Zirkin B R, Chang T S, Heaps J
J Cell Biol. 1980 Apr;85(1):116-21. doi: 10.1083/jcb.85.1.116.
Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.
先前的研究表明，蛋白水解活性与分离的兔精子细胞核相关，并且是硫醇诱导的细胞核体外解聚过程中发生的核鱼精蛋白降解的原因（Zirkin和Chang，1977；Chang和Zirkin，1978）。在本研究中，我们展示了旨在表征这种蛋白水解活性的实验结果。从兔精子细胞核中分离的碱性蛋白与5 mM二硫苏糖醇（DTT）和1% Triton X-100孵育不同时间后，在酸性尿素聚丙烯酰胺凝胶上呈现出迁移速度逐渐加快的条带，这反映了鱼精蛋白的逐步降解。最终，形成了一种特定且独特的肽条带模式。当用酯酶抑制剂对硝基苯基-对胍基苯甲酸酯（NPGB）处理精子细胞核以抑制与核相关的蛋白水解活性，然后除DTT和Triton X-100外再与几种外源蛋白酶之一孵育时，对于每种使用的外源蛋白酶都观察到了特征性的肽条带模式。对于胰蛋白酶、胰凝乳蛋白酶、链霉蛋白酶和木瓜蛋白酶，肽条带模式彼此不同，也不同于与核相关蛋白酶导致的鱼精蛋白降解的模式。相比之下，当兔顶体蛋白酶作为外源蛋白酶时，观察到的肽条带模式与与核相关蛋白酶的特征模式相同。这些结果直接证明，与兔精子细胞核相关且参与体外精子核解聚的蛋白酶类似顶体蛋白酶。
