Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin

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Abstract
Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzed the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It was isoelectric near pH 5.2 and had a MW of approximately 71,000. The enzyme also hydrolyzed the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but did not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzed the Pro7-Phe8 peptide bond in Bk. It was isoelectric at pH 4.9 and had a MW of approximately 68,000. Brain kininase B also hydrolyzed the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg and Phe-Ser-Pro-Phe-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. Kininase A and B hydrolyzed the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe- Arg-Ser-Val-Gln-Val. A large part of the C-terminal portion of bradykinin is apparently important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissile bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.

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