Purification and properties of alpha-glucan phosphorylase b from Indocibium guttattam
Soman, G.; Philip, G.
Indian Journal of Biochemistry and Biophysics 13(3): 202-207
1976
ISSN/ISBN: 0301-1208 PMID: 1010562 Document Number: 103225
.alpha.-Glucan phosphorylase b (a-D-1,4 glucan:orthophosphate .alpha.-D-glucosyl transferase, EC 2.4.1.1) was purified by a procedure involving ammonium sulfate fractionations, absorption on alumina C.gamma. gel and DEAE-cellulose chromatography. The purified enzyme showed an absolute requirement for AMP for activity and had specific activity comparable to that of rabbit skeletal muscle phosphorylase b. The enzyme had a MW of 2 .times. 105 and contained 2 mol of pyridoxal phosphate and 8-9 thiol groups. The enzyme was less stable as compared to the rabbit enzyme. AMP, EDTA or mercaptoethanol did not afford protection against inactivation. Heavy metal ions like Hg2+ and Ag+ and thiol-blocking reagents like p-hydroxymercuric benzoate and 5,5'-dithiobis-[2-nitrobenzoic acid] inactivated the enzyme. In contrast to the rabbit enzyme, inactivation of the fish enzyme by a thiol-blocking reagent was not reversed by cysteine. Modification of any thiol group with 5,5'-dithiobis-[2-nitrobenzoic acid] resulted in partial loss of activity.