Biochemical characterization of secreted proteases during growth in Tetrahymena pyriformis WH-14: comparison of extracellular with intracellular proteases.
Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and alpha-N-benzoyl-DL-arginine-rho-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000-24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3 and 1.4 mM for P-I, P-II, P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S-1 . M-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002364:Caseins / Q000378:metabolism; D000939:Epitopes; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D013769:Tetrahymena pyriformis / Q000201:enzymology* / Q000254:growth & development
D000596:Amino Acids; D002364:Caseins; D000939:Epitopes; C026018:azocasein; D010447:Peptide Hydrolases