Kulmacz R J, Wu K K
Department of Biological Chemistry, University of Illinois, Chicago 60612.
Arch Biochem Biophys. 1989 Feb 1;268(2):502-15. doi: 10.1016/0003-9861(89)90317-2.
Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [acetyl-14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme.(ABSTRACT TRUNCATED AT 400 WORDS)
前列腺素H合酶催化多不饱和脂肪酸转化为前列腺素、血栓素和前列环素的第一步反应。该酶通常结合在内质网膜上，但用去污剂溶解后可纯化至均一状态。通过检测合酶对蛋白酶降解的敏感性、血红素对这种蛋白酶敏感性的影响以及产生的蛋白水解片段的大小，比较了微粒体形式和纯去污剂溶解形式的合酶的拓扑结构。对于微粒体合酶，还确定了蛋白水解片段的定位。蛋白水解消化后对微粒体蛋白的分析包括通过聚丙烯酰胺凝胶电泳进行分离，并用针对纯合酶的多克隆抗体选择性检测合酶衍生的多肽。对于微粒体合酶和纯合酶，用胰蛋白酶孵育都会导致环氧化酶活性逐渐丧失，合酶亚基（70 kDa）裂解为38 kDa和33 kDa的两个片段。用蛋白酶K和胰凝乳蛋白酶孵育去污剂溶解形式的合酶也产生了非常相似的一对片段（38 kDa和33 kDa）。用胰蛋白酶孵育微粒体后，合酶的38 kDa和33 kDa片段都仍与膜结合；胰蛋白酶未从微粒体中释放出可溶形式的环氧化酶活性。此外，无论是胰蛋白酶还是蛋白酶K都未从其合酶已用[乙酰-14C]-阿司匹林标记的微粒体中释放出可溶的放射性标记肽。对于微粒体合酶，血红素的耗尽会增强对蛋白酶的敏感性（用蛋白酶K孵育90分钟后，66%的环氧化酶活性丧失），而添加血红素则会降低敏感性（仅20%的活性丧失），正如先前对去污剂溶解的合酶所证明的那样。在纯合酶与胰蛋白酶孵育的几个时间间隔中的每一个，合酶多肽的裂解程度与环氧化酶活性丧失的程度都有合理的良好相关性；在补充了不同量血红素的纯合酶消化物中也观察到了蛋白水解裂解与活性丧失之间的类似关系。（摘要截于400字）
