Val-Leu-Arg-p-nitroanilide

It has been postulated that the mammary kinin system may play a role in modulating mammary blood flow. Until the present study, the local release of bradykinin (BK) or other kinin system constituents into the mammary vasculature had not been reported and there were also conflicting findings on the action of BK on udder vasculature. Udders were removed from healthy lactating cows at slaughter. Pairs of ipsilateral quarters were perfused with Tyrode solution through the external pudendalis artery and drained via the cranial superficial epigastric vein. Mammary secretion was collected through teat cannulae. The perfusion pressure was linearly related to perfusate flux between 60 and 210 ml min(-1) and the flow rate was adjusted (110-150 ml min(-1)) to give a basal pressure of 85 mmHg. PO2, PCO2 and pH in the venous effluent perfusate stabilised at 157 +/- 10 mmHg, 50.1 +/- 2.4 mmHg and 7.1 +/- 0.03, respectively. The venous effluent contained immunoreactive BK and BK precursor, tissue kallikrein activity, and bradykinin-destroying enzyme. The concentration of BK stabilised at 378 +/- 48 pg (ml perfusate)(-1), that of trypsin-activated BK precursor was 679 +/- 59 pg BK equivalents ml(-1) and that of tissue kallikrein, measured as cleavage of D-Val.Leu.Arg-p-nitroanilide (D-Val.Leu.Arg-pNA), was 5.5 +/- 1.7 nmol p-NA h(-1) ml(-1). Arterial infusion of phenylephrine (0.49-490 microM) produced increases in perfusion pressure (vasoconstriction). Acetylcholine (ACh) (0.55-55 microM) and BK (0.1-10 microM) produced only vasodilatation. BK (EC50 = 1.00+/-0.04 microM) was a more potent vasodilator than ACh (EC50 = 9.57+/-0.49 microM). The basal BK concentration was 250 times below the threshold for vasoactivity. The udder produced a milk-like secretion, which was dependent on perfusate flow and contained a concentration of BK which remained unchanged from 60 to 180 min of perfusion (231 +/- 31 pg ml(-1)) unlike that in the venous effluent which doubled between 60 and 120 min. Thus, in addition to its secretion into milk, BK, together with its precursor and tissue kallikrein, is continuously released into the vasculature of the isolated, perfused, lactating bovine udder.

Background and purpose: Based on their ability to chelate metals, hydroxamate molecules and siderophores have been successfully used as metalloenzyme inhibitors. As the anthrax toxin lethal factor (LF) is a zinc (Zn)-metallopeptidase, an investigation of the ability of some small non-siderophore hydroxamate compounds, 5 hydroxamate-containing siderophores, and 1 catecholate siderophore was undertaken to determine whether these compounds would inhibit LF. In addition, salmon sperm protamine and ethylenediaminetetraacetic acid were investigated. Methods: A spectrophotometric assay of LF activity, based on its reaction with the substrate (Ac-gly-tyr-betaala-arg-arg-arg-arg-arg-arg-arg-arg-val-leu-arg-p-nitroanilide), was used to assess the degree of inhibition of LF by the putative inhibitors. Procedures were implemented to avoid iron contamination of the test solutions and non-ferrated siderophores and hydroxamates were used as potential inhibitors. Results: The hydroxamate-containing siderophores displayed limited capacities to inhibit LF, as did the low molecular weight hydroxamate compounds. In contrast, the catecholate siderophore enterobactin and the cationic polyamine salmon sperm protamine demonstrated notable inhibition of LF at concentrations ranging from approximately 10 to 200 microM. Conclusions: The polyamine salmon sperm protamine which mimics the target site of proteins cleaved by LF, was the most effective inhibitor of the molecules examined, while the small molecule hydroxamates and the hydroxamate siderophores were among the poorest. If chelation of the Zn of LF results in LF inhibition by the molecules examined, it is most likely secondary to binding of the putative inhibitors to the active site of LF.

An acidic kininogenin from Lachesis muta snake venom was purified to apparent homogeneity by a combination of gel filtration, isoelectric focusing and preparative gel electrophoresis. It was shown to be a highly stable serine protease (mol. wt 27,900; pI 5.4) capable of releasing bradykinin from low mol. wt bovine kininogen and of cleaving some synthetic chromogenic peptides with the following catalytic efficiencies (Kcat/Km, M-1.sec-1): N-benzoyl-Phe-Val-Arg-p-nitroanilide (1.92 x 10(4)); H-D-Val-Leu-Arg-p-nitroanilide (1.55 x 10(4)); N-acetyl-Phe-Arg-p-nitroanilide (3.98 x 10(2)); no hydrolysis was observed with N-benzoyl-Arg-p-nitroanilide. A marked and sustained hypotensive effect was recorded following i.v. injection of purified kininogenin into rats. Tachyphylaxis was observed after repeated i.v. injection of the enzyme, a phenomenon accompanied by a decrease of only 15% in the total circulating rat kininogen. Both the in vivo action and the enzymatic properties of the L. muta kininogenin indicate that this enzyme might be helpful for understanding the kinin-kininogen system.