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Cells possessing a known enzymic activity may be located by fluorescent probes designed to act as competitive inhibitors of this enzyme. We have prepared a series of dansyl N‐substituted guanidino derivatives which bind to the active centre of guanidinobenzoatase. 9‐Aminoacridine also acts as a competitive inhibitor and behaves similarly to these guanidino derivatives. These fluorescent probes have been used to locate tumour cells possessing this enzyme in thin sections of fixed tissue by employing fluorescent microscopy.

Human and bovine spermatozoa have been collected and washed repeatedly with isotonic saline to remove seminal plasma inhibitors and activate the acrosin. Then the acrosin activity of the cells was assayed with alpha-N-Benzoyl-DL-Arg-beta-naphthylamide (BANA). It was found that the surface-bound enzyme was not inhibited by high molecular weight inhibitors of trypsin but was markedly inhibited by low molecular weight trypsin inhibitors. Divalent metals (Zn++, Cu++, Hg++, Co++, Cd++) were all efficient inhibitors of acrosin on the washed cells. It was shown that the removal of zinc or copper from acrosin completely restored activity. It is proposed that the different levels of zinc in the male and female genital tract regulate acrosin activity. Aged cells released a soluble acrosin which was inhibited by serum and seminal plasma inhibitors of trypsin-like enzymes as well as by zinc ions in an identical manner to the surface-bound enzyme.

Summary.-Ehrlich ascites cells in mice have been shown to have a cell-surface trypsin-like neutral protease (TLNP) with proteolytic and p-naphthylamidase activity. This activity is inhibited by low-mol.-wt inhibitors of trypsin but not by 11 high-mol. -wt inhibitors of trypsin in free solution. We believe this lack of inhibition is due to protection given to the enzyme by the chemical environment of the cell surface. These cells were demonstrated to export a collagenase zymogen which has been shown to be activated by the cell-surface TLNP. When this protease was completely inhibited by low-mol.-wt inhibitors of trypsin, chymotrypsin was used to activate the collagenase zymogen exported by Ehrlich ascites cells. Examination of the products of collagenolysis at 15°C demonstrated the expected 3-and 4-length a-chain fragments derived from monomeric collagen, confirming that collagenase was one of the enzymes responsible for lysis of the collagen fibrils in the test system.

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