Effects of protease inhibitors on the autolysis and protease activities of Antartic krill.

Abstract

In order to determine which proteases are responsible for the autolysis of krill, the effects of several protease inhibitors on the autolysis and protease activities of krill were investigated. Homogenatesof whole bodies, and the cephalothorax and abdomen parts of frozen krill were equilibrated at 37°C at different pHs between 2 to 10 and allowed to stand for 16hr, following which the increase in the TCA soluble fraction was monitored. 14C-Hemoglobin (14C-Hb) hydrolyzing activity was also measured using each homogenate as a crude enzyme preparation. The degree of autolysis and the 14C-Hb hydrolyzing activity were maximumat pH 5-8 for the parts studied. The hydrolytic activity was highest in the cephalothorax, followed by that in the whole body and then the abdomen. The effects of inhibitors on the 14C-Hbhydrolyzing activity were examined, and it was seen that soybean trypsin inhibitor (STI), diisopropyl fluorophosphate (DFP) and leupeptin significantly inhibited the activity at neutral pH, and pepstatin, monoiodoacetic acid (IAAcid) and leupeptin Were effective at acidic pHfor all the parts. Investigation of the effects ofinhibitors on the autolysis at 20°C at pH 4 and 7 by SDS-polyacrylamide gel electrophoresis indicated that the autolysis of the cephalothorax and whole body at pH 7 was suppressed a little by STI and the autolysis of the abdomen and whole body at pH 4 was significantly inhibited by iodoacetamide (IAA) and leupeptin. These results suggest that the main proteases responsible for the autolysis of krill are trypsin like-proteases at neutral pH and cathepsins (B, H and L types) at acidic pH.