Abstract
Ehrlich ascites tumour cells grown in mice have a cell-surface trypsin-like neutral protease (TLNP) which is not inhibited by high-mol.-wt inhibitors of trypsin. This enzyme is inhibited by low concentrations of zinc, which may be removed by chelating agents, with the consequent return of enzymic activity. Gold, provided as the drugs aurothiomalate or auranofin, also inhibits TLNP. The gold can be removed from the enzyme by incremental addition of thiols. The mechanisms of gold transfer to the active site to cause inhibition and subsequent removal of gold with reactivation of TLNP, have been shown to be controlled by reversible thiol-exchange reactions.
Highlights
Summary.-Ehrlich ascites tumour cells grown in mice have a cell-surface trypsinlike neutral protease (TLNP) which is not inhibited by high-mol.-wt inhibitors of trypsin
It was demonstrated that the tumour cell surface TLNP was not inhibited by high-mol. wt proteininhibitors of trypsin in free solution
1978) and the reversible metal-ion inhibi- HC1 (BANA) and (b) even in the presence of tion of trypsin (Steven et al, 1979) suitable carriers, auranofin did not inhibit suggested that these types of compound trypsin in free solution under conditions might control cell-surface TLNP. where the cell surface TLNP was almost
Summary
Assay of trypsin-like neutral protease on Ehrlich ascites cells.-We used the fluorisystem which could transfer the gold from metric /3-naphthylamidase assay of Macthe sodium aurothiomalate to the active Donald et al (1966) as described for these centre of the enzyme (trypsin or TLNP) cells by Steven et al (1980) with the minor without precipitation of the substrate. We were unable to follow the return of active-site titratibility with MUGB in the presence of a fixed quantity of zinc and increasing molarity of EDTA, since EDTA interfered with the MUGB assay This difficulty was overcome by chelating zinc with sodium citrate rather than with EDTA; the re-activation of the TLNP could be followed by adding 3WpM MUGB (Fig. 2).
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