Samarel A M, Ogunro E A, Ferguson A G, Allenby P, Lesch M
J Clin Invest. 1982 Apr;69(4):999-1007. doi: 10.1172/jci110540.
Production of active lysosomal enzymes may involve limited proteolysis of inactive high molecular weight precursors. Precursor processing potentially regulates lysosomal enzyme activity. To test whether rabbit cardiac cathepsin D is first synthesized as a precursor and whether prolonged fasting (a condition affecting both cathepsin D and total cardiac protein turnover) influences precursor processing, rates of cathepsin D synthesis and processing were compared in left ventricular slices of control and 3-d-fasted rabbits incubated in vitro with [(35)S]methionine. (35)S-labeled cathepsin D was isolated by butanol-Triton X-100 extraction, immunoprecipitation, and dodecyl sulfate-polyacrylamide gel electrophoresis. Total cardiac protein synthesis was measured by tracer incorporation and normalized for differences in precursor pool size by direct measurement of [(35)S]aminoacyl-tRNA-specific radioactivity. Relative cathepsin D synthetic rates were obtained by comparing (35)S incorporation into cathepsin D with (35)S incorporation into all cardiac proteins. Enzyme processing was assessed in pulse-chase experiments and assayed by autoradiography. The results indicate that (a) rabbit cardiac cathepsin D is synthesized as a precursor (53,000 mol wt) that is processed to a 48,000-mol wt form, (b) rates of both cathepsin D and total cardiac protein synthesis are similar in control and fasted rabbits, suggesting that decreased enzyme degradation rather than increased synthesis is responsible for the elevated levels of cardiac cathepsin D in starvation, and (c) cathepsin D processing in hearts of fasted animals is incomplete, with accumulation of the precursor during pulse-chase experiments of 6 h duration. Based upon these results, a three-stage model for the regulation of cathepsin D activity in rabbit heart is proposed.
活性溶酶体酶的产生可能涉及对无活性高分子量前体的有限蛋白水解。前体加工可能调节溶酶体酶的活性。为了测试兔心脏组织蛋白酶D是否首先以前体形式合成，以及长期禁食（一种影响组织蛋白酶D和总心脏蛋白质周转的情况）是否影响前体加工，在体外将[（35）S]甲硫氨酸与对照兔和禁食3天的兔的左心室切片一起孵育，比较组织蛋白酶D的合成和加工速率。通过丁醇 - 曲拉通X - 100提取、免疫沉淀和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离[（35）S]标记的组织蛋白酶D。通过示踪剂掺入测量总心脏蛋白质合成，并通过直接测量[（35）S]氨酰 - tRNA特异性放射性对前体库大小的差异进行归一化。通过比较[（35）S]掺入组织蛋白酶D与[（35）S]掺入所有心脏蛋白质来获得相对组织蛋白酶D合成速率。在脉冲追踪实验中评估酶加工，并通过放射自显影进行测定。结果表明：（a）兔心脏组织蛋白酶D以前体（53,000道尔顿分子量）形式合成，该前体被加工成48,000道尔顿分子量的形式；（b）对照兔和禁食兔中组织蛋白酶D和总心脏蛋白质的合成速率相似，这表明饥饿时心脏组织蛋白酶D水平升高是由于酶降解减少而非合成增加所致；（c）禁食动物心脏中的组织蛋白酶D加工不完全，在持续6小时的脉冲追踪实验中前体积累。基于这些结果，提出了兔心脏中组织蛋白酶D活性调节的三阶段模型。
