Four Ca2+-dependent proteinase activities isolated from crustacean muscle differ in size, net charge, and sensitivity to Ca2+ and inhibitors.
Four Ca2+-dependent proteinase activities in lobster claw and abdominal muscle have been resolved by high-performance liquid chromatography on gel filtration and ion-exchange columns. These activities, which do not appear to be generated by autolytic or other degradative processes, differed from each other in molecular weight (peak I, Mr = 310,000; peak IIa, Mr = 125,000; peak IIb, Mr = 195,000; peak III, Mr = 59,000) and net charge, as indicated by elution from an ion-exchange column with a NaCl gradient. Although optimum activity occurred at 5-10 mM Ca2+ at pH 6.8, the enzymes differed in activation at lower Ca2+ concentrations. The concentrations required for half-maximal activation were 0.6 mM for peak III, 1 mM for peak I, 1.5 mM for peak IIa, and 2 mM for peak IIb. Only the peak III proteinase was active at 100 microM Ca2+; none were active at 10 microM and below. Although the lobster Ca2+-dependent proteinases were all inhibited, from 75 to 98%, by the cysteine proteinase inhibitors leupeptin, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine, and iodoacetamide, they showed differential responses to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor phenylmethanesulfonyl fluoride. Peak I was moderately (26%) inhibited by phenylmethanesulfonyl fluoride, whereas peaks IIb and III were inhibited 26 and 90%, respectively, by pepstatin. This is the first description of multiple forms of Ca2+-dependent proteinase that require Ca2+ at millimolar levels in any tissue, either vertebrate or invertebrate.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002154:Calpain / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007460:Iodoacetamide / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D008121:Nephropidae / Q000201:enzymology*; D010436:Pepstatins / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology
D002121:Calcium Channel Blockers; D007976:Leupeptins; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D010664:Phenylmethylsulfonyl Fluoride; D002154:Calpain; C032854:leupeptin; D002118:Calcium; C031375:pepstatin; D007460:Iodoacetamide