Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930

A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3 .4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mas s of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 8 0 kDa when the enzyme was heated to 100 degreesC in the presence of 2-merca ptoethanol before SDS-PAGE, suggesting that the native enzyme consists of t wo identical subunits and is folded in 2% SDS. The optimum pH, with glycyl- prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide su bstrates containing proline and, less efficiently, alanine in the P1 positi on. The enzyme was markedly inhibited by Cd2+, Zn2+, Hg2+, Co2+, and serine proteinase inhibitor di-isopropylfluorophosphate. (C) 2001 Elsevier Scienc e Ltd. All rights reserved.