Sucrase activity and its kinetic properties in peritrophic membrane, and in membrane-bound and soluble fractions of midgut in the silkworm, Bombyx mori

Abstract

The contribution of sucrase in the peritrophic membrane to the hydrolysis of sucrose in the midgut of the silkworm, Bombyx mori was evaluated. Sucrase activities per individual insect and its kinetic properties were determined in the peritrophic membrane and membrane-bound and soluble fractions of midgut between days 2 and 6 in the fifth instar. The activity of the sucrase in the peritrophic membrane relative to membrane-bound sucrase of midgut increased, from day 2 to day 6, approximately 5-fold; from 1:334 to 1:71 determined in sodiumpotassium-phosphate buffer, which appeared to indicate an increasing significance of the sucrase in the peritrophic membrane during this developmental period. The sucrase hi the peritrophic membrane showed high activity at alkaline pH, 8.0–11.0 as well as at pH 6.5–8.0. A shift towards higher activity was observed from day 4 to day 6, at pH 5.0–6.5 in all three sucrases, but at pH 8.0–11.0, a smaller shift was observed in the sucrase in the peritrophic membrane. The Km of the sucrase in the peritrophic membrane was 1.2 mM and an apparent substrate activation was observed, which was similar to the characteristics of the membrane-bound sucrase of midgut. Determination of sucrase activity in the presence of various metal ions at 1 and 10 mM disclosed that most of the metal ions exerted inhibitory effects, notably in Hg2+, Cu2+ and Zn2+. Similar effects were observed with the sucrase in the peritrophic membrane and soluble sucrase of midgut. The effects of Tris and Tricine, and phlorizin, phloretin and ouabain on the activity of the membrane-bound sucrase were investigated. Among them, Tris was the most potent inhibitor followed by Tricine; 40 and 30% of the control activity were observed at final 32 and 150 mM, respectively. Phlorizin and ouabain exerted no effect, whereas Phloretin caused the activity to reduce to 80% of the control at the final 400 mM.