Trehalase from the dormant spore of Dictyostelium discoideum

Abstract

Following purification of dormant spores of the cellular slime mold Dictyostelium discoideum, trehalase (α,-α′-trehalose-1- d-glucohydrolase, EC 3.2.1.28) activity was solubilized by a freeze-thaw cycle followed by passage of the crude homogenate through a French pressure cell. Enzyme specific activity for spore trehalase was 1.8 nmol/min/mg, a value no greater than 10% of that observed for the crude myxamoebal enzyme. Assay of samples of myxamoebal and spore enzyme singly and after they were mixed together resulted in values additive for trehalase activity from the two cell types. Enzymatic activity was stable to repeated freeze-thaw cycles, storage at 6°C, and temperatures up to about 50°C. Based upon analysis of kinetic data with Arrhenius plots, theQ 10 and energy of activation were estimated (between 30 and 40°C) as 1.95 and 12.0 ± 0.5 kcal/mol, respectively. The pH and temperature optima for maximal activity were 5.5 and 46 to 50°C, respectively. The apparentK m for spore trehalase was 1.2 m M trehalose as estimated from Lineweaver-Burk double-reciprocal plots of initial velocity data versus substrate concentration. Electrophoretic characterization of the spore enzyme indicated the presence of a single peak of trehalase and that the latter had a relative mobility equal to that of the enzyme purified from vegetative myxamoebae (i.e., isozyme I). The developmentally regulated form of trehalase, i.e., isozyme II, was not detectable.