Purification and characterization of the maize amyloplast stromal 112-kDa starch phosphorylase

A plastidic 112-kDa starch phosphorylase (SP) has been identified in the am yloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymol ogical and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch ad sorption, Q-Sepharose, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-po lyacrylamide gel electrophoresis. Anti-SP antibodies recognized the purifie d 112-kDa SP enzyme and N-terminal amino acid sequence analysis confirmed t hat the purified enzyme is the amyloplast stromal 112-kDa SP. Analysis of t he purified enzyme by Superose 6 gel filtration chromatography indicated th at the native enzyme consisted of two identical subunits. The pH optimum fo r the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolyt ic direction. SP activity was inhibited by thioreactive agents, diethyl pyr ocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 and 16.9 kcal/mol, respec tively, and the enzyme was thermally labile above 50 degreesC. Results of k inetic experiments indicated that the enzyme catalyzes its reaction via a s equential Ri Ri mechanism. The K-m value for amylopectin was eight-fold low er than that of glycogen. A kinetic analysis indicated that the phosphoroly tic reaction was favored over the synthetic reaction when malto-oligosaccha rides (4 to 7 units) were used as substrates. The specificity constants (V- max/K-m) of the enzyme measured in either the synthetic or the phosphorolyt ic directions increased with increasing chain length. (C) 2001Academic Pres s.