Samarel A M, Worobec S W, Ferguson A G, Lesch M
J Biol Chem. 1984 Apr 25;259(8):4702-5.
Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.
兔心脏组织中的组织蛋白酶D以分子量为48,000的多种异构体形式存在。其形成机制及其在心脏蛋白质降解中的功能作用尚不清楚。我们之前已经证明，组织蛋白酶D最初以分子量为53,000的前体形式合成，该前体在心脏溶酶体内通过有限的蛋白水解作用加工成分子量为48,000的酶的活性形式。为了确定活性组织蛋白酶D的多种形式是否源自共同的前体，在脉冲（15或30分钟）和脉冲追踪（30或150分钟）实验中，用[35S]甲硫氨酸标记离体灌注的Langendorff兔心脏。通过丁醇/Triton X-100提取和用抗组织蛋白酶D IgG-琼脂糖进行免疫吸附来分离新合成的组织蛋白酶D，并且通过二维电泳和荧光自显影分离异构体形式。在15分钟和30分钟的脉冲灌注后，35S标记的组织蛋白酶D以单一前体形式出现（分子量 = 53,000，等电点 = 6.6）。在30分钟脉冲和30分钟追踪后，前体被修饰以产生多种前体形式，所有这些形式的分子量均为53,000，但等电点不同（6.6 - 6.0）。在30分钟脉冲和150分钟追踪灌注后，检测到前体和经蛋白水解加工的活性组织蛋白酶D的多种形式（分子量 = 48,000，等电点 = 6.2 - 5.6）。35S标记的、经蛋白水解加工的活性组织蛋白酶D形式与心脏组织中存在的主要组织蛋白酶D形式共迁移。亚细胞分级分离和在氯喹存在下的灌注表明，组织蛋白酶D的多种前体形式起源于非溶酶体的细胞内区室。因此，活性组织蛋白酶D的多种形式源自共同的高分子量前体，并且它们的合成发生在心脏溶酶体内前体的有限蛋白水解之前。
