METHODS

Muscle extracd8. For assay of glucose or pentose heart muscle was extracted with boiling water as described by Battaglia & Randle (1960).

Glycogen synthesis. This was assayed by the change in glycogen concentration during perfusion of rat heart or incubation of rat hemidiaphragms. The initial glycogen was measured in heart muscle on separate hearts washed through with 10-15 ml. of medium. With hemidiaphragms one member of eachpair was used for assayof the initial glycogen concentration and the other incubated and used for assay of the final glycogen concentration. Glycogen was assayed in muscle as follows. Muscle was dissolved in 30%(w/v) KOH (2-4 ml./g.) and crude glycogen was precipitated and washed as described by Walaas & Walaas (1950). The crude glycogen was then hydrolysed in 2 ml. of 2N-H2SO4 for 3 hr. on a boiling-water bath. After cooling, the hydrolysate was diluted and brought to pH 7 by the addition of 4 ml. of M-sodium phosphate buffer, pH 7, and 2 ml. of 2N-NaOH and water to 10 ml. Glycogen glucose was thenassayed on a suitable sample by the glucoseoxidase method (Huggett & Nixon, 1957). In some experiments glycogen synthesis was measured in perfused heart with [6-14C] glucose as follows. The crude glycogen was precipitated and washed as described above, and then dissolved in 5-10 ml. of water. A sample (1-2 ml.) of this solution was hydrolysed and glycogen glucose assayed to obtain the glycogen concentration as described above. The glycogen in the remainder of the solution was purified by precipitation of contaminating material at pH 4-0-4-5 (adjusted with glass electrode and 2N-H2SO4) followed by shaking with an equal volume of CHCl3 and a drop of octan-2-ol and removal of the aqueous phase after centrifugation (contaminating material is precipitated at the interface). In control experiments the aqueous phase was shown to yield (after dialysis against distilled water and precipitation with ethanol and drying of the pre-cipitated glycogen in vacuo) glycogen that on analysis contained, by wt., 96-99% of glucose. The aqueous phase was hydrolysed and analyses were made of glucose (see above) and radioactivity (after plating and assay in a Nuclear-Chicago gas-flow counter). The specific activityof medium glucose was measured in the same way. The net incorporation of medium glucose into muscle glycogen (in mg. of glucose/g. wet wt. of tissue) is given by: Glycogen conen.(mg./g.) sp. activity of purified glycoven