Shi H, Cenedella R J
Department of Biochemistry, Kirksville College of Osteopathic Medicine, MO 63501.
J Lipid Res. 1993 Dec;34(12):2177-82.
We have attempted to map the regional distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme activity and protein mass along the radial axis of the ocular lens. Because lens plasma membrane is extremely rich in cholesterol and the lens must synthesize this cholesterol, the activity of HMGR could be a key factor controlling lens membrane formation. Lenses of young rats were divided into discrete fractions by gradual dissolution in a tergitol-containing buffer; each fraction was then equated to a specific arc of the radius based upon its protein content. Aliquots of each fraction were assayed for HMGR enzyme activity and protein mass. Relative protein mass was quantitatively estimated by Western blotting using a monoclonal antibody to HMGR with immunoreactivity detected by enhanced chemiluminescence. Lens HMGR possessed a molecular mass of about 97 kDa and localized in the cell's insoluble fraction. Peak levels of both HMGR enzyme activity and protein mass were found in the outer 5% of the lens radius; levels of both decreased precipitously from there to the outer 10% radius mark. This distribution paralleled synthesis of the membrane's cholesterol, phospholipid, and intrinsic protein. We conclude that the abrupt cessation of plasma membrane synthesis in the ocular lens could involve loss of HMGR activity over a narrow arc of the lens radius, and that this activity loss is due to disappearance of enzyme protein.
我们试图绘制3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）的酶活性及蛋白质含量沿晶状体径向轴的区域分布情况。由于晶状体质膜富含胆固醇，且晶状体必须合成这种胆固醇，因此HMGR的活性可能是控制晶状体膜形成的关键因素。将幼鼠的晶状体置于含有辛烷基酚聚氧乙烯醚的缓冲液中逐步溶解，从而分成不同的组分；然后根据各组分的蛋白质含量，将其对应于半径上的特定弧度。对每个组分的等分试样进行HMGR酶活性及蛋白质含量的测定。通过使用针对HMGR的单克隆抗体进行蛋白质免疫印迹法，并采用增强化学发光法检测免疫反应性，对相对蛋白质含量进行定量估算。晶状体HMGR的分子量约为97 kDa，定位于细胞的不溶性组分中。HMGR的酶活性及蛋白质含量的峰值均出现在晶状体半径外5%的区域；从该区域到半径外10%的标记处，二者的含量均急剧下降。这种分布与膜胆固醇、磷脂及内在蛋白的合成情况平行。我们得出结论，晶状体质膜合成的突然停止可能涉及在晶状体半径的狭窄弧度内HMGR活性的丧失，且这种活性丧失是由于酶蛋白的消失所致。
