Elsevier

Carbohydrate Research

Volume 227, 6 April 1992, Pages 241-255
Carbohydrate Research

(1 → 4)-α-d-Glucan synthesis by a chloroplastic phosphorylase isolated from spinach leaves in independent of added primer

Dedicated to Professor David Manners.
https://doi.org/10.1016/0008-6215(92)85075-BGet rights and content

Abstract

The chloroplastic phosphorylase from spinach leaf was partially purified using ammonium sulfate fractionation and chromatography on DE-52 and Mono-Q columns. The enzyme was capable of synthesizing (1 → 4)-α-d-glucan in the absence of added glucan primer, with a lag in time that was shortened by increasing enzyme concentration or by adding bovine serum albumin and/or sodium citrate. The product of the unprimed activity formed a blue complex with iodine-iodide reagent (peak of absorption between 600 and 620 nm) that was insoluble in 5% trichloroacetic acid and in 1% (w/v) KCl in 75:25 methanol-water. Umprimed phosphorylase activity was greatly stimulated by citrate and less by other anions like malate and succinate. Addition of branching enzyme to the assay medium stimulated the unprimed reaction two-fold, or 42-fold if assayed in the presence of 0.1m citrate. In the primed phosphorylase reaction, the presence of 0.8m citrate decreased the Km for glycogen and amylopectin from 1.8 and 0.17 mg/mL to 0.12 and 0.055 mg/mL, respectively. The phosphorylase fraction (after f.p.l.c.) was found to contain 27 nmol of “anhydroglucose” per mg of protein. It is concluded that the chloroplast phosphorylase from spinach leaves in capable of synthesizing (1 → 4)-α-d-glucan in the absence of added glucan primer, possibly by elongating an endogenous primer that is associated with the enzyme in a non-covalent fashion.

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