A unique protease responsible for selective degradation of a yolk protein in Bombyx mori. Purification, characterization, and cleavage profile.
A unique protease responsible for selective degradation of a yolk protein in Bombyx mori. Purification, characterization, and cleavage profile.
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DOI：
10.1016/s0021-9258(19)35459-6
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发表时间：
1988-01
期刊：
影响因子：
--
通讯作者：
L. S. Indrasith;T. Sasaki;O. Yamashita
中科院分区：
文献类型：
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作者：
L. S. Indrasith;T. Sasaki;O. Yamashita
卵黄蛋白是家蚕卵巢发育过程中合成和积累的一种卵黄蛋白，不仅是胚胎发育的营养来源，也是卵黄系统有限降解重组的营养来源。以纯化的鸡蛋特异性蛋白为底物，在胚胎中鉴定了一种负责其有限水解酶的蛋白酶，并对其进行了纯化。该酶表观分子量为30,500，一个亚基为29,000道尔顿。它以精氨酸或赖氨酸残基上的羰基键对合成底物进行水解，并被二异丙基氟磷酸盐、苯基甲烷磺酰氟和亮氨酸酶强烈抑制，表明它是一种类胰蛋白酶。与其他蛋黄蛋白相比，该酶对鸡蛋特有蛋白具有极高的特异性(超过2000倍)。完整的卵子特异蛋白分两步被切割成三个片段：第一个步骤释放一个8.7 kDa的多肽作为最终产物和一个55 kDa的多肽中间体，第二个中间体被切割成36 kDa和17.2 kDa的多肽。通过将这些多肽的NH2末端氨基酸序列与完整的鸡蛋特异性蛋白的序列进行比较，发现该酶首先在Lys-Asn处切割，然后在Arg-Asp处切割。蛋白水解酶活性在胚胎发育中期突然出现，并在幼虫分化完成时急剧增加。
A yolk protein, egg-specific protein, synthesized and accumulated in the developing ovaries of Bombyx mori serves not only as the nutritive source for embryogenesis but also for the reorganization of the yolk system through limited degradation. Using the purified egg-specific protein as a substrate, a protease responsible for its limited hydrolysis was identified in embryonating eggs and purified to homogeneity. The protease had an apparent molecular mass of 30,500 with one subunit of 29,000 daltons. It hydrolyzes synthetic substrates at carbonyl bonds of Arg or Lys residues, and the hydrolysis is strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and leupeptin, suggesting that it is a trypsin-like protease. The protease shows an extremely high degree (over 2,000-fold) of specificity for egg-specific protein compared to other yolk proteins. Intact egg-specific protein is cleaved into three fragments in two steps; the first releases a 8.7-kDa peptide as an end product and a 55-kDa peptide intermediate, and in the second the intermediate is cleaved into 36- and 17.2-kDa peptides. By relating the NH2-terminal amino acid sequences of these peptides to the sequence of the intact egg-specific protein, the protease was shown to cleave first at a Lys-Asn site and secondly at Arg-Asp. Proteolytic activity abruptly appears mid-way in embryogenesis and increases steeply during completion of larval differentiation.