Rodent kinin-forming enzyme systems--II. Purification and characterization of an acid protease from Murphy-Sturm lymphosarcoma.
An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was purified 640-fold by extraction of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation, DEAE-Sephadex A-50 batch adsorption, QAE-Sephadex A-50 column chromatography, Sephadex G-200 gel filtration, and CM-32 cellulose chromatography. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. Two protease fractions obtained by Sephadex G-200 gel filtration had identical molecular weights of 39,500-41,000 and were similar in other physico-chemical and kinetic characteristics. The purified enzyme showed three major isozymic forms (alpha, beta and gamma) with isoelectric points (pI) of 5.2, 5.5 and 5.8, respectively, and nearly identical amino acid compositions. The enzyme had a high moles percent of both aspartic and glutamic acids. The carbohydrate moiety of the enzyme contained 2 moles of N-acetylglucosamine and 8 moles of mannose per mole of enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0 with a sharp decline of activity on either side of the pH optimum. The protease activity was very stable above pH 3.4. The Km values for the purified enzyme fractions A and B were 31.17 and 31.19 microM, respectively, and the corresponding Vmax values were 6.17 and 5.5 microM tyrosine per mg per min at 37 degrees and pH 3.0. The enzyme was inhibited strongly by pepstatin (Ki = 31 X 10(-9)M and alpha = 0.1). The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents/mg substrate after a 2-hr incubation at 37 degrees.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D010450:Endopeptidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007705:Kinins / Q000378:metabolism; D008228:Lymphoma, Non-Hodgkin / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009368:Neoplasm Transplantation; D051381:Rats; D011919:Rats, Inbred Strains; D012513:Sarcoma, Experimental / Q000201:enzymology*
D000596:Amino Acids; D002241:Carbohydrates; D007705:Kinins; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases