G. Fereyroux et al., THE HUMAN PANCREATIC ALPHA-AMYLASE ISOFORMS - ISOLATION, STRUCTURAL STUDIES AND KINETICS OF INHIBITION BY ACARBOSE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1388(1), 1998, pp. 10-20
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.