Ma H, Shih M, Hata I, Fukiage C, Azuma M, Shearer T R
Department of Oral Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Exp Eye Res. 1998 Aug;67(2):221-9. doi: 10.1006/exer.1998.0515.
mRNA for a newly discovered isoform of calpain, termed Lp82, was recently discovered in young rat lens. The purpose of the present experiments was to test for expression of Lp82 protein. Casein zymography after incubation with calcium was used to detect Lp82 proteolytic activity in regions of lenses from young rats. Lp82 protein was detected by immunoblotting or by ELISA after DEAE-5PW chromatography using a polyclonal antibody generated to a peptide sequence in Lp82. Northern blot analysis assessed expression of Lp82 mRNA. Four results demonstrated expression of Lp82 protein; (1) immunoblot reactivity at the predicted molecular mass of 82 kDa, (2) a unique band of calcium-activated lysis in casein zymograms, (3) partial purification and retention of activity from a single Lp82 peak on DEAE-5PW chromatography, and (4) positive immunoblotting and Northern blot analysis only in lens and not in other rat tissues. These results showed that Lp82 protein is lens-preferred, relatively abundant in young rats (especially nucleus), and enzymatically active. Proteolysis of crystallins in the nucleus of young rat lens during normal maturation and cataract formation, formerly attributed solely to m-calpain, may in fact be due to concerted action of both lens Lp82 and ubiquitous m-calpain.
最近在幼鼠晶状体中发现了一种新的钙蛋白酶亚型的信使核糖核酸（mRNA），称为Lp82。本实验的目的是检测Lp82蛋白的表达情况。用钙孵育后的酪蛋白酶谱法检测幼鼠晶状体区域中Lp82的蛋白水解活性。使用针对Lp82中一段肽序列产生的多克隆抗体，通过免疫印迹法或在DEAE - 5PW色谱分析后用酶联免疫吸附测定法（ELISA）检测Lp82蛋白。Northern印迹分析评估Lp82 mRNA的表达。四项结果证明了Lp82蛋白的表达：（1）在预测的82 kDa分子量处有免疫印迹反应性；（2）酪蛋白酶谱中有一条独特的钙激活裂解带；（3）在DEAE - 5PW色谱上从单个Lp82峰进行部分纯化并保留活性；（4）仅在晶状体而非其他大鼠组织中免疫印迹和Northern印迹分析呈阳性。这些结果表明，Lp82蛋白在晶状体中优先表达，在幼鼠（尤其是晶状体核）中相对丰富且具有酶活性。在幼鼠晶状体核正常成熟和白内障形成过程中，以前仅归因于m - 钙蛋白酶的晶状体蛋白水解作用，实际上可能是由于晶状体Lp82和普遍存在的m - 钙蛋白酶的协同作用。
