Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

Publisher Website
Google Scholar
Abstract
The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. An alkaline (pH 10.5) extraction of cytokinase from rabbit kidney lysosome-microsome fraction, followed by chromatography on DEAE-cellulose at pH 7.6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. Cytokinase is a protein with E0.1%1 cm = 0.87 at 280 m[mu], and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S20,w 2.9-3.1 s and molecular weight 50,000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH 8 and pH 9, and is maximally active and stable at pH 8.5. It is inactivated by heat at 78[degree]. Cytokinase and human urokinase have the same Km value and are inhibited in a partially competitive manner by [epsilon]-aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.