Purification and Characterization of the Maize Amyloplast Stromal 112-kDa Starch Phosphorylase
| Autor: | Bruce P. Wasserman, George M. Carman, Ying Yu, Helen He Mu |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Phosphorylases
Starch Amylopectin Immunoblotting Size-exclusion chromatography Biophysics Phenylglyoxal Zea mays Biochemistry Catalysis Adenosine Diphosphate Glucose chemistry.chemical_compound Enzyme activator Cations Diethyl Pyrocarbonate Amyloplast Phosphorylation Molecular Biology Anion Exchange Resins chemistry.chemical_classification Gel electrophoresis Starch phosphorylase Chromatography Chemistry Sepharose Temperature Hydrogen-Ion Concentration Chromatography Agarose Enzyme Activation Kinetics Resins Synthetic Glucose Enzyme Models Chemical Ammonium Sulfate Chromatography Gel Electrophoresis Polyacrylamide Gel Adsorption |
| Zdroj: | Archives of Biochemistry and Biophysics. 388:155-164 |
| ISSN: | 0003-9861 |
| DOI: | 10.1006/abbi.2000.2267 |
| Popis: | A plastidic 112-kDa starch phosphorylase (SP) has been identified in the amyloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymological and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch adsorption, Q-Sepharose, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-polyacrylamide gel electrophoresis. Anti-SP antibodies recognized the purified 112-kDa SP enzyme and N-terminal amino acid sequence analysis confirmed that the purified enzyme is the amyloplast stromal 112-kDa SP. Analysis of the purified enzyme by Superose 6 gel filtration chromatography indicated that the native enzyme consisted of two identical subunits. The pH optimum for the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolytic direction. SP activity was inhibited by thioreactive agents, diethyl pyrocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 and 16.9 kcal/mol, respectively, and the enzyme was thermally labile above 50 degrees C. Results of kinetic experiments indicated that the enzyme catalyzes its reaction via a sequential Bi Bi mechanism. The Km value for amylopectin was eight-fold lower than that of glycogen. A kinetic analysis indicated that the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (4 to 7 units) were used as substrates. The specificity constants (Vmax/Km) of the enzyme measured in either the synthetic or the phosphorolytic directions increased with increasing chain length. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect