ISOLATION AND STRUCTURAL STUDIES OF THE INTACT SCRAPIE AGENT PROTEIN
ISOLATION AND STRUCTURAL STUDIES OF THE INTACT SCRAPIE AGENT PROTEIN
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DOI：
10.1016/0003-9861(87)90380-8
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发表时间：
1987-11-01
影响因子：
3.9
通讯作者：
POTEMPSKA, A
中科院分区：
文献类型：
--
作者：
BOLTON, DC;BENDHEIM, PE;POTEMPSKA, A
通过使用蛋白酶 K 消化的方法纯化痒病剂，产生蛋白质产物 PrP-27-30，其表观质量为 27-30 kDa (D. C. Bolton 等人 (1982) Science 218, 1309-1311 ；S. B. Prusiner 等人 (1982) Biochemistry 21, 6942-6950)。相比之下，33-37 kDa 的糖蛋白 HaSp33-37 是通过不使用蛋白酶消化的程序从受痒病影响的仓鼠大脑中分离出的主要蛋白质成分。含有 HaSp33-37 的纯化级分每毫克蛋白质含有 > 1011 LD50 单位的痒病剂。 HaSp33-37 的蛋白酶 K 消化产生的产物与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹无法区分的 PrP-27-30。确定了HaSp-33-37的前22个残基的氨基酸序列。该序列与经信号蛋白酶处理后预测的 PrP-27-30 前体 N 末端的序列一致 (K. Basler 等人 (1986) Cell 46, 417-418 ；N. K. Robakis 等人 (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381)。 HaSp33-37用N.α.-甲苯磺酰基-L-苯丙氨酸氯甲基酮-胰蛋白酶消化，产生29-32kDa的蛋白质片段；消化后，该部分保留了完整的生物活性。 29-32kDa蛋白质的氨基末端序列对应于HaSp33-37和PrP-27-30的氨基末端之间的中间位置。我们得出结论，HaSp33-37 是瘙痒病原蛋白的完整形式，而 PrP-27-30 是在分离瘙痒病原期间引入蛋白酶 K 时通过蛋白酶 K 降解产生的。
Purification of the scrapie agent by methods using digestion with proteinase K yields a protein product, PrP-27-30, with an apparent mass of 27-30 kDa (D. C. Bolton et al. (1982) Science 218, 1309-1311; S. B. Prusiner et al. (1982) Biochemistry 21, 6942-6950). In contrast, a 33-37 kDa glycoprotein, HaSp33-37, was the major protein component isolated from scrapie-affected hamster brain by a procedure that did not use protease digestion. The purified fractions containing HaSp33-37 had > 1011 LD50 units of the scrapie agent per milligram of protein. Proteinase K digestion of HaSp33-37 gave a product indistinguishable from PrP-27-30 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The amino acid sequence of the first 22 residues of HaSp-33-37 was determined. The sequence coincided with that predicted for the N-terminus of the precursor to PrP-27-30 (K. Basler et al. (1986) Cell 46, 417-418; N. K. Robakis et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6377-6381) after processing by signal protease. HaSp33-37 was digested with N.alpha.-tosyl-L-phenylalanine chloromethyl ketone-trypsin to produce a 29-32 kDa protein fragment; following digestion this fraction retained complete biological activity. The amino terminal sequence of the 29-32 kDa protein corresponded to a position intermediate between the amino termini of HaSp33-37 and PrP-27-30. We conclude that HaSp33-37 is the intact form of the scrapie agent protein and that PrP-27-30 is produced by proteinase K degradation when this enzyme is introduced during isolation of the scrapie agent.