Use of different derivatives of D-Val-Leu-Arg for studying kallikrein activities in cat submandibular glands and saliva

Abstract

Glandular kallikrein shows a special selectivity for D-Val-Leu-Arg-4-methoxy-2-naphthylamide in comparison with other potential oligopeptide substrates and it provides a useful histochemical substrate, although the reaction may not always be specific. However, in cat submandibular saliva, a biochemical assay using the closely related D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin (AFC) as substrate, which affords more sensitive detection, showed that soya bean trypsin inhibitor causes no inhibition. This indicates that there are unlikely to be contaminating enzymes competing for the substrate in this body fluid. Support for this observation has been gained by the useful new enzyme overlay membrane technique for fluorescent assessment of reactive bands of enzymes after isoelectric focusing, using membranes of cellulose acetate impregnated with D-Val-Leu-Arg-AFC. Comparison of results after isoelectric focusing of purified cat submandibular kallikrein with samples of cat submandibular saliva confirmed that the substrate is monospecific for kallikrein in saliva of the cat. This knowledge has enabled us to start assessing the dynamics of the secretion of kallikrein by the gland. Testing individual drops of saliva has shown that an amazingly rapid mobilization of kallikrein occurs in high concentrations on sympathetic nerve stimulation. The corresponding oligopeptide-based inhibitor D-Val-Leu-Arg-chloromethyl ketone was found to be strongly inhibitory of the amidase reaction by kallikrein but showed a low specificity for kallikrein. Nevertheless, its effects have been tested in vivo by the intravascular route and it caused an increase in the resting salivary vascular resistance whether administered close-arterially or intravenously. Thus, it would seem that a kallikrein-like protease does influence the background tone in the vessels and the source of this enzyme is thought to be mast cells.