophobic chromatography, together with the fact that part of the enzyme activity was found in the fat cake of adipocyte homogenate suggests the hydrophobic nature of rat adipocyte PAF-AH. The enzyme activity was distinct from the Ca2+-dependent and independent phospholipase A2, the lysophospholipase, the lecithin:cholesterol acyltransferase (LCAT) and from non-specific acetylhydrolases. We partially purified PAF-AH from rat adipocyte cytosolic fraction and the purified enzyme revealed a major protein band of 66 kDa and a minor one of 37 kDa on SDS/PAGE. The purified PAF-AH has an apparent Km value of 4.9 microM and the enzyme activity was inactivated by PMSF and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and moderately stimulated by dithiothreitol (DTT). Furthermore, in this study we identified PAF in rat adipocytes and determined its concentration.
Chroni
A
A
Department of Chemistry (Biochemistry), University of Athens, Greece.
Mavri-Vavayanni
M
M
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D001681:Biological Assay; D001792:Blood Platelets / Q000187:drug effects; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D004822:Epididymis; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000201:enzymology
D010972:Platelet Activating Factor; D010741:Phospholipases A; D054467:Phospholipases A2; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase