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Biochim Biophys Acta. 1998 Oct 14;1388(1):10-20.

The human pancreatic alpha-amylase isoforms: isolation, structural studies and kinetics of inhibition by acarbose.

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1
Laboratoire de Biochimie et Biologie de la Nutrition, CNRS-ESA 6033, Faculté des Sciences et Techniques de St. Jérôme, Université d'Aix-Marseille, 13397 Marseille cedex 20, France.

Abstract

A rapid method is proposed for isolating the two main components of human pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6. 6). The molecular mass of HPA I (55862+/-5 Da) and that of HPA II (55786+/-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 (55788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics 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 in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

PMID:
9774702
[Indexed for MEDLINE]

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