FACTORS AFFECTING METABOLISM in vitro 119 



proteins (Perlman, 1955) including those from tissues (Kennedy 

 and Smith, 1954) and certain enzymes (p. 14), its recognition 

 as a component of brain phosphoprotein appears to estabhsh the 

 latter as a definite entity. 



Attempts to isolate the phosphoprotein itself by methods less 

 degradative than those previously employed led to the discovery 

 that guanosine di- and triphosphates were also involved in the 

 rapid response to electrical impulses (Heald, 19576). Here, tissues 

 were first fixed in the chloroform methanol extractant of Folch et 

 al. (1951) and after removal of the phospholipids the residues were 

 washed with saturated ammonium sulphate to remove inorganic 

 phosphate and other soluble phosphates. From the residue the 

 fraction increasing in specific radioactivity was extractable at 

 pH 11-0 and analysis of the extracts revealed an identity with 

 guanosine nucleotides. As with the phosphoprotein, no change in 

 quantity was found on stimulation, the levels remaining at 

 0-5 /Ltmoles/g wet wt. The residue after alkaline extraction still 

 contained a phosphoprotein (Heald, 1958) though the increase in 

 radioactivity was not as great as had been expected. Reasons for 

 this may relate to an eff"ect of the alkaline conditions upon stability 

 (Heald, 19576). In addition to guanosine phosphates small 

 quantities of adenosine di- and triphosphates were also found. 

 These did not change in specific radioactivity when the tissue slice 

 was electrically stimulated in contrast to the changes noted with 

 adenosine triphosphate in trichloracetic acid extracts. Since 

 washing with ammonium sulphate was continued until no further 

 nucleotide absorbing at 250 m^a could be detected it is possible 

 that these adenine nucleotides represent a group which is meta- 

 bolically distinct. 



Change in specific radioactivity without a change in quantity is 

 suggestive of the participation of these phosphates in a series of 

 linked reactions involving phosphorylation and dephosphorylation. 

 A simple system connecting such changes with those in phospho- 

 creatine and inorganic phosphate is represented diagrammatically 

 in Fig. 14, which is an elaboration of ideas previously presented 

 in somewhat less detail Heald (1956). In a wider context 

 it also represents a more detailed picture of specific points in a 

 general scheme such as that discussed by Brink (1957). Thus, 

 electrical impulses are considered to alter structures associated 



