Anti-AMY1 (native) polyclonal antibody

C-terminal processing of low pl barley alpha-amylase (AMY1) results in multiple forms in malt, aleurone protoplasts, and transformed yeast. Expression of an AMY1 cDNA in yeast thus leads to four secreted forms with distinct pl values between 4.7 and 5.1 and essentially identical M(r). AMY1-1 and AMY1-2 lacking the C-terminal Arg-Ser are generated by carboxypeptidase in vitro from AMY1-3 and AMY1-4, respectively. In vivo processing is due to the KEX1-encoded yeast carboxypeptidase. AMY1-2 and AMY1-4 are fully active, whereas AMY1-1 and AMYl-3 retain 3-4% activity toward p-nitrophenyl maltoheptaoside and have one fewer SH group, due to reaction with glutathione. AMY1-1-AMY1-4 are indistinguishable from malt AMY1 with respect to Ca2+, substrate-, and beta-cyclodextrin-binding as well as recognition by three monoclonal antibodies and limited proteolysis by proteinase K. Transient AMY1 precursors present in barley aleurone protoplasts were trapped by addition of serine carboxypeptidase inhibitors, indicating that endogenous carboxypeptidase participates in the maturation of AMY1 during germination. Three pairs of precursor/mature AMY1 forms are recognized, presumably corresponding to the three genes encoding AMY1. Malt carboxypeptidase II can convert in vitro the precursors isolated from protoplasts into processed enzyme, and AMY1 from malt accordingly lacks the C-terminal heptapeptide. This report thus demonstrates posttranslational protein modification by carboxypeptidase in higher plants.

This review on barley alpha-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsites to the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barley alpha-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding sites have been identified. Engineering of outer subsites -6 and +4 alters action patterns and relative specificities. Thus, compared to wild-type, Y105A AMY1 (subsite -6) shows 140%, 15%, and <1% and T212Y (subsite +4) 32%, 370%, and 90% activity towards starch, maltodextrin, and maltoheptaoside, respectively. The enzyme kinetic properties and modeled maltododecaose complexes suggest binding mode multiplicity. Following ail initial hydrolytic cleavage of amylose, an average of 1.9 bonds are cleaved per enzyme-substrate encounter, defining a degree of multiple attack (DMA) of 1.9. DMA increased to 3.3 for Y105A and decreased to 1-1.7 for other subsite mutants. The fusion of a starch-binding domain to AMY1 raised the DMA to 3.0 and increased the amount of higher oligosaccharide products. Remarkably, the subsite mutants had unchanged distribution of released oligosaccharides of DP 5-9, but the profiles differed for the shorter products. A recently identified surface binding site, found exclusively in AMY1, involves the conserved Tyr(380) which has no effect on the DMA, but proved critical for beta-cyclodextrin binding as shown by mutational and surface plasmon resonance analyses. Accordingly, AMY2 has lower affinity for beta-cyclodextrin. Hydrolysis of amylopectin proceeds via a fast and a slow reaction rate, with beta-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr(380) in activity on amylopectin. Barley seeds produce different proteinaceous inhibitors acting specifically on insect, animal or plant alpha-amylases. Rational mutagenesis of barley alpha-amylase/subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the importance of ionic bonds for inhibitory activity.