Callahan J W, Khalil M, Philippart M
Pediatr Res. 1975 Dec;9(12):908-13. doi: 10.1203/00006450-197512000-00009.
Sphingomyelinase was obtained in excellent yield from liver and brain by homogenization with 0.05 M citrate-phosphate buffer, pH 4.5, containing 0.25% Triton-X-100 (v/v) followed by dialysis of the supernatant fluids against 1% glycine. Total recovery of enzyme was slightly less with tissue from Niemann-Pick disease compared with control tissue. Isoelectric focusing of liver and brain extracts was successfully used to resolve several species of sphingomyelinase. Three (I-III) of the five species were partially characterized. Enzyme I (pI 4.6) had a pH optimum of 4.8-5.0 in acetate buffer and a Km value of 0.026 mM. Both sphingomyelinases I and II were the major enzymes, whereas III, IV, and V were found at lower levels. Of the two major species in normal liver and brain (I and II), species I alone persisted in liver from the two cases of type C, while species III, IV, and V were present. In brain, only species II was decreased but the resolution of the brain enzymes was less satisfactory.
通过用含有0.25% Triton-X-100（体积/体积）的0.05 M柠檬酸-磷酸盐缓冲液（pH 4.5）对肝脏和脑组织进行匀浆，随后将上清液对1%甘氨酸进行透析，可获得高产率的鞘磷脂酶。与对照组织相比，来自尼曼-皮克病组织的酶总回收率略低。肝脏和脑组织提取物的等电聚焦成功用于分离几种鞘磷脂酶。五个种类中的三个（I-III）得到了部分表征。酶I（pI 4.6）在醋酸盐缓冲液中的最适pH为4.8-5.0，Km值为0.026 mM。鞘磷脂酶I和II都是主要的酶，而III、IV和V的含量较低。在正常肝脏和脑组织中的两种主要种类（I和II）中，仅种类I在两例C型病例的肝脏中持续存在，同时存在种类III、IV和V。在脑组织中，只有种类II减少，但脑酶的分离效果不太令人满意。
