Changes in kinetic parameters and total activity of midgut sucrase in the silkworm, Bombyx mori during larval-pupal-adult development

Abstract

1. 1. Developmental changes in total activity of midgut sucrase in the silkworm, Bombyx mori were determined with two separate assay systems which were prepared based on kinetic parameters of midgut sucrase from larvae and pharate adults. 2. 2. pH Optima were at 6.0–6.6 for the fifth instar larvae and at pH 5.6 and 6.9 for the pharate adults. K m values toward sucrose at pH 5.6 were 10.0 mM for larval enzyme and 2.3 mM for pharate adult enzyme. For assay of the larval type of enzyme, a final concentration of 100 mM substrate at pH 6.0 was employed and for pharate adult type of enzyme, 15 mM substrate at pH 5.6. 3. 3. To determine the moment that midgut sucrase changes electrophoretically from larval to pharate adult type, electrophoresis of midgut tissue homogenates on native polyacrylamide gel at pH 8.9 was conducted during silkworm development and the change was observed on the day of larval-pupal ecdysis. 4. 4. Sucrase activity was low in the fourth instar. From the fifth instar it increased steeply to reach a maximum level at day 4–6 with V m = 87.0 μmoles of glucose/min per individual midgut, which was approximately 9-fold greater than the level at day 0, fifth instar. Activity then decreased rapidly to a low level until larval-pupal ecdysis and the level was maintained until 1 day before adult emergence. 5. 5.In the fifth instar larvae, the membrane-bound fraction of midgut tissue homogenate exhibits low but significant sucrase activity, consisting of up to 10% of total activity. pH-Activity relationships of membrane-bound and soluble enzymes were similar in the pH range between pH 5.5 and 7.5 but the K m value for sucorse was 7.7 mM and 16.6 mM for membrane-bound and soluble enzyme, respectively. In high substrate concentration the membrane-bound enzyme showed activation by substrate whereas the soluble enzyme showed substrate inhibition.