Lxxv. Studies in Glycolysis Ii. the First Stages of Glycolysis in Muscle Extract;

IN a previous communication [Kendal & Stickland, 1937, 1] it was shown that if rabbit muscle is minced and thoroughly washed with water, and then extracted several times with dilute Na2HPO4, the later extracts (usually the 4th) contain much of the glycolytic enzyme system but are freer from coenzymes than extracts prepared by earlier methods. This was shown by the fact that on addition of boiled muscle extract a rapid formation of lactic acid from polysaccharide took place, while on addition of Mg+ +, adenosinetriphosphate, cozvmase and hexosediphosphate practically no lactic acid was formed. It was also shown that if Mg++ and adenosinetriphosphate alone were added to such extracts considerable quantities of phosphate were esterified, although no lactic acid was formed. The present communication deals with the nature of the esters formed under these conditions. The initial esterification process in the glycolytic enzyme system prepared by the method of Meyerhof [1925] has been studied by Parnas and his collaborators [Parnas & Baranowski, 1935; Ostern et al. 1936]. They found that a direct reaction between glycogen and inorganic phosphate takes place, giving rise to the Embden ester. Later Parnas & Mochnacka [1936] showed that by suitable treatment (electrodialysis at pH 8 2, or autolysis at 200 followed by long dialysis) extracts could be obtained which no longer phosphorylysed glycogen except on addition of adenylic acid or inosinic acid. Since the original extracts were thought to be free from adenosine phosphates it was supposed that they must have contained inosinic acid. As will be seen later esterification may be brought about by a concentration of adenosinetriphosphate or adenylic acid much too low to be detectable by chemical methods. Cori & Cori [1936; 1937] discovered, as an intermediate step in the formation of the Embden ester, a new ester which they identified as an aldose-l-monophosphate, and later as phosphoric acid o-glucoside. This ester was formed under the action of washed muscle or pulp or ordinary dialysed (17 hr.) aqueous muscle extract, and was rapidly transformed by the same enzyme preparations into the ordinary Embden ester. The addition of adenylic acid increased the rate of formation of this new ester. Our experiments with the new enzyme preparations confirm and extend the results of Parnas and of Cori & Cori.