Rabbit tissue peptidases that hydrolyse the peptide hormone bradykinin. Differences in enzymic properties and concentration in rabbit tissues

Abstract

The distribution and properties of neutral peptidases acting on the peptide hormone bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) were determined in several rabbit tissues. The supernatant and particulate fractions prepared from tissue homogenates (25000g for 60min) were studied. Bradykinin inactivation (kininase activity) was measured by bioassay with the isolated guinea-pig ileum. The sites of peptide-bond cleavage were determined in the amino acid analyser, which permits detection and measurement of amino acids and peptides derived from bradykinin. The results indicate that kininases are present in a wide range of concentrations in different tissues, kidney and lung having the most activity. Kininases present in different tissues were distinguished on the basis of sensitivity to the effects of EDTA, dithiothreitol and ZnCl2 and by the site of peptide-bond hydrolysis in bradykinin.

Purification of rabbit brain endooligopeptidases and preparation of antienzyme antibodies

“…Previous reports from our laboratory indicated that, when bradykinin is incubated with purified brain endooligopeptidase A or B, the major peptides formed are Arg1 -* Phe5 and Arg1 -* Pro7, respectively (Oliveira et al, 1976). Since these fragments are slowly degraded by brain peptidases present in tissue homogenate (Cicilini et al, 1977;Coelho et al, 1981), the amount of Arg1 -* Phe5 and Arg1 -* Pro7 formed when bradykinin is incubated with enzyme preparations was used to evaluate the relative activity of brain endooligopeptidases A and B, respectively, present in the early stage of enzyme purification. The quantitative recovery of bradykinin products formed by the action of brain peptidases clearly shows that purified brain endooligopeptidases A and B are free from any other peptidases acting on bradykinin or on its products.…”

The renal kallikrein-kinin system.

“…Kininases I and II, described earlier in connection with the plasma kallikrein, are also widely distributed in tissues (Cicilini et al, 1977;Erdos, 1976). Kidney has very high kininase activity, most of which is kininase II .…”

Inhibition of rabbit tissue kininase by anti-(endo-oligopeptidase A) antibodies

“…Crude enzyme preparations from rabbit tissue homogenates were prepared by the method described by Cicilini et al (1977). Tissue homogenates were centrifuged at 25000g for lh.…”

“…One property that distinguishes these neuropeptide-metabolizing brain endopeptidases from most proteinases (Barrett, 1977) is the fact that their activity is apparently restricted towards oligopeptides (Camargo et al, 1979a). The existence in other tissues of enzymes exhibiting properties similar to those of brain endo-oligopeptidases was indicated by Cicilini et al (1977) and Camargo et al (1979b) on the basis of sites of cleavage in the bradykinin molecule as well as of the resistance of peptides attached to agarose to hydrolysis by tissue kininases. In the present paper we describe the development of monospecific antibodies against brain endo-oligopeptidase A and the use of antibodies to study the distribution in rabbit tissues of an immunoreactive endopeptidase that hydrolyses bradykinin at the same peptide bond as does brain endo-oligopeptidase A.…”