New specific assays for tonin and tissue kallikrein activities in rat submandibular glands. Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva.

At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH 4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH 9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using D-Val-Leu-Arg-AFC (DVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not DVLR-AFC. Estimates of the Km values of tissue kallikrein for DVLR-AFC and tonin for ZVKKR-AFC were found to be similar (approx. 20 microM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 microM aprotinin is due to tonin and a similar percentage of the DVLR-AFC hydrolysing activity in the presence of 10 microM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.