Photoaffinity labeling of Crotalus atrox phospholipase A2 by a substrate analogue.
Photoaffinity labeling of Crotalus atrox phospholipase A2 by a substrate analogue.
复制标题
通过底物类似物对响尾蛇磷脂酶 A2 进行光亲和标记。
DOI：
10.1021/bi00504a030
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发表时间：
1981
期刊：
影响因子：
2.9
通讯作者：
Law,JH
中科院分区：
文献类型：
--
作者：
Huang,KS;Law,JH
摘要：磷脂酰乙醇胺(PE类似物)的耐光性类似物L，是以非同位素和~(14)C放射性标记形式，以L构型(天然磷脂构型)和外消旋形式合成的。当未标记的外消旋化合物在Crotalus trex的磷脂酶A2存在下被光解时，观察到广泛的酶失活。Ca~(2+)和耐光性化合物胶束的形成刺激了失活速度。磷脂酰乙醇胺的二己基醚类似物可保护酶免于失活。磷脂酶A2在L或外消旋14C标记的耐光性PE类似物存在下照射时，可产生共价标记的多肽。活性化合物标记多肽的N-末端区域(残基1-15)和中心区域(残基43-97)，而L化合物只标记N-末端区域。阿曲霉磷脂酶A2第10位的唯一蛋氨酸允许溴化氰降解，这表明L化合物标记的氨基酸仅限于N-末端的前10个氨基酸残基，磷脂酶A2对L磷脂具有绝对专一性，D-磷脂是竞争性抑制物。这些研究的结果强调了磷脂的头基区在磷脂酶-底物相互作用中的重要性，并表明底物的两个光学异构体在
Kuo-Sen Huang* and John H. Law* abstract: A photolabile analogue of phosphatidylethanolamine (photolabile PE analogue), l, 2-di-0-hexylglycero-3-(ethyl diazomalonamidoethyl phosphate), was synthesized in nonisotopic and 14C-radiolabeled form and in both the L configuration (that of the naturally occurringphospholipids) and the racemic form. When the unlabeled racemic compound was photolyzed in the presence of phospholipase A2 of Crotalus atrox, extensive enzyme inactivation was observed. The rate of inactivation was stimulated by Ca2+ and by formation of micelles of the photolabile compound. The dihexyl ether analogue of phosphatidylethanolamine protected the enzyme from inactivation. Phospholipase A2 gave rise to a covalently labeled polypeptide when irradiated in the presence of either l or racemic 14C-labeled photolabile PE analogue. The ra-cemic compound labeled both the N-terminal region (residues 1-15) and the central region (residues 43-97) of the polypeptide while the L compound labeled only the N-terminal region. The lone methionine at position 10 of the C. atrox phospholipase A2 permitted degradation by cyanogen bromide, which showed that labeling by the L compound was restricted to the first ten amino acid residues at the N-terminal end.Phospholipase A2 has an absolute specificity for L-phospho-lipids, and D-phospholipids are competitive inhibitors. The results of these studies underscore theimportance of the head-group region of the phospholipid in phospholipase-substrate interactions and suggest that the two optical isomers of the substrate may be rather differently oriented on the