人组织蛋白酶D

Human cathepsin D.

作者信息

Barrett A J

出版信息

Adv Exp Med Biol. 1977;95:291-300. doi: 10.1007/978-1-4757-0719-9_17.

Abstract

Cathepsin D was purified from human liver by a procedure involving autolysis, acetone fractionation, and chromatography on ion-exchange media and organomercurial-sepharose. Multiple forms of the enzyme were then separated by preparative isoelectric focusing. The molecular weight of the protein was found to be 43,000. Its amino acid composition was determined and it was shown to be a glycoprotein. When treated with sodium dodecyl sulphate or chaotropic agents (without reduction) all forms of the enzyme tested gave components of about 28,000 and 14,000 molecular weight. Specific antisera were raised against the enzyme, and the characteristics of immunoinhibition were investigated. Immuno-inhibition of rabbit cathepsin D within living macrophages was shown to interfere with degradation of some proteins endocytosed by the cells. The antisera against human and rabbit cathepsin D were used in immunofluorescent localization of the enzyme in sites of tissue damage in which cathepsin D might be implicated. The characteristics of inhibition of human cathepsin D by pepstatin were established. At pH values below 5, KD values of 5 x 10(-10)M were determined and pepstatin was shown to be an excellent titrant for cathepsin D. In the range pH 5-6.4 DK increased steeply and it was concluded that the binding site for substrate and inhibitor was abolished by a conformational change in the enzyme molecule in which three protons are lost.

摘要

组织蛋白酶D是从人肝脏中纯化得到的，其纯化过程包括自溶、丙酮分级分离以及在离子交换介质和有机汞琼脂糖上进行色谱分离。然后通过制备性等电聚焦分离该酶的多种形式。发现该蛋白质的分子量为43,000。测定了其氨基酸组成，并表明它是一种糖蛋白。用十二烷基硫酸钠或离液剂（不进行还原处理）处理时，所测试的所有形式的酶都产生了分子量约为28,000和14,000的组分。制备了针对该酶的特异性抗血清，并研究了免疫抑制的特性。结果表明，兔组织蛋白酶D在活巨噬细胞内的免疫抑制作用会干扰细胞内吞的某些蛋白质的降解。用人和兔组织蛋白酶D的抗血清对该酶在可能涉及组织蛋白酶D的组织损伤部位进行免疫荧光定位。确定了胃蛋白酶抑制剂对人组织蛋白酶D的抑制特性。在pH值低于5时，测定的KD值为5×10⁻¹⁰M，并且表明胃蛋白酶抑制剂是组织蛋白酶D的一种优良滴定剂。在pH 5 - 6.4范围内，DK急剧增加，得出的结论是，酶分子发生构象变化并失去三个质子时，底物和抑制剂的结合位点被消除。