84 GENERAL METABOLISM in vitro 



respiration in their preparations. Treatment of mitochondria so as 

 to alter the permeabiHty of the membrane permits oxidation of the 

 intermediates of the tricarboxyhc acid cycle (Shepherd, 1953; 

 Gallagher et ai, 1956) and it is possible that the conditions used by 

 Brody and Bain permitted the entry of citric acid. Similar con- 

 siderations may be used in assessing the phosphorylation which is 

 said to accompany the metabolism of acetate since attempts to 

 demonstrate the oxidation of acetate by cerebral slices or in 

 dispersions were unsuccessful (Webb and Elliot, 1951 ; Elliot et al., 

 1942). The high phosphorous/oxygen ratio of 3-6 found for the 

 oxidation of a-ketoglutarate is unusual. The errors inherent in 

 measurements of oxidative phosphorylation over short periods of 

 time have been commented upon by Slater and Holton (1954) who 

 pointed out that the time taken for the contents of the reaction 

 vessels to reach temperature equilibrium with the water in the 

 thermostat bath is longer than is generally realized and can lead to 

 artificially high phosphorous/oxygen ratios. 



Although in preparations such as described above the synthesis 

 of labile phosphates is measured by trapping the phosphate or 

 glucose-6-phosphate the presence of added trapping agents is not 

 essential for phosphorylation to proceed. Thus, Lee and Eiler 

 (1953) using brain dispersions free from glucose and added 

 creatine, were able to demonstrate a rapid incorporation of added 

 radioactive phosphate into adenosine triphosphate during the 

 oxidation of succinate. As with mitochondrial preparations phos- 

 phorylation was completely inhibited by anaerobiosis. 



The establishment, in disintegrated preparations, of adenosine 

 triphosphate as the major energy-rich phosphate first formed during 

 oxidative phosphorylation is paralleled by its formation in intact 

 cerebral slices. Thus in slices of guinea pig cerebral cortex meta- 

 bolizing glucose, radioactive phosphate from the medium was 

 incorporated most rapidly into the y-phosphorus of adenosine 

 triphosphate followed closely by incorporation into phospho- 

 creatine (see p. 117). Incorporation into the hexose monophos- 

 phates was less rapid, followed in order of decreasing specific 

 radioactivities by adenylic acid and diphosphopyridine nucleotide 

 (Heald 1956^). With the exception of inorganic phosphate, these 

 phosphates accounted for 70-80% of the radioactivity present in 

 the tissue extracts. On the basis of product precursor relationships 

 described by Zilversmit et al. (1943) it seems probable that the 



