Purification and Characterization of a Secreted Protease from Tetrahymena pyriformis

Abstract

A simple major protease, sevreted into the medium during growth of Tetrahymana pyriformis strain W, has been purified about 4000‐fold by (NH4)2SO4 precipitation, ion‐excange chromatogrphy, gel filtrtion and affinity chromatography on organomercurial‐Sepharose. Thje purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a monomeric protein with a molecular weight of 22000–23000. Amino acid alalysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena proteaseconsists of mutiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5–8.0 with α‐N‐benzoyl‐Dl‐arginie‐P‐nitroanilide and with azocasein, while it was in the lower pH range (4.5–5.5) for denatuired hemoglobins. The purified enzyme was inhibited by componds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena. Copyright © 1983, Wiley Blackwell. All rights reserved