TITLE Purification and characterization of a cytosolic starch phosphorylase from etiolated rice seedlings
AUTHOR Jen-Hung HSU
Biochemistry Laboratory, Department of Agricultural Chemistry, and Department of Biochemical Science and Technology, National Taiwan University, No.1 Roosevelt Rd., Sec 4, Taipei 106, Taiwan
Chien-Chih YANG
Biochemistry Laboratory, Department of Agricultural Chemistry, and Department of Biochemical Science and Technology, National Taiwan University, No.1 Roosevelt Rd., Sec 4, Taipei 106, Taiwan
Jong-Ching SU
Biochemistry Laboratory, Department of Agricultural Chemistry, and Department of Biochemical Science and Technology, National Taiwan University, No.1 Roosevelt Rd., Sec 4, Taipei 106, Taiwan
Ping-Du LEE*
Biochemistry Laboratory, Department of Agricultural Chemistry, and Department of Biochemical Science and Technology, National Taiwan University, No.1 Roosevelt Rd., Sec 4, Taipei 106, Taiwan
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ABSTRACT Starch phosphorylase1 (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 45oC~50 oC. In the synthetic reaction for Glc 1-P, the Km value was 2.1 mM, and the Vmax value was 5.85 U mg-1. In the phosphorolytic direction for orthophosphate, the Km value was 3.8 mM. Pho1 has a higher affinity for amylopectin, glycogen, soluble starch and dextrin than for maltooligosaccharide (6 to 10 glucose units). In addition, the Km 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+, Hg2+ and Zn2+ also reduce the enzyme activity. However, thiol reagents activate Pho1 activity, suggesting that sulfhydryl-group(s) may be required for enzyme stability.
KEYWORD Affinity chromatography; Dextrin; Glucan; Rice; Starch phosphorylase;
ARTICLE INFO Botanical Bulletin of Academia Sinica, Volume 45 Number 3 July 2004, page 187-196, 10 pages
PUBLISHER Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China