Saini S S, Peterson J W, Chopra A K
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1019, USA.
Biochem Biophys Res Commun. 1997 Sep 18;238(2):436-42. doi: 10.1006/bbrc.1997.7295.
Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.
合成蜂毒肽可抑制多种来源的分泌型磷脂酶A2（PLA2）的酶活性，这些来源包括蜜蜂和蛇毒、牛胰腺以及类风湿性关节炎患者的滑液，且不受底物（如[14C] - 磷脂酰胆碱或磷脂酰乙醇胺囊泡以及[3H] - 油酸标记的大肠杆菌）的影响。Lineweaver - Burk分析表明，蜂毒肽是蜂毒PLA2的非竞争性抑制剂，使最大反应速度（Vmax）从200单位/分钟/毫克蛋白降至50单位/分钟/毫克蛋白。米氏常数（Km）保持不变（0.75纳摩尔）。蜂毒肽以30:1的摩尔比（蜂毒肽:酶）抑制约50%的纯化蜂毒PLA2活性。由于酶动力学表明存在PLA2 - 蜂毒肽相互作用，因此使用蜂毒肽 - 琼脂糖亲和柱从人血清中纯化PLA2。此外，还开发了一种酶联测定法，使用抗生物素蛋白 - 过氧化物酶和包被有生物素化蜂毒肽的酶联免疫吸附测定（ELISA）板来定量生物流体中的PLA2活性。这些观察结果可能具有潜在的治疗意义，同时也为PLA2的分离和定量提供了便利的基础。
