CELLULAR BIOCHEMISTRY 



by adenylic acid, the concentration needed (1 X 10"^ M) is 

 higher than that found in muscle. Epinephrine, when added to 

 liver slices or to diaphragm muscle incubated aerobically in 

 phosphate-Ringer's solution, causes a rapid increase in the con- 

 centration of active phosphorylase and at the same time the 

 glucose output of the liver slices and the lactic acid production 

 by the diaphragm are increased (19), No effect of epinephrine 

 has so far been described in a cell-free system. In order to 

 measure the relative amounts of the two forms of phosphorylase 

 in resting muscle, special precautions are necessary. The tech- 

 nique recently used involved freezing the gastrocnemius muscle 

 in situ in amytalized rats. The muscle was rapidly homogenized 

 in the cold in a Waring Blendor in a solution containing 0.001 

 M versene and 0.02 M fluoride, which inhibits the intercon- 

 version enzymes but has no effect on phosphorylase activity. 

 Under these conditions rat muscle was found to contain 30 to 

 50 per cent phosphorylase a, the remainder being phosphory- 

 lase b.* Following the subcutaneous or intravenous injection 

 of epinephrine the phosphorylase a content of muscle rose to 

 nearly 100 per cent within a few minutes, whereas the total 

 phosphorylase content remained unchanged. In isolated rat 

 diaphragm Sutherland (19) observed an increase in phos- 

 phorylase a content within 4 minutes after the addition of epi- 

 nephrine. Unexplained is the mechanism of the reversal of phos- 

 phorylase action. This occurs in an isolated rat diaphragm 

 which is actively synthesizing glycogen from the glucose in the 

 medium. As soon as epinephrine is added, glycogen break- 

 down exceeds glycogen synthesis so that there is either no gain 

 or an actual loss of glycogen (20). The hexosemonophosphate 



* Similar values were obtained when muscle not frozen in situ was homog- 

 enized within a few seconds after excision from the animal. When fluoride 

 was omitted from the solution used for homogenization, almost all of the 

 phosphorylase was present in the inactive or b form. An amount of homogenate 

 corresponding to 4 to 5 mg. of muscle was used in the phosphorylase tests. 

 The activity in a test without adenylic acid, expressed as per cent of the 

 activity with adenylic acid, corresponds to the per cent of phosphorylase a. 



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