Activation of the guinea pig granulocyte NAD(P)H-dependent superoxide generating enzyme: localization in a plasma membrane enriched particle and kinetics of activation.
Activation of the guinea pig granulocyte NAD(P)H-dependent superoxide generating enzyme: localization in a plasma membrane enriched particle and kinetics of activation.
复制标题
豚鼠粒细胞 NAD(P)H 依赖性超氧化物生成酶的激活：质膜富集颗粒中的定位和激活动力学。
作者：
H. Cohen;M. Chovaniec;W. Davies
研究了豚鼠粒细胞超氧（O2-）生成系统的亚细胞定位、激活动力学和底物特异性。通过温和超声处理和差速离心从粒细胞制备膜富集颗粒（足状体）。这些podosomes富集三倍的已知质膜标记物，5'-核苷酸酶，和腺苷酸环化酶。由静息粒细胞制成的足体具有非常少的NAD（P）H依赖性O2-产生。由毛地黄皂苷刺激的细胞制成的足状体同样富含膜标记物，但NAD（P）H依赖性O2产生增加15至20倍。NADPH的KmAPP是NADH的十分之一，但Vmax相同。毛地黄皂苷刺激的全细胞O2-生产的动力学平行的酶活性的变化，在这些podosomes。温度影响这种酶的活化速率和程度。酶的最适pH值、活化的最适pH值和全细胞O2-产生的最适pH值均为7.5。如果细胞用葡萄糖和氰化物处理，酶活性增加，在用2-脱氧葡萄糖（2-DOG）处理的细胞中酶活性被抑制，并且需要钙的存在来活化。这些效应与粒细胞O2-产生的效应相似。因此，粒细胞O2生成酶系统位于富含质膜标记物的部分上，并且粒细胞产生的动力学与该酶的活化速率和量直接相关。
The subcellular localization, kinetics of activation, and substrate specificity of the guinea pig granulocyte superoxide (O2-) generating system was investigated. Membrane-enriched particles (podosomes) were made from granulocytes by mild sonication and differential centrifugation. These podosomes are enriched threefold for known plasma membrane markers, 5'-nucleotidase, and adenylate cyclase. Podosomes made from resting granulocytes have very little NAD(P)H-dependent O2- production. Podosomes made from cells stimulated with digitonin are equally enriched for membrane markers but have a 15- to 20-fold increase in NAD(P)H-dependent O2- production. The KmAPP for NADPH is one-tenth that for NADH, but the Vmax is the same. The kinetics of digitonin-stimulated whole-cell O2- production parallel the changes in enzyme activity in these podosomes. Temperature affects both the rate and extent of activation of this enzyme. The pH optimum for the enzyme, the pH optimum for activation, and the pH optimum for whole-cell O2- production are all 7.5. Enzyme activity is increased if the cells are treated with glucose and cyanide, inhibited in cells treated with 2-deoxyglucose (2-DOG), and requires the presence of calcium for activation. These effects are similar to those found for granulocyte O2- production. Thus, the granulocyte O2- generating enzyme system is located on a fraction enriched for plasma membrane markers, and the kinetics of granulocyte production are directly related to the rate and amount of activation of this enzyme.