FACTORS AFFECTING METABOLISM in vitro 123 



phorylation of adenosine diphosphate by inosine triphosphate 

 (Krebs and Hems, 1955) the reaction is extremely slow (about 

 0-5/xmoles/g hr~^) and does not contribute to the formation of 

 adenosine triphosphate in aqueous dispersions of rat brain; the 

 reaction between cytidine triphosphate and adenosine diphosphate 

 also appears to proceed only slowly (McMurray et al., 1957). 



Evidence to support a sequence such as that of Fig. 14, as a 

 major pathway of energy metabolism of the tissue during excitation 

 at present rests upon the finding that these components together 

 with inorganic phosphate were the only phosphates changing 

 rapidly upon brief application of electrical pulses to the intact 

 tissue. For such a sequence to be prominent the rates of reaction 

 must be high. So far, accurate data in intact tissue are known only 

 for phosphocreatine and inorganic phosphate. In dispersions, 

 enzymes operating at similar rates are phosphocreatine phospho- 

 kinase (3600jLtmoles/g hr"^, Narayanaswami, 1952) and adenosine 

 triphosphatase (1600/Ltmoles/g hr"^. Gore, 1951). However, the 

 latter enzyme does not appear to be involved, for in systems where 

 it is inhibited with sodium fluoride the labelling of phospho- 

 protein is increased (Heald and Stancer, 1960). The rate of 

 metabolism of phosphoprotein has been computed at a rough 

 minimum value of 400 /xmoles/g hr~^ (Heald, 1958). It is possibly 

 significant that brain contains a phosphoprotein phosphatase 

 which operates at rates up to 60/xmoles/g hr"^ with casein as a 

 substrate (Feinstein and Volk, 1949). The nature of the phospho- 

 protein is largely unknown, but it is envisaged as being an inter- 

 mediate capable of accepting and donating energy-rich phosphate, 

 analogous to the reaction described for phosvitin (Rabinowitz and 

 Lipmann, 1958) rather than an enzyme the activity of which is 

 increased by the general increase in metabolism of the tissue. For 

 this role the energy of the phosphate group(s) must be readily 

 transferable and the group(s) are presumably labile. In this 

 connection the finding of the phosphorus attached to o-phos- 

 phorylserine is an anomaly for the oxygen-phosphorus link in this 

 amino acid is remarkably stable (Plimmer, 1941). The normal 

 procedures for isolation of phosphorylserine involve an acid 

 hydrolysis which is well known to catalyse rearrangements such as 

 the transfer of a group linked to a nitrogen atom to one linked to 

 an oxygen atom. Suggestions as to how such rearrangements may 

 occur to yield o-phosphorylserine from phosphoprotein enzymes 



