Purification and characterization of a cytosolic starch phosphorylase from etiolated rice seedlings

Abstract

Starch phosphorylasel (Pho1) from etiolated rice (Oryza sativa L. cv. Tainong 67) seedlings was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B anion exchange chromatography, and dextrin-Sepharose 4B affinity chromatography. The purification fold was 299, and the enzyme activity recovery was about 21%. The molecular mass of the native Pho1 on Superose 12 gel filtration was 145 kDa. The subunit molecular weight as determined by SDS-PAGE was 85 kDa. The enzyme has an optimum pH of 5 and an optimum reaction temperature of about 45℃~50℃. In the synthetic reaction for Glc 1-P, the k(subscript m) value was 2.1 mM, and the V(subscript max) value was 5.85 U mg ^-1. In the phosphorolytic direction for orthophosphate, the k(subscript m) value was 3.8 mM. Phol has a higher affinity for amylopectin, glycogen, soluble starch and dextrin than for maltooligosaccharide (6 to 10 glucose units). In addition, the k (subscript m) value for amylopectin was ninefold lower than for dextrin. Cyclohexaamylose, cycloheptaamylose, cyclooctaamylose, and maltotetrose were inhibitors of Pho1. Mannose 1-P, Fru 6-P, ADPGlc, UDPGlc, AMP, IMP and PEP also inhibit Pho1. The metal ions Ag (superscript +), Hg(superscript 2+), and Zn(superscript 2+) also reduce the enzyme activity. However, thiol reagents activate Phol activity, suggesting that sulfhydryl-group(s) may be required for enzyme stability.