A protease that increases during a period of enzymic and metabolic adjustment in Tetrahymena.

The specific activity of a neutral protease (assayed at pH 8, using azocasein as substrate) in Tetrahymena doubled or tripled within a few hours after the onset of shaking of statically grown, stationary phase cultures. The increase occurred during a period when several peroxisomal enzymes were decreasing. The increase was prevented by actinomycin D or cycloheximide, both of which also prevented the decrease in peroxisomal enzymes. Protease activity towards hemoglobin at pH 3.6 increases during this period, but to a lesser extent, while activity towards BANA (α-N-benzoyl-d,l-arginine 2-naphthylamide) was almost unchanged. The three protease activities have been partially purified by gel filtration and affinity chromatography, and are indistinguishable on this basis. Chromatography on DEAE-Sephadex yields three peaks having activity towards BANA but not towards hemoglobin and azocasein, and two peaks having activity towards all three substrates. The activities towards azocasein and hemoglobin are also indistinguishable on the basis of sensitivity to a variety of inhibitors, to temperature, and chromatography on CM Sephadex. The partially purified protease has an absolute sulfhydryl requirement when azocasein is used as substrate and is inhibited by leupeptin, chymostatin, TLCK, TPCK, and iodacetamide but not by pepstatin or PMSF. Activity towards BANA is much more susceptible to these inhibitors than is that towards azocasein. About half of the activity towards azocasein sediments with the large particle (40,000g-min) fraction. The distribution between two components of this fraction resembles that of a lysosomal marker. However, the activity did not follow the distribution of marker enzymes of any of the typical cell organelles when either subfraction was centrifuged through a sucrose density gradient, nor did it follow; the distribution pattern of the other two protease activities. Much of the activity, in fact, remained at the top of the gradient, even after repeated washings of the particulate fraction or fractionation in the presence of a membrane-stabilizing agent or a protease inhibitor. The protease or proteases appears to be in part responsible for the rapid loss of enzyme activity that is characteristic of Tetrahymena homogenates. The existence of a protease that can attack cellular enzymes at physiological pH suggests that extralysosomal breakdown of proteins can occur in a eukaryotic cell and may be of importance in the regulation of cellular enzyme levels.