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.