biologia plantarum

Biologia plantarum 36:409, 1994 | DOI: 10.1007/BF02920941

Purification and characterization of phosphorylase from tubers ofDioscorea dumentorum

U. Olooha¹, E. N. Ugochukwu¹

¹ Department of Biochemistry, University of Benin, Benin City, Nigeria

Phosphorylase from tubers ofDioscorea 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.

Received: February 8, 1993; Accepted: May 11, 1993; Published: September 1, 1994  Show citation