ISOTOPIC ASSAY FOR NADPH OXIDASE ACTIVITY AND SOME CHARACTERISTICS OF ENZYME FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES
ISOTOPIC ASSAY FOR NADPH OXIDASE ACTIVITY AND SOME CHARACTERISTICS OF ENZYME FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES
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DOI：
10.1172/jci107981
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发表时间：
1975-01-01
影响因子：
15.9
通讯作者：
MCCALL, CE
中科院分区：
文献类型：
--
作者：
DECHATELET, LR;MCPHAIL, LC;MCCALL, CE
已经开发了用于测量由6-磷酸葡萄糖酸脱氢酶反应形成的NADP的量的NADPH伊索德酶的同位素测定。在适当的条件下，NADP的存在量与[1- 14 C]6-磷酸葡萄糖酸释放的14 CO2的量成正比。因为这种检测使用放射性同位素，它比传统的酶检测灵敏得多。如通过该测定所测量的，人颗粒NADPH氧化酶在CN负存在下具有活性，被Mn-2+刺激，并且具有5.5的pth最适值。从已被允许摄取酵母聚糖的细胞中分离的颗粒一致地表现出比从静息细胞或用未经预调理的酵母聚糖攻击的细胞中分离的颗粒更高的酶活性。在宽范围的底物浓度下观察到这种效应，并且不能通过不同样品之间蛋白质浓度的差异来解释。如果使用完整的匀浆代替分离的颗粒，则仅在吞噬细胞的匀浆中可以观察到酶活性，甚至仅在高浓度的NADPH下才能观察到酶活性。这表明细胞内可能存在酶的抑制剂。对一例慢性肉芽肿病患者进行了研究。当比较静息细胞和吞噬细胞的颗粒时，患者细胞的tnadph氧化酶活性没有差异。相比之下，两个对照患者的颗粒中的酶活性在吞噬作用后加倍。这些结果与NADPH氧化酶在伴随人中性粒细胞吞噬作用的呼吸爆发启动中的作用一致。
An isotopic assay for NADPH ixodase that measures the amount of NADP formed by the 6-phosphogluconate dehydrogenase reaction has been developed. Under appropriate conditions, the amount of NADP present is directly proportional to the amount of 14CO2 released from [1-14C]6-phosphogluconic acid. Because this assay employs radioisotopes, it is far more sensitive than conventional assays for the enzyme. The human granule NADPH oxidase, as measured by this assay, is active in the presence of CN minus, is stimulated by Mn-2+, and has a pth optimum of 5.5. Granules isolated from cells that have been allowed to ingest zymosan consistently exhibited more enzyme activity than did granules isolated from either resting cells or cells challenged with zymosan that was not preopsonized. This effect was observed over a wide range of substrate concentrations and could not be explained by differences in protein concentrations between the various samples. If whole homogenates are used in place of isolated granules, the enzyme activity can be observed only with a homogenate of phagocytizing cells and even then only at a high concentration of NADPH. This suggests that an inhibitor of the enzyme might be present within the cell. One patient with chronic granulomatous disease was studied. There was no difference in tnadph oxidase activity of the patients' cells when granules from resting and phagocytizing cells were compared. In contrast, the enzyme activity in granules from two control patients doubled upon phagocytosis. These results are consistent with a role for NADPH oxidase in the initiation of the respiratory burst accompanying phagocytosis by human neutrophils.