| pubmed-article:16661543 | pubmed:abstractText | The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V(max) for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K(m) for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K(m) values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V(max) values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K(m) values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K(m) value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity. | lld:pubmed |
