Prolylcarboxypeptidase (angiotensinase C): purification and characterization of the enzyme from Xanthomanas maltophilia.
Prolylcarboxypeptidase (Angiotensinase C, EC 3.4.16.2) was purified to homogeneity from cell free extracts of Xanthomonas maltophilia by ammonium sulfate fractionation and sequential chromatographies on DEAE-Toyopearl, Sephadex G-150, FPLC-Hiload Superdex 200 pg, and FPLC-Hitrap SP columns, with an activity recovery of 15%. The molecular weight of the enzyme was found to be 330,000 by gel filtration and 83,000 by SDS-PAGE, suggesting a tetrameric form for the native enzyme. It had an optimum pH of 8.5 and stability between pH 8.0 and 11.0. The isoelectric point of the enzyme was 6.6. The enzyme hydrolyzed Pro-X bonds when proline was in the penultimate position from the carboxyl terminal. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), while phenylmethylsulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), iodoacetamide, and metal chelators had no effect.
Suga
K
K
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Ito
K
K
Tsuru
D
D
Yoshimoto
T
T
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D005591:Chemical Fractionation; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007460:Iodoacetamide / Q000494:pharmacology; D007525:Isoelectric Focusing; D007531:Isoflurophate / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011392:Proline / Q000378:metabolism; D014974:Xanthomonas / Q000201:enzymology*; D020245:p-Chloromercuribenzoic Acid
D002614:Chelating Agents; D002729:Chloromercuribenzoates; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D020245:p-Chloromercuribenzoic Acid; D011392:Proline; D002268:Carboxypeptidases; C022448:lysosomal Pro-X carboxypeptidase; D000645:Ammonium Sulfate; D007460:Iodoacetamide