Discussion 219 



DISCUSSION 



Lehninger: Is acyl phosphatase present in the ascites cell extracts? 

 Harary has proposed a mechanism for dephosphorylation of ATP 

 through 1 : 3-diphosphoglycerate (Harary, I. (1957). Biochim. biophys. 

 Acta, 26, 434). 



Racker: Krimsky in our laboratory has studied the hydrolysis of 

 1 : 3-diphosphoglycerate. The acyl phosphatase described by Prof. 

 Lipmann many years ago was found to cleave 1 : 3-diphosphoglycerate. 

 In fact, 1 : 3-diphosphoglycerate is so labile that, when a high enough 

 concentration of it is maintained, the rate of non-enzymic hydrolysis is 

 quite rapid. In the presence of 3-phosphoglycerate (PGA), PGA kinase 

 and an ATP-regenerating system, the spontaneous hydrolysis of 1 : 3- 

 diphosphoglycerate is quite appreciable so that it gives the appearance 

 of the presence of an active ATPase which is dependent on PGA. 



One can readily demonstrate in extracts of ascites tumour cells a 

 PGA-activated "ATPase activity". Whether the observed rate of 

 phosphate liberation is entirely due to spontaneous hydrolysis of 1 : 3-di- 

 phosphoglycerate, or whether other mechanisms contribute (e.g. 

 specific acyl phosphatases) has not been established. It may be added 

 that in addition to the heat-stable enzyme, skeletal muscle contains a 

 heat-labile phosphatase with somewhat different properties. A heat- 

 labile enzyme was also observed by Harary to be present in liver. 



Chance: This model is largely based upon experiments with ascites 

 tumour cells in which we have demonstrated three different metabolic 

 events following glucose addition (Chance, B., and Hess, B. (1959). 

 Science, in press): (1) the activation of respiratory and phosphorylative 

 activity upon adding glucose; (2) an inhibition of intracellular ATP 

 utilization by a lowering of the intracellular ATP level ; (3) an inhibition 

 of both respiration and glucose utilization when the added glucose . 

 exceeds the available ATP store. The latter regulation is related to, 

 but more intense than, that which is recognized as the Crabtree effect. 

 It is the purpose of the model to demonstrate whether or not these 

 regulations can be manifestations of the same process of metabolic 

 control. 



The model contains four basic functions as indicated by the four 

 main blocks of Fig. lA. First, phosphorylation of glucose; second, 

 glycolytic phosphorylations of ADP ; third, oxidative phosphorylations 

 of ADP; fourth, ATP utilization. In these experiments, the glucose 

 concentration is initially zero, but the system contains a pyruvate 

 store, an inorganic phosphate store and two ATP stores, one associated 

 with the glucose phosphorylation enzymes and the other associated 

 with the oxidative phosphorylative system. Respiratory metabolism 

 is limited by the rate of ATP utilization, the rate of which is set not 

 only by the needs of the cell for synthesis, transport, etc., but also by 

 the rate at which ATP is made available from store II to store I (the 

 assignment of endogenous ATP utilization to ATP directly expendable 

 from store I is not wholly arbitrary since regulation 2 mentioned above 

 appears to require that this be so). 



