Biochemical and molecular biological studies of starch phosphorylase from etiolated rice seedlings

• Main Authors: Jen-Hung Hsu, 許仁弘
• Other Authors: Ping-Du Lee, Ph.D.
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/91512025465968007387

博士 === 國立臺灣大學 === 農業化學研究所 === 91 === Abstract Starch phosphorylase (H-RSP) 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 300 and the enzyme activity recovery was about 21 %. The molecular mass of the native H-RSP on Superose 12 gel filtration was 143 kDa. The molecular mass of subunit 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℃. H-RSP has a higher affinity for amylopectin, glycogen, soluble starch and dextrin rather than for maltooligosaccharide (6 to 10 glucose units). In addition, the Km value for amylopectin was nine-fold lower than for dextrin. Cyclohexaamylose, cycloheptaamylose, cyclooctaamylose, and maltotetrose were inhibitors of H-RSP. Mannose-1-P, Fru 6-P, ADPGlc, UDPGlc, AMP, IMP and PEP also inhibite H-RSP. The metal ions Ag+, Hg2+ and Zn2+ also reduce the enzyme activity. However, thiol reagents activate H-RSP activity, suggesting that sulfhydryl-group(s) may be required for enzyme stability. In the synthetic reaction, the Km value for Glc 1-P was 2.1 mM. The Km for orthophosphate of H-RSP in the phosphorolytic direction was 3.83 mM. The Km value for Glc 1-P was lower than for orthophosphate. In the phosphorolytic direction, H-RSP favored soluble starch more than glycogen as substrate. The Km values were 0.76 and 1.19 mg mL-1, respectively, which is in contrast with the synthetic reaction. Based on the results, the kinetics data indicates that H-RSP favors synthetic direction over phosphorolytic direction. Therefore, in the etiolated rice seedlings, H-RSP may play role in starch synthesis. RSP cDNA clones were obtained from etiolated rice seedlings. The H form RSP full length cDNA is detected as 2,502 bp long whereas the full length of L form cDNA is 2,788 bp in length. The similarities between the L and H forms RSP in nucleic acid and protein level are 63.4% and 73.6%, respectively. Among the plants, the homology of H form RSP cDNA between rice and wheat is highest, and with 89.2% identity. Whereas, the L form rice RSP is more similar to the type II L form SP of potato leaf. In protein sequence composition, the L form differs from the H form with an 80 amino acids insertion sequences called Loop 80 (L80). The L80 insertion located at the starch binding site, which likely accounts for the differences in their substrate specificity. In addition, there is no protein degrading PEST sequences was found in the L80.