Abstract
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 200pg, and FPLC-Hitrap SP columns, with an activity recovery of 15%. The molecular weight of the enzyme was found to be 330, 000 by gelfiltration 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.
