Partial characterization and activity distribution of proteases along the intestine of grass carp, Ctenopharyngodon idella (Val.)

A series of studies based on biochemical assays and electrophoretical observations has been conducted in order to investigate activity distributions and partially characterize various types of proteinases in the digestive tract of grass carp, Ctenopharyngodon idella (Val.). The casein digestion assays revealed that the presence of acidic proteinase had the highest activity at pH 2.5-3.0 and the alkaline proteinases at pH 10.0. The acidic proteinase activity distribution was found to decrease gradually from the oesophagus to the anus. Pepstatin A and EDTA inhibited the acidic proteinases activity. The SDS-substrate-PAGE showed that crude extraction of grass carp intestine contained an acidic proteinase active component with molecular mass of 28.5 ku. The substrate-PAGE at neutral pH condition showed the presence of two acidic proteinase active components. The activity distribution of alkaline proteinase was found to slightly fluctuate along the intestine. And the whole intestine had very high activity. The inhibition assays and substrate specificity assays showed that trypsin was the main active component of the alkaline proteinases. The SDS-substrate-PAGE further showed that the crude extraction of grass carp intestine had four types of alkaline proteinase with molecular mass of 26.4, 30.8, 43.0 and 105.0 ku respectively. They were characterized to be trypsin (26.4, 30.8 and 43.0 ku) and un-serine proteinase (105.0 ku) respectively. No chymotrypsin was detected.