Purification and characterization of proline-beta-naphthylamidase, a novel enzyme from pig intestinal mucosa.
An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.
Takahashi
T
T
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo.
Ikai
A
A
Takahashi
K
K
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D002241:Carbohydrates; D011480:Protease Inhibitors; D000626:Aminopeptidases