Purification and characterization of a trypsin-like protease from the culture supernatant of Actinobacillus actinomycetemcomitans Y4.
We purified and characterized a protease from Actinobacillus actinomycetemcomitans. The protease was isolated from the culture supernatant by sonication in phosphate-buffered 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The protease was purified by acetone precipitation, followed by column chromatography with Arginine Sepharose 4B, DEAE Sepharose CL-6B, Sephacryl S-200HR and HiTrap Q. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protease showed a clear band at approximately 50 kDa. The protease showed trypsin-like activity with hydrolytic activity for the synthetic substrates N alpha-benzoyl-DL-arginine p-nitroanilide (BApNA) and N alpha benzoyl-DL-lysine p-nitroanilide (BLpNA). The activity of the protease was stable at pH 7.0 to approximately 8.0. The activity of the protease was inhibited by leupeptin, phenylmethylsulfonyl fluoride (PMSF), and EDTA, but was not affected by dithiothreitol (DTT), cysteine, 2-mercaptoethanol, pepstatin or soybean trypsin inhibitor. These data suggest that this protease is a serine protease or metallo protease. This enzyme extensively degraded collagen type I and fibronectin.
Wang
P L
PL
Department of Pharmacology, Osaka Dental University, Hirakata, Japan. wang@cc.osaka-dent.ac.jp
Shirasu
S
S
Shinohara
M
M
Daito
M
M
Fujii
T
T
Kowashi
Y
Y
Ohura
K
K
D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology*; D001587:Benzoylarginine-2-Naphthylamide / Q000378:metabolism; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017077:Culture Media, Conditioned; D011480:Protease Inhibitors; C098918:benzoyllysine nitroanilide; D001587:Benzoylarginine-2-Naphthylamide; D010450:Endopeptidases; D014357:Trypsin; D008239:Lysine