Machang'u R S, Prescott J F
Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph.
Can J Vet Res. 1991 Oct;55(4):332-40.
Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)
从马红球菌ATCC 33701的培养上清液中分离出胆固醇氧化酶（CO）和胆碱磷酸水解酶（CPH）这两种胞外酶，并检测了它们的溶血和细胞毒性活性。纯化过程包括硫酸铵分级沉淀、离子交换和凝胶过滤色谱法。通过金黄色葡萄球菌β毒素预致敏的绵羊红细胞（SRBC）的协同溶血作用，测定CO的纯化倍数为32.8倍，产率为0.3%。胆固醇水悬浮液的氧化也证明了CO的酶活性。CO的活性可被浓度可逆抑制。通过对对硝基苯基磷酰胆碱的水解，测定CPH的纯化倍数为412.4倍，产率为1.7%。两种胞外酶的纯度通过免疫印迹法得以确认。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上，CO的分子量（Mr）为60 kD，CPH的Mr为65 kD。胆碱磷酸水解酶不水解鞘磷脂。然而，在浓缩培养上清液中显示出鞘磷脂酶C（SMC）活性。SMC与CPH活性的这种分离表明马红球菌产生两种不同的磷脂酶C胞外酶，一种是CPH，另一种是SMC。CO和CPH单独或联合使用，即使随后将细胞在4℃冷藏（“热-冷”处理），也不会裂解天然SRBC。纯化的CO可裂解β毒素致敏的SRBC。即使在高浓度下，CPH对这种致敏的SRBC也仅显示出轻微的溶血活性。CO和CPH联合裂解β毒素致敏的SRBC仅显示出轻微的相加效应，而非协同效应。（摘要截短于250字）
