title: Purification and characterization of the maize amyloplast stromal 112-kDa starch phosphorylase.
abstract: 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.
authors.affiliation: Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08901, USA.
authors.forename: H H
authors.initials: HH
authors.lastname: Mu
authors.forename: Y
authors.initials: Y
authors.lastname: Yu
authors.forename: B P
authors.initials: BP
authors.lastname: Wasserman
authors.forename: G M
authors.initials: GM
authors.lastname: Carman
mesh_terms: D000245:Adenosine Diphosphate Glucose / Q000378:metabolism; D000327:Adsorption; D000645:Ammonium Sulfate / Q000494:pharmacology; D000687:Amylopectin / Q000378:metabolism; D000837:Anion Exchange Resins / Q000737:chemistry; D002384:Catalysis; D002412:Cations; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D008956:Models, Chemical; D010658:Phenylglyoxal / Q000737:chemistry; D006005:Phosphorylases / Q000737:chemistry* / Q000302:isolation & purification*; D010766:Phosphorylation; D012117:Resins, Synthetic; D012685:Sepharose / Q000737:chemistry; D013213:Starch / Q000737:chemistry; D013696:Temperature; D003313:Zea mays / Q000201:enzymology*
chemical_list: D000837:Anion Exchange Resins; D002412:Cations; D012117:Resins, Synthetic; D000245:Adenosine Diphosphate Glucose; C042855:Mono Q; D013213:Starch; D012685:Sepharose; D000687:Amylopectin; D006005:Phosphorylases; D005947:Glucose; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D000645:Ammonium Sulfate