T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen γ

Abstract

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 Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms 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.

Kallikrein rK10‐induced kinin‐independent, direct activation of NO‐formation and relaxation of rat isolated aortic rings

“…injections of rKI0, may indicate a limited activation, since rKlO, unlike rKl, did not have an effect on systemic BP. This is in agreement with previous results where rK10 was found to be a rather inefficient T-kininogenase, (Gutman et al, 1988;Berg et al, 1991) compared to the high/lowmolecular kininogenase rKl (Maier et al, 1983). This may explain the lack of a systemic effect of rKlO, in spite of the fact that T-kininogen is the main kininogen in rat plasma (Okamoto & Greenbaum, 1983b).…”

“…T-kinin, like bradykinin, contracts rat uterus and guinea-pig ileum, and induces hypotension (Okamoto & Greenbaum, 1983b). However, kinetic studies have shown that rK10 is not a very efficient kininogenase (Gutman et al, 1988;Berg et al, 1991) compared to human urinary kallikrein (Maier et al, 1983), and its role in vasomotor regulation may be questionable. In the present study, we therefore wanted to characterize further the biological effects of rK10 with respect to its ability to alter vascular resistance, either directly like rK9, trypsin, and thrombin, or through the release of kinins, like rKl.…”

“…Another enzyme of the kallikrein family, rKIO (Berg et al, 1992), previously named antigen gamma , endopeptidase K (Gutman et al, 1988), proteinase B (Kato et al, 1987), or T-kininogenase (Xiong et al, 1990), so far found in the rat SMG only, resembles rKl in its ability to release kinin. However, rKlO differs from rKl in that it releases T-kinin (Ile-Serbradykinin) from T-kininogen (Okamota & Greenbaum, Author for correspondence at: Department of Physiology, Box 1103, Blindern, 0317 Oslo, Norway. 1983a; Barlas et al, 1987;Gutman et al, 1988;Berg et al, 1991) a substrate present in the rat only (Gutman et al, 1988), whereas rKl, in the rat, releases bradykinin from high and low molecular weight kininogen (Alhenc-Gelas et. al., 1981;Kato et al, 1985).…”

Transcriptomic and proteomic analysis of liver and muscle alterations caused by surgical stress in rats

“…Another protein related to the proteolytic cascades and found to be increased in stressed rats was T-kininogen-1 precursor, the major kininogen in rat plasma. This protein is hydrolysed by trypsin, a serine protease, to the vasoactive peptide T-kinin, with a vasodilator action similar to bradykinin and kallidin [45,46]. Conversion of T-kininogen into T-kinin by the serine protease trypsin is regulated by SERPINs, mainly α 1 -antitrypsin, which was also increased in stressed rats.…”

Processing of Epidermal Growth Factor in the Rat Submandibular Gland by Kallikrein-Like Enzymes

“…The kallikrein-like enzymes, rK1 , rK2 , rK7 (Berg et al, 1992c), rK9 (Berg et al, 199213) and rKlO (Berg et al, 1991), were purified from the rat submandibular gland as previously described.…”