C-terminal Amidating Enzyme Isolated And Purified

Carboxyl-terminal a-Amidating Enzyme is an enzyme existing in the animals' body widely, which can catalysis proteins and polypeptides to be amidated. There are many proteins and polypeptides with bioactivities in the organism. And most of them are hormone,neurotransmitter,toxin,tropho-element, which have important physiological functions .Before they have bioactivities, their carboxyl terminals shoμld be amidated in the biosynthesis process .And most of the polypeptides with physiological activities are effective drugs, eg. Calcitionin,Pancreaotropic homone. Therefore it is very significant to research C-AE in the field of forming polypeptide drugs.So far, all the amidating enzymes are received from the organism of the mammals. Those amidating enzymes from different mammals almost have no differences. But the sequence and spatial structure of the amino acids have strong conservatisms. So we decided to use the arthropods, which are fairly different from mammals, as raw material to receive a C-AE with new sense.In this study, we received a electrophoresis pure C-AE from the body of Buthus martensii Karsch through some different isolation methods. We can establish a absolutely new system to detect the activity of the Amidating Enzyme , taking the Dansyl-Tyr-Va1-Gly as the substrate and using the capillary electrophoresis as the detecting device.Its molecular weight is 37.2kDa by measurement of SDS-polyacrylamide gel electrophoresis. Its isoelectric point was 6.14 determined by isoelectric focusing electrophoresis.Its maximum UV adsorptionwave-length is 278.4 nm.And the result of glycoprotein staining showed that it wasn't a glycoprotein .The optimal pH is from 6.5 to 8.5 and the best reaction tempertaure is about 30℃.Cu²⁺ ,vitamin C and catalase can be used as its helper factors and the proper concentration of each listed above is 5μmol/L,3mmol/L,2.5μg/ml respectively.