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 mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two 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 substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate.
Koreeda
Y
Y
Department of Operative Dentistry, Nihon University School of Dentistry at Matsudo, 271-8587, Chiba, Japan.
Hayakawa
M
M
Ikemi
T
T
Abiko
Y
Y
D000409:Alanine / Q000378:metabolism; D002104:Cadmium / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000494:pharmacology; D003374:Coumarins; D004151:Dipeptides; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007531:Isoflurophate / Q000494:pharmacology; D008623:Mercaptoethanol; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D018720:Prevotella / Q000145:classification / Q000201:enzymology*; D011392:Proline / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D003374:Coumarins; D004151:Dipeptides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D002104:Cadmium; D007531:Isoflurophate; D003035:Cobalt; D008623:Mercaptoethanol; C057970:glycylprolyl-4-methylcoumaryl-7-amide; D011392:Proline; D018819:Dipeptidyl Peptidase 4; D008628:Mercury; D015032:Zinc; D000409:Alanine