On the relation between a stearoyl-specific transacylase from bovine testis membranes and a copurifying acyltransferase.
On the relation between a stearoyl-specific transacylase from bovine testis membranes and a copurifying acyltransferase.
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关于来自牛睾丸膜的硬脂酰特异性转酰基酶与共纯化酰基转移酶之间的关系。
DOI：
10.1021/bi971749y
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Glomset,JA
中科院分区：
文献类型：
--
作者：
Hollenback,D;Glomset,JA
牛睾丸膜含有一种辅酶a依赖的转酰基酶，它可以催化硬脂酰基团从磷酸甘油优先转移到溶血磷脂酸和溶血磷脂酰肌醇的2-酰基分子物种[Itabe et al.,(1992)]。医学杂志。[j].化学与工程学报，1997,18(2):357 - 357。我们现在已经将这种酶纯化了1000倍，并证明它与酰基转移酶结合。纯化的转酰基酶可以使用磷脂酸、磷脂酰肌醇或磷脂酰肌醇-4-磷酸作为酰基供体，并催化硬脂酰基团的转移，而不是棕榈酰基或油酰基。相比之下，纯化的酰基转移酶使用酰基辅酶A作为酰基供体，并且对硬脂酰基转移没有这种偏好。此外，磷脂酰肌醇-4,5-二磷酸对这两种酶的抑制程度和机制也不同。然而，这两种酶在几个方面是相似的：它们使用相同的酰基受体，当它们一起测定时，会竞争酰基受体sn-2-油基溶血磷脂酸；它们平行失去活性，除非加入阴离子磷酸甘油酯或硬脂酰辅酶A来稳定；它们对热和ph的敏感性相似。解释这些结果的一种方法是假设转酰基酶反应发生在两个连续的步骤中：第一步是硬脂酰特异性，其中硬脂酰基从1-硬脂酰-2-酰基磷酸甘油酯转移到辅酶a，第二步是相对非酰基链特异性，其中硬脂酰基从硬脂酰辅酶a转移到2-酰基溶磷甘油酯。根据这条推理线，我们使用的转酰基酶测定测量了两个步骤的净效应，而酰基转移酶测定只测量了第二步的效果。
Bovine testis membranes contain a coenzyme A-dependent transacylase that can catalyze the preferential transfer of stearoyl groups from phosphoglycerides tosn-2-acyl molecular species of lysophosphatidic acid and lysophosphatidylinositol [Itabe et al., (1992)J. Biol. Chem. 267, 15319−15325]. We have now purified this enzyme 1000-fold and shown that it copurifies with an acyltransferase. The purified transacylase can use phosphatidic acid, phosphatidylinositol, or phosphatidylinositol-4-phosphate as an acyl donor and catalyzes the transfer of stearoyl groups in preference to palmitoyl groups or oleoyl groups. In contrast, the purified acyltransferase uses acyl-coenzyme A as an acyl donor and shows no such preference for stearoyl group transfer. Furthermore, phosphatidylinositol-4,5-bisphosphate inhibits the two enzymes to different extents and by different mechanisms. Nevertheless, the enzymes are similar in several respects: they use the same acyl acceptors and, when assayed together, compete for the acyl acceptor,sn-2-oleoyl lysophosphatidic acid; they lose activity in parallel unless stabilized by the addition of an anionic phosphoglyceride or stearoyl-coenzyme A; and they show similar sensitivities to heat and pH. One way to explain these results is to postulate that the transacylase reaction occurs in two successive steps: a stearoyl-specific first step in which a stearoyl group is transferred from ansn-1-stearoyl-2-acyl phosphoglyceride to coenzyme A, and a relatively non-acyl-chain-specific second step in which a stearoyl group is transferred from stearoyl-coenzyme A to ansn-2-acyl lysophosphoglyceride. According to this line of reasoning, the transacylase assay that we have used measures the net effect of both steps, whereas the acyltransferase assay measures only the effect of the second step.