Metal content and biochemical analyses of a recombinant collagenase PrtV from Vibrio parahaemolyticus.
PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagen-like sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.
Yu
M S
MS
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei.
Yap
M N
MN
Lee
C Y
CY
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D008670:Metals / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D003012:Microbial Collagenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D009532:Nickel / Q000032:analysis; D009842:Oligopeptides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014736:Vibrio parahaemolyticus / Q000201:enzymology* / Q000235:genetics; D015032:Zinc / Q000032:analysis
D008670:Metals; D009842:Oligopeptides; D011994:Recombinant Proteins; C031821:2-furanacryloyl-leucyl-glycyl-prolyl-alanine; D009532:Nickel; D003094:Collagen; D003012:Microbial Collagenase; D015032:Zinc