title: Hemocyanin-derived phenoloxidase activity with broad temperature stability extending into the cold environment in hemocytes of the hair crab Erimacrus isenbeckii.
abstract: Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.
authors.forename: Sang Gu
authors.initials: SG
authors.lastname: Kim
authors.forename: Byung Wook
authors.initials: BW
authors.lastname: Jung
authors.forename: Hahyung
authors.initials: H
authors.lastname: Kim
mesh_terms: D000818:Animals; D003386:Brachyura / Q000201:enzymology*; D002396:Catechols / Q000378:metabolism; D002458:Cell Fractionation; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006433:Hemocyanins / Q000378:metabolism; D006434:Hemocytes / Q000737:chemistry / Q000201:enzymology; D006358:Hot Temperature; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D014442:Monophenol Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D017385:Sequence Homology; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013682:Teichoic Acids / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D002396:Catechols; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D013682:Teichoic Acids; D012967:Sodium Dodecyl Sulfate; D007980:Levodopa; C009900:lipoteichoic acid; D006433:Hemocyanins; D014442:Monophenol Monooxygenase; D014357:Trypsin; C034221:catechol