Purification and characterization of arginine carboxypeptidase produced by Porphyromonas gingivalis.
Arginine carboxypeptidase was isolated from the cytoplasm of Porphyromonas gingivalis 381 and purified by DEAE-Sephacel column chromatography, followed by high-performance liquid chromatography on DEAE-5PW and TSK G2000SW(XL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of three major bands at 42, 33, and 32 kDa with identical N-terminal sequences. By Western blotting analysis and immunoelectron microscopy, the arginine carboxypeptidase was found to be widely distributed in the cytoplasm and on the surface of the outer membrane. The open reading frame corresponding to the N-terminal amino acids of the arginine carboxypeptidase was detected by a search of the sequence of the P. gingivalis W83 genome. This sequence showed homology with mammalian carboxypeptidases (M, N, and E/H) and included a zinc-binding region signature, suggesting that the enzyme is a member of the zinc carboxypeptidase family. The purified enzyme was inhibited by EGTA, o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and some metal ions, such as Cu(2+), Zn(2+), and Cd(2+). On the other hand, Co(2+) activated the enzyme. The enzyme released arginine and/or lysine from biologically active peptides containing these amino acids at the C terminus but did not cleave substrates when proline was present at the penultimate position. These results indicate that the arginine carboxypeptidase produced by P. gingivalis is an exo type of metallocarboxypeptidase. This enzyme may function to release arginine in collaboration with an arginine aminopeptidase, e.g., Arg-gingipain, to obtain specific amino acids from host tissues during the growth of P. gingivalis.
Masuda
Kaname
K
Department of Preventive Dentistry, School of Dentistry, The University of Tokushima, Tokushima, Japan.
Yoshioka
Masami
M
Hinode
Daisuke
D
Nakamura
Ryo
R
D015097:3-Mercaptopropionic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D025901:Carboxypeptidase B2 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D013379:Substrate Specificity
C029729:2-mercaptomethyl-3-guanidinoethylthiopropionic acid; D015097:3-Mercaptopropionic Acid; D025901:Carboxypeptidase B2