PURIFICATION AND PARTIAL CHARACTERIZATION OF ALPHA-AMYLASE ALLOZYMES FROM THE LESSER GRAIN BORER, RHYZOPERTHA-DOMINICA

Alpha-Amylase was purified from adults of the lesser grain borer, Rhyzopertha dominica (F.), by ammonium sulfate precipitation, glycogen complex formation, and gel filtration chromatography. Specific activity increased from 16 AU/mg protein in the crude extract to 705 AU/mg protein in the final sample (1 AU = 1 mg maltose hydrate/min at 30-degrees-C). Two major protein bands, active in starch zymograms, were present at R(m) 0.71 and 0.79 when the sample was examined by polyacrylamide gel electrophoresis (PAGE) on 7.5% gels. In addition, several minor proteins that had alpha-amylase activity were also present. Molecular masses of the two major allozymes were estimated to be 57 and 55 kDa under dissociating conditions. Isoelectric points of the allozymes were at pH 3.4 and 3.5. The amylases were most active at pH 7 and the presence of 20 mM NaCl resulted in a 10.7-fold increase in V(max).K(m) for soluble starch was 0.127%. Saline extracts of wheat ("Florida 302") were 2- and 3-fold more inhibitory on a weight basis towards the amylases from R. dominica than were extracts prepared from two cultivars of triticale, "Morrison" and "CT-4161", respectively. Interaction of purified alpha-amylase inhibitors from wheat, inhibitor-0.28 and a sample of the inhibitor-0.19 family of isoinhibitors, with the alpha-amylases from R. dominica was studied. Complex formation between the amylases and inhibitor-0.28 was demonstrated by PAGE, although the protein-protein complexes that formed were not completely stable during electrophoresis. K(i) values were estimated to be 2.6 nM for inhibitor-0.28 and 2.9 nM for inhibitor-0. 19. Binding of these inhibitors to alpha-amylases from R. dominica was not as tight compared with the interaction of these inhibitors with amylases from Sitophilus weevils and Tenebrio molitor.