CHARACTERIZATION OF KININOGENASE ACTIVITY OF AN ACIDIC PROTEINASE ISOLATED FROM HUMAN KIDNEY

An acidic proteinase was purified from human kidney cortex. The enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtrati on, and isoelectric points of 5.2 and 6.1. The optimum pH for hydrolys is of bovine hemoglobin was about 3.5. Reverse-phase KPLC analysis of the incubation mixture of the enzyme with human plasma showed the pres ence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. The specific activities were 2.91 mu g MLBK equivalent.mg(-1).min(-1) at pH 3.5 and 2.15 mu g MLBK equivalent.mg(-1).min(-1) at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstati n A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage point s. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz- Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Le u-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Ar g-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred a t the Arg-Ser linkage. -Pro-GIy-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Ed dnp was hydrolyzed by the renal acidic proteinase and yielded the pept ide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinetic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-E ddnp (K-m=0.69+/-0.08 mu M; K-cat=0.052+/-0.0095 s(-1); K-cat/K-m= 0.0 75+/-0.005 mu M-1.s(-1)) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K-m=1. 56+/-0.16 mu M; K-cat=0.0048+/-0.0001 s(-1); K-cat/K-m=0.003+/-0.0003 mu M-1.s(-1)). Human liver cathepsin D had no activity on C-terminal s equences and human pepsin hydrolyzed them at the Ser-Ser bond. The res ults suggest that the renal acid proteinase is distinct from human pep sin and human liver cathepsin D and releases MLBK from human kininogen .