FACTORS AFFECTING METABOLISM in Vttro 107 



only substrate which appears fully capable of meeting the energy 

 requirements of the tissue in vitro. 



Lack of Oxygen and Metabolic Inhibitors 



It was shown above that glucose was the substrate most actively 

 supporting phosphorylation in cell-containing tissues in vitro. 

 Information regarding the extent to which the various stages of its 

 metabolism are necessary for this support can be obtained by 

 inhibiting or restricting one particular enzymic system more than 

 another. This can be achieved by studying metabolism under 

 anaerobic conditions, where glycolysis is the sole energy-producing 

 system or by the addition of various metabolic inhibitors such as 

 fluorides, iodoacetates, malonates, cyanides, azides and 2 : 4- 

 dinitrophenol. 



With cerebral slices metabolizing glucose, replacement of an 

 oxygen atmosphere by nitrogen markedly reduces the levels of 

 phosphocreatine (Mcllwain, \9S%) and the incorporation of 

 radioactive phosphate into the phospholipids of rat and cat cerebral 

 slices (Schachner et al., 1942; Strickland, 1954). Incorporation 

 into the ribose nucleic acids (Findlay et al., 1953) and the phos- 

 phoprotein fraction (Findlay et al., 1954) is also reduced. Under 

 these conditions, glycolysis, although greatly increased was 

 nevertheless insufficient to support the normal phosphorylation, 

 processes. Analogous results were obtained with cyanides which 

 at 10~2 M abolished incorporation of phosphate into the phospho- 

 lipids, nucleic acids and phosphoproteins. At this concentration 

 oxygen uptake is known to be almost completely suppressed whilst 

 lactic acid production is markedly increased (Dixon and Elliot, 

 1929; Dixon, 1940, Stern, et al., 1952). Since the primary action 

 of cyanide is the inhibition of the cytochrome oxidase system this 

 situation is comparable with a lack of oxygen. Agents such as 

 fluorides and iodoacetates at 10~2_10~^ M yielded similar results. 

 Malonate at lO"^ M has no eflFect tipon the oxygen uptake or lactic 

 acid production of slices metabolizing glucose (Heald, 1953) and 

 similarly did not alter the incorporation of radioactive phosphate 

 into the phospholipids (Strickland, 1954). However, Tsukada et 

 al. (1958), also working with cerebral slices, have reported that 

 incorporation of radioactive phosphate into the acid-soluble 

 phosphates and residual organic phosphate fraction was decreased 

 by 80-90% in the presence of 10~2 M malonate. It is difficuh to 



