Purification and characterization of glycogen phosphorylase b from the breast muscle of fruit bat, <i>Eidolon helvum</i> Kerr.

Abstract

The kinetic and physicochemical properties of glycogen phosphorylase b from the breast muscle of the fruit bat, Eidolon helvum Kerr were investigated in order to obtain some information about the possible physiological role of the enzyme in meeting the energy requirements of the bat muscle either at the initiation of or during flight. Glycogen phosphorylase b was purified from fruit bat breast muscle to apparent homogeneity with specific activity of approximately 37 units/mg of protein and a yield of 4 %. The enzyme was completely dependent on AMP for activity; hence it was designated phosphorylase b. The native and subunit molecular weights of the enzyme as determined by gel filtration on Sephadex G-200 and SDS-polyacrylamide gel electrophoresis were 187,000  12,600 Da and 90,500  1,200 Da respectively, thus implying that it is a dimeric protein. The Michealis-Menten constants, Km, of the enzyme for glycogen and glucose-1-phosphate were 0.06 mg/ml and 6.94 mM respectively, while apparent Km for AMP was 0.08 mM. The turnover number, kcat, was 65.56 s-1. Although sodium fluoride (NaF) and sodium sulphate (Na2SO4) on their own activated the enzyme, it was observed that in the presence of AMP, only sodium sulphate (Na2SO4) caused activation at low concentration. The optimum pH for the fruit bat muscle phosphorylase b activity was 6.4. In conclusion, the overall results of this study showed that the fruit bat muscle glycogen phosphorylase b is physically and catalytically similar to the enzyme from other mammalian sources.

International Journal of Biological & Chemical Sciences Vol. 1 (2) 2007: pp. 99-107