Kojima T, Yanagihara Y, Mifuchi I
Microbiol Immunol. 1984;28(3):291-302. doi: 10.1111/j.1348-0421.1984.tb00681.x.
Hemolysis by leptospiral hemolysin was strongly inhibited by bovine serum. The inhibitory activity was observed in the chloroform-methanol-soluble fraction of bovine serum. The inhibitor was eluted in a complex lipid fraction and was separated into two fractions (Fr. I and II) by silicic acid column chromatography. Fractions I and II inhibited approximately 75% and 95%, respectively, of hemolysis by leptospiral hemolysin. Fraction I was identified as phosphatidylethanolamine (PdE) by silica gel thin-layer chromatography (TLC). Two kinds of phospholipids (PLs) were detected in Fr. II by TLC. One was resistant to alkaline treatment and was identified as sphingomyelin (Spm), and the other was sensitive to such treatment and was identified as phosphatidylcholine (PdC). PLs, such as Spm, PdC, phosphatidylglycerol, PdE, phosphatidylserine and cardiolipin, inhibited hemolysis by leptospiral hemolysin, but phosphatidylinositol did not show any inhibitory activity. PLs lacking the amino group in the polar backbone of the molecules were more effective. From experiments using erythrocytes of various kinds of animals, it was revealed that the hemolytic sensitivity of mammalian erythrocytes to leptospiral hemolysin depended on the Spm content in the erythrocyte membrane. On the other hand, phospholipase C (PLase C) activity with Spm and PdC as substrates was detected in the culture supernatant of Leptospira. Therefore, leptospiral hemolysin was presumed to be PLase C, perhaps sphingomyelinase. The inhibitors of leptospiral hemolysin present in bovine serum were identified as PLs. PLs in bovine serum were suggested to function as inhibitors of the interaction between leptospiral hemolysin and the surface of the erythrocyte membrane.
钩端螺旋体溶血素引起的溶血作用受到牛血清的强烈抑制。在牛血清的氯仿 - 甲醇可溶部分中观察到了这种抑制活性。该抑制剂在复合脂质部分被洗脱，并通过硅酸柱色谱分离为两个部分（部分I和部分II）。部分I和部分II分别抑制钩端螺旋体溶血素引起的约75%和95%的溶血作用。通过硅胶薄层色谱（TLC）鉴定部分I为磷脂酰乙醇胺（PdE）。通过TLC在部分II中检测到两种磷脂（PLs）。一种对碱性处理有抗性，被鉴定为鞘磷脂（Spm），另一种对这种处理敏感，被鉴定为磷脂酰胆碱（PdC）。诸如Spm、PdC、磷脂酰甘油、PdE、磷脂酰丝氨酸和心磷脂等磷脂抑制钩端螺旋体溶血素引起的溶血作用，但磷脂酰肌醇未表现出任何抑制活性。分子极性主链中缺乏氨基的磷脂更有效。通过使用各种动物红细胞的实验表明，哺乳动物红细胞对钩端螺旋体溶血素的溶血敏感性取决于红细胞膜中的Spm含量。另一方面，在钩端螺旋体的培养上清液中检测到以Spm和PdC为底物的磷脂酶C（PLase C）活性。因此，推测钩端螺旋体溶血素为PLase C，可能是鞘磷脂酶。牛血清中存在的钩端螺旋体溶血素抑制剂被鉴定为PLs。提示牛血清中的PLs可作为钩端螺旋体溶血素与红细胞膜表面相互作用的抑制剂发挥作用。
