Phosphorylases isolation from potato

Earlier studies on the properties of phosphorylases isolated from various sources have indicated that their subunits are similar in size with about 100,000 daltons.15-17 The reaction proceeds in a rapid equilibrium random Bi-Bi mechanism as has been shown by kinetic studies with rabbit skeletal muscle phosphorylases a18-20 and b,21,22 rabbit liver enzyme,23 potato tuber enzyme,24 and the enzyme from E. coli.25) In contrast, the substrate specificities for various glucans differ considerably depending on the enzyme sources. The rabbit muscle enzyme has high affinity for branched glucans such as glycogen and amylopectin but low affinity for amylose and maltodextrin.26,27 The potato tuber enzyme can act on amylose, amylopectin, and maltodextrin but only poorly on glycogen,28,29 while the E. coli enzyme shows high affinity for maltodextrin.10 ... 

Table 12-2 Analysis of Glucose-1-Phosphate Isolated from Potato Phosphorylase Reaction pmol of Reducing /xmol of Inorganic Equivalents/0.1 ml Phosphate/0.1 ml...

The continuing interest of Bourne in the chemistry of polysaccharides and associated enzymes originated from the work of Haworth and Peat directed towards the enzymic synthesis and degradation of starch. The impetus for this work was given by the discovery, made by C. S. Hanes in 1940, that a phosphorylase isolated from the potato and pea effects the synthesis, from D-glucosyl phosphate, of starch, later shown (by Haworth, Heath, and Peat) to be amylose. In his first paper (with Haworth and Peat) in 1944, Bourne described the isolation of the Q-enzyme which, in conjunction with phosphorylase, effects the conversion of D-glucosyl phosphate into the major component of whole starch, namely, amylopectin. He had discovered the Q-enzyme in a fraction discarded by previous workers. Already, the quintessence of his mind was revealed in this work meticulous attention to detail, and perception of essentials. 

Phosphorylase can be easily isolated from potatoes and, after purification, used to catalyze the polymerization of glucose-1-phosphate in order to obtain linear polysaccharide chains with a-(l— 4) glycosidic linkages, as can be seen in figure 5. 

Both described enzymes are isolated from natural sources. Phosphorylase is isolated from potatoes whereas the glycogen branching enzyme is produced by various bacterial sources. Depending on the source the properties of the products and the reaction conditions may differ. 

Potato phosphorylase (1) was isolated from potato tubers by precipitation with ammonium sulphate according to the method of Ziegast et al. (9). Further purification was carried but by hydrophobic interaction... 

Soon after Cori and Cori showed the existence of a branching factor, Haworth, Peat, and Bourne reported the isolation from potato juice of an enzyme fraction termed Q-enzyme, which, in association with potato phosphorylase, produced a polysaccharide having the properties of amylopectin. Peat and his collaborators also presented evidence that the Q-enzyme was capable of converting linear amylose to branched amylopectin without the participation of inorganic phosphate in the reaction. They therefore concluded that the Q-enzyme is a nonphos-phorolytic enzyme. Besides being present in the potato, Q-enzyme has also been found in the wrinkled pea,the broad bean, Neisseria perfiava, and Polytomella coeca. " ... 

From Solarium tuberosum, Slabnik and Frydman isolated a unique phosphorylase that has no requirement for the primer addition for formation of an amylopectin-like polysaccharide in a cell-free system. The properties of this enzyme were found to differ from those of the usual potato phosphorylase this new enzyme is assumed to be a glycoprotein, the glycosidic component of which acts as the primer. The activity of this phosphorylase disappeared at 55 , in contrast to the usual phosphorylase activity (which withstands this temperature). There was also good correlation between formation of polysaccharide and appearance of inorganic phosphate in the absence of primer. The polysaccharide formed de novo" was shown to be an eflScient primer for starch synthesis. ... 

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