THE HUMAN PANCREATIC ALPHA-AMYLASE ISOFORMS - ISOLATION, STRUCTURAL STUDIES AND KINETICS OF INHIBITION BY ACARBOSE

A rapid method is proposed for isolating the two main components of hu man pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA 1 (7.2), the main component, was determined using an isoelectr ofocusing method and found to differ from that of HPA II (6.6). The mo lecular mass of HPA I (55 862 +/- 5 Da) and that of HPA II (55 786 +/- 5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55 788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further i nvestigated by performing limited proteolysis. Two fragments with an a pparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilism, whereas digestionwith p apain yielded two cleaved fragments with molecular masses of 38 kDa an d 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the pres ence of the calcium chelator EDTA is in the L3 loop (B domain). The ki netics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate . The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All i n all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences wer e observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be e xpected in view of the high degree of structural identity previously f ound to exist between human and porcine pancreatic amylases, the prese nt data show that the inhibitory effects of acarbose on the kinetic be havior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by PIPA as well as by PPA in b oth cases requires two carbohydrate binding sites in addition to the c atalytic site. (C) 1998 Elsevier Science B.V. All rights reserved.