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The following D. discoideum glycosidases were studied: beta-hexosaminidase (EC 3.2.1.30 and EC 3.2.1.17), alpha-mannosidase (EC 3.2.1.24), beta-glucosidase (EC 3.2.1.21), beta-galactosidase (EC 3.2.1.23) and alpha-glucosidase (EC 3.2.1.20) or glucoamylase (EC 3.2.1.3). beta-hexosaminidase and beta-glucosidase were purified to greater than 90% homogeneity, alpha-mannosidase was purified to two protein components both with enzymic activity; whereas alpha-glucosidase and beta-galactosidase could not be separated and the purified preparation contained several components. The biochemical and biophysical properties of the purified enzymes are described. Antibodies against these enzymes were prepared and the immunological properties of purified and crude samples of glycosidases from various stages of the slime mould life-cycle were examined. These antibody preparations were used as tools to isolate isotopically labelled enzymes from crude extracts in experiments which show that beta-hexosaminidase and glucosidase are synthesized de novo and are stable during growth and the first few hours of development. From these studies and others it is concluded that the rates of accumulation of assayable enzyme activity of beta-hexosaminidase and glucosidase reflects precisely the rates of de novo enzyme synthesis; the effect of enzyme degradation, inhibition or activation being negligible. It is concluded that the marked Increases in the levels of beta-hexosaminidase, glucosidase and galactosidase activities per cell during the life-cycle of D. discoideum are determined by changes in the rate of general protein synthesis and degradation and changes in the rate of cell division rather than changes in the rate of enzmye synthesis. For alpha-mannosidase changes in the rate of enzyme synthesis may also be a factor. Studies on the physiological role of the glycosidases indicate that they have an intracellular digestive function during various stages of the life-cycle; during growth some of the enzymes act on the exogenous food source within food vacuoles, and during development some of the enzymes act on endogenous material, probably' within autophagic vacuoles. |
