Identification of a major PAF acetylhydrolase in human serum/plasma as a 43 kDa glycoprotein containing about 9 kDa asparagine-conjugated sugar chain(s).
Platelet-activating factor (PAF) acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the beta-radioluminescence of [3H]acetyl-PAF or by the convenient method for determining PAF acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain(s) is a major enzyme in human serum/plasma.
Akiyama
M
M
Department of Pharmaco-Biochemistry School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422.
Sugatani
J
J
Suzuki
T
T
Suzuki
Y
Y
Miwa
M
M
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008567:Membranes, Artificial; D008970:Molecular Weight; D010741:Phospholipases A / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism*; D011145:Polyvinyls; D005465:Fluorocarbon Polymers
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D008567:Membranes, Artificial; D010741:Phospholipases A; D010972:Platelet Activating Factor; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers