Ox brain aminopeptidases. Further purification of three bovine enzymes.

Abstract

Four peptidases capable of acting upon Leu∙Gly∙Gly have been separated from bovine brain extracts, and three have been highly purified by ammonium sulfate fractionation and by chromatography on DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. A Mn2+-stimulated aminopeptidase (E2) was purified 180-fold over an initial 16 300 × g supernatant fraction and has a M.W. of approximately 117 250 as estimated by sucrose density gradient centrifugation. Two peptidases which are inhibited by heavy metals (E1P1 and E1P2) were purified approximately 2683-fold and 2461-fold over the initial extracts and have approximate M.W.'s of 61 500 and 85 700, respectively. E1P1 and E1P2 have Km's of 0.36 and 0.38 mM, respectively. The pH optima for E1P1, E1P2, and E2 are 6.6–7.8, 7.2–8.1, and 7.8–9.6. E2 is more heat-labile than E1P1 and E1P2 since it is completely inactivated by heating at 55° for 30 min at pH 6.5 under which conditions the latter two enzymes retain full activity. The ΔEa for the hydrolysis of Leu∙Gly∙Gly was calculated to be 10 800, 13 420, and 15 480 for E1P1, E1P2, and E2. E1P1 and E1P2 hydrolyze tripeptides but do not cleave dipeptides. E2 cleaves tripeptides, and only cleaves Leu∙Gly, of a series of dipeptides examined, having a specificity differing notably from leucine aminopeptidase. All the enzymes were inactive towards leucine-p-nitroanilide and leucine-β-naphthylamide, suggesting that they should not be classed among the arylamidases. On the basis of differential heat stability, it is estimated that a peptidase sedimented by ammonium sulfate at 20% saturation contains 40% of the original tripeptidase activity; E2 represents 30% of the activity; E1P1 and E1P2 contribute 12 and 18%, respectively, to the tripeptidase activity in the original extracts.