从墨菲-斯特姆淋巴肉瘤中纯化和鉴定一种酸性蛋白酶及血管肽激肽

Bedi G S, Balwierczak J, Back N

Adv Exp Med Biol. 1983;156 (Pt B):705-26.

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was isolated, purified and characterized. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. The purification was carried out in seven steps involving homogenization of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation to 60% saturation, DEAE-Sephadex batch treatment, chromatography on a QAE-Sephadex column, gel filtration on Sephadex G-200 and finally chromatography on CM-32 cellulose. The purification scheme resulted in a 640-fold purification and a homogeneous preparation as confirmed by disc gel electrophoresis and Ouchterlony immunodifussion. Sephadex G-200 elution profiles indicated two different protease fractions containing protease activity which showed a single band with identical relative mobility on polyacrylamide gel electrophoresis in the presence and absence of SDS, and also showed immunological cross-reactivity against anti-acid protease antiserum raised in the rabbit against one of the fractions. The acid protease is presumed to be a single protease which has a tendency to aggregate. An apparent molecular weight of 39,500 was estimated by gel filtration on Sephadex G-200, and 41,000 by polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis showed that the acid protease was comprised of a major band with a molecular weight of 4,000 and two fainter bands with molecular weights of 27,000 and 12,000. The purified enzyme showed three major isozymic forms (alpha, beta, gamma) which had isoelectric points (pI) of 5.2, 5.5 and 5.8 respectively, and had near identical amino acid compositions. The carbohydrate moiety contained 2 mol of N-acetylglucosamine and 8 mol of mannose per mol enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0. The protease activity was very stable above pH 3.4. The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents per mg substrate after 2 hr incubation at 37 degrees. The vasopeptide was purified to electrophoretic homogeneity by gel filtration on Sephadex G-50 and chromatography on a column of CM-Sephadex. The amino acid composition of vasopeptide was Ser2, Gly1, Pro4, Ile1, Leu1, Phe2, Arg2.

从大鼠墨菲 - 斯特姆淋巴肉瘤（MSLS）肿瘤中分离、纯化并鉴定了一种酸性蛋白酶。该蛋白酶可水解牛血红蛋白，与纯化的大鼠血浆激肽原一起孵育时会形成血管肽激肽。纯化过程分七个步骤进行，包括肿瘤组织匀浆、用冰醋酸进行酸沉淀、硫酸铵沉淀至60%饱和度、DEAE - 葡聚糖批量处理、在QAE - 葡聚糖柱上进行色谱分离、在Sephadex G - 200上进行凝胶过滤，最后在CM - 32纤维素上进行色谱分离。纯化方案实现了640倍的纯化，经圆盘凝胶电泳和奥克特洛尼免疫扩散证实为均一制剂。Sephadex G - 200洗脱图谱显示有两个不同的含有蛋白酶活性的组分，在有无SDS的情况下，聚丙烯酰胺凝胶电泳显示它们具有相同相对迁移率的单一条带，并且对兔抗其中一个组分产生的抗酸性蛋白酶抗血清也显示出免疫交叉反应性。推测该酸性蛋白酶是一种有聚集倾向的单一蛋白酶。通过在Sephadex G - 200上进行凝胶过滤估计其表观分子量为39,500，通过聚丙烯酰胺凝胶电泳估计为41,000。SDS - 聚丙烯酰胺凝胶电泳显示该酸性蛋白酶由一条分子量为4,000的主要条带和两条分子量分别为27,000和12,000的较淡条带组成。纯化后的酶显示出三种主要的同工酶形式（α、β、γ），其等电点（pI）分别为5.2、5.5和5.8，且氨基酸组成几乎相同。每摩尔酶的碳水化合物部分含有2摩尔的N - 乙酰葡糖胺和8摩尔的甘露糖。消化牛血红蛋白的最适pH约为3.0。蛋白酶活性在pH 3.4以上非常稳定。在37℃孵育2小时后，酸性蛋白酶从纯化的大鼠血浆激肽原中释放激肽的初始速率很快，2小时后达到460纳克缓激肽当量/毫克底物的平台期。通过在Sephadex G - 50上进行凝胶过滤和在CM - 葡聚糖柱上进行色谱分离，将血管肽纯化至电泳均一性。血管肽的氨基酸组成为Ser2、Gly1、Pro4、Ile1、Leu1、Phe2、Arg2。