Purification and characterization of a digestive cysteine proteinase from the American lobster (Homarus americanus).
A new cysteine proteinase was isolated from the digestive juice of the American lobster (Homarus americanus). The enzyme was purified by a combination of affinity and ion-exchange chromatography and gel filtration. The cysteine proteinase accounted for 80% of the proteolytic activity in the lumen of the hepatopancreas. The most potent heavy-metal inhibitors were Hg, Cu, and Ag ions. Inhibition by organic proteinase inhibitors, including E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane] and activation of the enzyme by 2-mercaptoethanol and dithiothreitol are characteristic of cysteine proteinases. Several similarities to papain are noted and include the N-terminal sequence, of which 22 of the first 28 amino acids are identical. Some notable differences are the higher Mr of 28,000 compared with 23,350 for papain, and the low isoelectric point (pI 4.5) of the lobster enzyme. The effects of pH and temperature on catalytic activity of the lobster proteinase were studied with benzyloxycarbonylalanine p-nitrophenyl ester as the substrate. The kcat./Km value was effectively temperature-independent between 10 and 60 degrees C. The pH-activity profile for the lobster enzyme revealed four apparent protonation states, of which only two are active.
Laycock
M V
MV
Atlantic Research Laboratory, National Research Council, Halifax, Nova Scotia, Canada.
Hirama
T
T
Hasnain
S
S
Watson
D
D
Storer
A C
AC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors; D004355:Drug Stability; D005612:Freeze Drying; D005750:Gastric Juice / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008121:Nephropidae / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D000596:Amino Acids; D015853:Cysteine Proteinase Inhibitors; D008670:Metals; D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases