Inhibition of Free and Bound Trypsin‐Like Enzymes

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Abstract
The inhibition of .beta.-naphthylamidase activity of free trypsin, trypsin-Sepharose and a trypsin-like neutral protease on the surface of tumor cells [mouse Ehrlich ascites cells] have been studied in independent systems and with mixtures of free trypsin plus surface-bound trypsin. High-molecular-weight (protein) inhibitors of free trypsin are less effective inhibitors of trypsin-Sepharose and fail to inhibit the cell surface neutral protease. Inhibition of mixtures of free trypsin plus trypsin-Sepharose follows independent kinetic plots for each component. The free trypsin is reacted before any Sepharose-bound trypsin reacts with high-molecular-weight inhibitors. Low-molecular-weight inhibitors of trypsin inhibit bound trypsin equally well. Papain-derived peptides from high-molecular-weight inhibitors of trypsin inhibit free trypsin, trypsin-Sepharose and the cell-surface neutral protease almost equally well. Fluorescence microscopy has shown that a high-molecular-weight inhibitor of trypsin does not bind to the tumor cell-surface neutral protease, but it does bind to trypsin-Sepharose. The cell-surface neutral protease was capable of activation of latent .beta.-naphthylamidase activity in the presence of excess extracellular inhibitors of free trypsin. The mechanism by which trypsin-Sepharose remains partially active in the presence of excess inhibitor necessary to inhibit an equivalent quantity of free trypsin has been discussed. A search for inhibitors which are selectively active against the cell-surface neutral protease and have no action on trypsin-like enzymes in free solution must take into account the modifying effects of the cell surface on neutral protease activity.

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