Klein K, Rudy B, McIntyre J O, Fleischer S, Trommer W E
Fachbereich Chemie, Universität Kaiserslautern, Erwin-Schroedinger-Strasse, Federal Republic of Germany.
Biochemistry. 1996 Mar 5;35(9):3044-9. doi: 10.1021/bi952173k.
The interaction of phospholipid with (R)-3-hydroxybutyrate dehydrogenase, a phosphatidylcholine-requiring membrane enzyme, has been studied using ESR spectroscopy of spin-labeled lipids, both as ordered multibilayers and in lipid vesicle suspensions (liposomes). Partially oriented phospholipid multibilayers were prepared from lipid vesicles composed of a 1:1 mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Vesicles containing (R)-3-hydroxybutyrate dehydrogenase yielded active preparations of the enzyme in such multibilayers. With increasing protein/lipid ratio, the order of the multibilayers was disrupted as monitored by ESR spectroscopy with a spin-labeled analogue of PC, 5-doxyl-PC (5 mol %, 10% of total PC) as a probe. The outer peak separation of 5-doxyl-PC varied with the lipid/protein ratio. The lower the ratio, the larger was the separation, with higher activity enzyme being more effective in exerting this effect. When 5-doxylstearic acid was substituted for 5-doxyl-PC or when the enzyme was inactive, the 2A(zz) value stayed practically constant at its lower limit (about 54 G). Multilayers composed of 81% PE, 11% diphosphatidylglycerol (DPG), and 8% 5-doxyl-PC (no unlabeled PC present) gave similar results. With this lipid mixture, the maximal 2A(zz) value (about 61 G) was reached at lower protein/lipid ratios, although the enzymic activity of (R)-3-hydroxybutyrate dehydrogenase is reduced to 40% in this system. The outer peak separation also depended on the presence of the coenzyme, NAD+, and 2-methylmalonate. The latter enhances binding of NAD+ about 100-fold by forming a ternary complex. With this ternary complex, the 2A(zz) values were increased unless the maximal values had been reached already in the absence of coenzyme. In all these experiments only a single ESR spectral component was observed. Similar results were obtained for the enzyme in liposomes, although the effect was less pronounced apparently due to the higher mobility of the probe. It is concluded that PC is motionally restricted by (R)-3-hydroxybutyrate dehydrogenase and yet is in rapid exchange with the bulk lipid on the ESR time scale. PC is required for formation of tight and functional complexes with NAD [Rudy et al. (1989) Biochemistry 28, 5354-5366], and such complexes strengthen the interaction of the enzyme with PC.
利用自旋标记脂质的电子顺磁共振波谱法,对磷脂与(R)-3-羟基丁酸脱氢酶(一种需要磷脂酰胆碱的膜酶)之间的相互作用进行了研究,该研究分别在有序多层膜和脂质囊泡悬浮液(脂质体)中进行。部分定向的磷脂多层膜由磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)按1:1混合组成的脂质囊泡制备而成。含有(R)-3-羟基丁酸脱氢酶的囊泡在这种多层膜中产生了有活性的酶制剂。随着蛋白质/脂质比例的增加,用PC的自旋标记类似物5-二氧代-PC(5摩尔%,占总PC的10%)作为探针,通过电子顺磁共振波谱监测发现多层膜的有序性被破坏。5-二氧代-PC的外峰间距随脂质/蛋白质比例而变化。该比例越低,间距越大,活性越高的酶在发挥这种作用时越有效。当用5-二氧代硬脂酸替代5-二氧代-PC或酶无活性时,2A(zz)值实际上保持在其下限(约54 G)不变。由81%的PE、11%的二磷脂酰甘油(DPG)和8%的5-二氧代-PC(不存在未标记的PC)组成的多层膜给出了类似的结果。对于这种脂质混合物,在较低的蛋白质/脂质比例下达到了最大2A(zz)值(约61 G),尽管在该系统中(R)-3-羟基丁酸脱氢酶的酶活性降低到了40%。外峰间距还取决于辅酶NAD+和2-甲基丙二酸的存在。后者通过形成三元复合物使NAD+的结合增强约100倍。有了这种三元复合物,2A(zz)值会增加,除非在不存在辅酶的情况下已经达到了最大值。在所有这些实验中只观察到了一个电子顺磁共振光谱成分。对于脂质体中的酶也得到了类似的结果,尽管由于探针的流动性较高,这种效应显然不太明显。得出的结论是,PC的运动受到(R)-3-羟基丁酸脱氢酶的限制,但在电子顺磁共振时间尺度上仍与大量脂质快速交换。PC是与NAD形成紧密且功能性复合物所必需的[鲁迪等人(1989年)《生物化学》28卷,5354 - 5366页],并且这样的复合物加强了酶与PC的相互作用。