Purification of rabbit brain endooligopeptidases and preparation of anti-enzyme antibodies.
Endooligopeptidase A was purified approximately 3 000-fold and endooligopeptidase B approximately 1200-fold from the 25 000g supernatant fraction of rabbit brain homogenate. The purified enzymes were homogeneous on the basis of acrylamide gel electrophoresis under denaturing and nondenaturing conditions, isoelectric focusing, immunochemical criteria, and specific activities of the elution profile of gel filtration on Sephadex G-100. The only peptide bond cleaved by endooligopeptidase A in bradykinin, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, is Phe5-Ser6, whereas endooligopeptidase B hydrolyzes the Pro7-Phe8 peptide bond of bradykinin and the Pro3-Gly4 bond of des-Phe8-Arg9-bradykinin. The specific activity of the homogeneous enzymes using bradykinin as substrate was 1087 nmol min-1 mg-1 for endooligopeptidase A and 292 nmol min-1 mg-1 for endooligopeptidase B. Gel filtration suggested molecular weights of 75 000 and 68 000 for endooligopeptidases A and B, respectively. Sodium dodecyl sulfate gel electrophoresis suggested that each endooligopeptidase consisted of a single polypeptide chain with molecular weights of 74 000 and 69 000 for the A and B enzymes, respectively. Purified endooligopeptidase A or B injected into goats produces monospecific antisera directed against each enzyme. The antibody prepared against each endooligopeptidase did not react with or inhibit the activity of the other enzyme.
D000818:Animals; D000936:Antigen-Antibody Complex; D001920:Bradykinin; D001921:Brain / Q000201:enzymology*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007106:Immune Sera; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D007527:Isoenzymes; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; D003546:Cysteine Endopeptidases; C028307:endopeptidase B; D001920:Bradykinin