Akiyama M, Sugatani J, Suzuki T, Suzuki Y, Miwa M
Department of Pharmaco-Biochemistry School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422.
J Biochem. 1998 May;123(5):786-9. doi: 10.1093/oxfordjournals.jbchem.a022005.
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.
通过对经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离的蛋白质进行电转印，在聚偏二氟乙烯（PVDF）膜上鉴定出人血清/血浆中的血小板活化因子（PAF）乙酰水解酶。在膜上43 kDa区域检测到该酶活性，表现为[3H]乙酰-PAF的β-放射发光降低，或者通过测定PAF乙酰水解酶活性的简便方法（三氯乙酸沉淀法）来检测。用N-糖苷酶F处理后，该酶活性转移至PVDF膜上的34 kDa区域。另一方面，经银染的SDS-PAGE分析显示，仅观察到一条对应分子量为53 kDa的条带。用N-糖苷酶F处理53 kDa蛋白后，其分子量变为43 kDa（蛋白A）。蛋白A的NH2末端32个氨基酸序列与Tjoelker等人（《自然》374, 549 - 553, 1995）报道从NH2末端缺失54个氨基酸的异源酶的序列完全一致。即使对经N-糖苷酶F消化的酶进行胰蛋白酶处理后，其PAF-AH活性仍保留在PVDF膜上的34 kDa区域，但污染蛋白A消失。此外，大多数血清PAF-AH保留在接骨木凝集素（SSA）琼脂糖柱上，并用半抗原糖乳糖洗脱。这些结果表明，由34 kDa蛋白和约9 kDa天冬酰胺结合糖链组成的PAF乙酰水解酶是人类血清/血浆中的主要酶。
