PURIFICATION AND CHARACTERIZATION OF PHOSPHORYLASE FROM TUBERS OF DIOSCOREA-DUMENTORUM

Phosphorylase from tubers of Dioscorea dumentorum was fractionated by (NH4)2SO4 precipitation and further purified to apparent homogeneity. Two forms of phosphorylase, Dd1 and Dd2 were obtained with different K(m), optimum temperatures and pH values. They obeyed Michaelis-Menten kinetics, they were activated by AMP, Mg2+ and Ca2+ but inhibited by ATP, ADP, ADP-glucose and Na2SO4. The molecular masses found for Dd1 and Dd2 were 102 000 and 195 000 respectively. SDS gel electrophoresis indicated that Dd2 is a dimer and Dd1 is a monomer. Both phosphorylase forms contained pyridoxal-5'-phosphate as their prosthetic group, which was essential for their activity. Dd2 is more active in starch synthesis while Dd1 is more active in starch degradation. The affinity of the enzyme forms for P(i) is not a measure of the rate of glucan degradation. The decline in activities of the enzymes between pH 5.5 and 7.5 was due to unsuitable ionic forms of the enzymes and not due to irreversible denaturation. Both forms of phosphorylase showed double displacement reaction mechanism in direction of phosphorolysis, while in direction of synthesis, sequential mechanism was indicated. Indoleacetic acid activated the enzyme forms in direction of starch degradation but acted as an inhibitor in direction of glucan synthesis. The results of this study suggest that Dd2 may have some synthetic function while Dd1 has a degradative role. The effect of pH on the phosphorylase activity suggests histidine as the amino acid residue around the active site that might be involved in enzyme catalysis.