Dryjański M, Otlewski J, Wilusz T
Institute of Biochemistry, University of Wrocław, Poland.
Acta Biochim Pol. 1990;37(1):169-72.
A new serine proteinase was isolated from Cucurbita ficifolia seeds by the purification procedure, which includes: extraction, salting out with ammonium sulphate, chromatography on CM-cellulose. Sephacryl S-300 gel filtration and h.p.l.c. on DEAE-2SW TSK column. The enzyme was homogeneous both in native and SDS PAGE. Three independent methods showed its molecular mass to be approximately 77 kDa. The enzyme was inhibited by specific serine proteinase organic inhibitors, and was active in the presence of inhibitors specific for other proteinase classes. Surprisingly, squash proteinase exhibited a very high and broad pH optimum with a maximum at 10.7. It hydrolysed many different peptide bonds in B-chain of insulin and was able to cleave four bonds in endogenous serine proteinase inhibitor (CMTI).
通过以下纯化步骤从黑籽南瓜种子中分离出一种新的丝氨酸蛋白酶，该步骤包括：提取、硫酸铵盐析、CM-纤维素柱色谱、Sephacryl S-300凝胶过滤以及在DEAE-2SW TSK柱上进行高效液相色谱。该酶在天然聚丙烯酰胺凝胶电泳和十二烷基硫酸钠聚丙烯酰胺凝胶电泳中均表现为均一性。三种独立方法显示其分子量约为77 kDa。该酶被特异性丝氨酸蛋白酶有机抑制剂抑制，并且在其他蛋白酶类别的特异性抑制剂存在下具有活性。令人惊讶的是，南瓜蛋白酶表现出非常高且宽泛的最适pH值，在pH 10.7时达到最大值。它能水解胰岛素B链中的许多不同肽键，并且能够切割内源性丝氨酸蛋白酶抑制剂（CMTI）中的四个键。
