Abstract
Two molecular species of proline endopeptidase (PEP I and II) were concurrently isolated from normal human plasma. They were clearly separated on ammonium sulfate fractionation of plasma followed by chromatographies on DEAE-Sephadex and Con A-Sepharose and thereafter were partly purified by separate but almost identical procedures which included chromatographies on Sephacryl S-300, hydroxyapatite and carbobenzoxy-Gly-Pro-AH-Sepharose. The apparent molecular weights of PEP I and PEP II were 130 and 62 kilodaltons, respectively, as judged by gel filtration on TSK-Gel G 4000 SW. PEP I had a high affinity for Con A-Sepharose, but PEP II did not. Both enzymes were apparently highly specific for peptides such as angiotensin I, bradykinin, substance P and succinyltrialanine p-nitroanilide, cleaving them after proline or alanine residues. However, both enzymes were inert toward elastin and albumin. The Km and optimal pH values toward succinylglycyl-L-proline 4-methylcoumaryl-7-amide were 2.3 mM and 5.5-6.5 for PEP I and 0.71 mM and 6.0-7.0 for PEP II. Both enzymes were extremely sensitive to diisopropyl fluorophosphate, but no effect was observed with phenylmethanesulfonyl fluoride, aprotinin, soybean trypsin inhibitor, iodoacetamide or ethylenediaminetetraacetate. Elastatinal significantly inhibited PEP II while PEP I activity was only slightly affected. In addition, PEP II was strongly inactivated by p-chloromercuribenzoate and divalent metal ions such as Hg2+ and Zn2+, whereas PEP I was only slightly inactivated by these reagents. Thus, the enzymes seem to be quite distinct. PEP II appears to be very similar to PEPs purified previously from brain, pituitary and kidney tissues in terms of enzymatic properties, physicochemical properties and substrate specificity. On the other hand, PEP I is different from other PEPs in molecular nature and some enzymatic properties, but is indistinguishable in substrate specificity and enzyme classification.
