Action pattern, kinetical properties and electrophoretical studies of an alpha-amylase present in midgut homogenates from rhynchosciara americana (diptera) larvae

Abstract

1. 1. Rhynchosciara americana midgut amylase binds chloride ( K d = 1.6 mM at 37°C ) resulting in a 34-fold increase in the K cat without affecting the K m, but causing a shift in the optimum pH of the enzyme from 6.8 to 8.0. 2. 2. The ions Br −, NO 3 − and I − also activate the amylase but with a decreasing efficiency. 3. 3. An Arrhenius plot of activity versus temperature show that the amylase display only one slope with a corresponding apparent energy of activation of 4.8 Kcal/mole. 4. 4.|The amylase is a calcium-dependent metalloenzyme which shows a pure competitive inhibition by Hg 2+ and a pure non-competitive inhibition by Cu 2+ with K i respectively of 0.21 mM and 0.23 mM. 5. 5.|The resistance to the sulfhydryl reagents p-mercuribenzoate and iodoacetate together with the high Hg 2+ K i suggest that sulfhydryl groups are not essential for the midgut amylase activity. 6. 6. Curves of blue-reducing values showed the midgut amylase is an α-amylase with a multiple attack degree between 1.7 and 6. 7. 7. Polyacrylamide gel electrophoresis of midgut homogenates shows the existence of two major isoamylases one of which is largely predominant. 8. 8. The results are compared with α-amylases from several sources.