T-KININOGENASE ACTIVITY OF THE RAT SUBMANDIBULAR-GLAND IS PREDOMINANTLY DUE TO THE KALLIKREIN-LIKE SERINE PROTEASE ANTIGEN-GAMMA

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen-gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen-gamma. No other T-kininogen catalytic enzymes were observed. Antigen-gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen-gamma had a K(m) of 29 +/- 4-mu-m and a k(cat)/K(m) of 140 m-1 . s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10-mu-g of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42200-mu-g of kinin/h per mg respectively. Thus antigen-gamma is a weak kininogenase. The isoelectric point of antigen-gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen-gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91%, and 87%, amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.