Isolation and characterization of a trypsin from the slipper lobster, Thenus orientalis (Lund).
Trypsin has been isolated and purified from the digestive glands of the slipper lobster, Thenus orientalis. It is a glycoprotein with a molecular mass of approximately 35 kDa as judged by both SDS-PAGE and gel filtration. The N-terminal amino acid sequence has strong homology to crustacean trypsins. This is confirmed by the cross-reaction of crustacean trypsins with antibodies to the T. orientalis enzyme. Despite a 40% identity with the bovine trypsin N-terminal sequence, there was no cross-reaction with the mammalian serine proteases. The optimum kcat and kcat/Km values for N-alpha-benzoylarginine-p-nitroanalide were 0.91 s-1 and 9.7 x 10(3) M-1 s-1, respectively, with this specificity constant being lower than those reported for other crustacean trypsins. Inhibition studies indicated the presence of serine and histidine at the active site and pKa of the catalytic histidine residue was found to be 5.7 in the free enzyme and 4.7 in the Michaelis complex.
Johnston
D
D
Department of Molecular Sciences, James Cook University of North Queensland, Townsville, Australia.
Hermans
J M
JM
Yellowlees
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D001665:Binding Sites; D004064:Digestive System / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000201:enzymology*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors
D014361:Trypsin Inhibitors; D001586:Benzoylarginine Nitroanilide; D014357:Trypsin