Porcine Liver Succinyltrialanine p-Nitroanilide Hydrolytic Enzyme. Its Purification and Characterization as a Post-Proline Cleaving Enzyme

摘要: Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, hydroxylapatite columns. The homogeneous as judged polyacrylamide gel electrophoresis in the presence absence sodium dodecyl (SDS). pI 4.9 dis electrofocusing molecular weight calculated to be 72,000 filtration G-150 column 74,000 SDS-polyacrylamide electrophoresis. Acidic amino acids amounted 17.2% total acid residues, basic ones, 12.9%. No hexosamine detected. STANA-hydrolytic showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide 6.5 succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), stable between 6 7 dithiothreitol. This hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, several proline-containing natural peptides addition p-nitroanilide, but unable hydrolyze substrates aminopeptidases, dipeptidylaminopeptidase IV, trypsin, chymotrypsin. Elastatinal chymostatin were effective inhibitors their IC50 values 8.7 micrograms/ml 18.2 micrograms/ml, respectively. completely inhibited 10(-7) M p-chloromercuribenzoic (pCMB), p-chloromercuriphenylsulfonic (pCMPS), 10(-4) diisopropyl phosphofluoridate (DFP), not 1 mM E-64, which is known an inhibitor specific thiol proteinase. easily inactivated agitation Vortex mixer, its recovered compounds such dithiothreitol, 2-mercaptoethanol cysteine. effects substantially identical when measured either or succinyl-Gly-Pro-MCA substrate. These results indicate that post-proline cleaving [EC 3.4.21.26].