Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase.
1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10.5) extraction of cytokinase from rabbit kidney lysosome-microsome fraction, followed by chromatography on DEAE-cellulose at pH7.6 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. 3. Cytokinase is a protein with E(0.1%) (1cm.)=0.87 at 280mmu, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S(20,w) 2.9-3.1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8.5. It is inactivated by heat at 78 degrees . 4. Cytokinase and human urokinase have the same K(m) value and are inhibited in a partially competitive manner by in-aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.
D000818:Animals; D000933:Antifibrinolytic Agents; D002417:Cattle; D002482:Cellulose; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005343:Fibrinolytic Agents; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010958:Plasminogen; D011817:Rabbits; D012756:Sheep; D012995:Solubility; D013053:Spectrophotometry; D013300:Streptokinase / Q000032:analysis* / Q000037:antagonists & inhibitors; D013552:Swine
D000933:Antifibrinolytic Agents; D005343:Fibrinolytic Agents; D010958:Plasminogen; D002482:Cellulose; D013300:Streptokinase