174 Appendix: analytical methods 



the same reaction. Methods somewhat similar to those of Tower 

 and Thorn et al. have been described by Morton (1958) but have 

 not yet been appHed to tissue extracts. The di- and triphospho- 

 pyridine nucleotides are estimated by coupling with cytochrome-c 

 reductase and measuring the rate of reduction. The oxidized forms 

 are reduced by alcohol dehydrogenase and glucose- 6- phosphate 

 dehydrogenase. In this latter determination the nucleotides were 

 extracted with hot OT iVHCl, or 04 A^NaOH (Glock and McLean, 

 1955). In the other methods extracts were made either in perchloric 

 acid, most of which was removed as the potassium salt before 

 analysis, or in trichloracetic acid which was later removed with 

 ether. 



The use of enzymes has provided a specific method for the esti- 

 mation of radioactivity in the y-phosphorus of adenosine and 

 guanosine nucleotides. This is particularly valuable when investi- 

 gating rates of incorporation of radioactive phosphate. The 

 nucleotides are first separated by paper chromatography or 

 ionophoresis, eluted from the paper and incubated with a potato 

 apyrase preparation made according to the method of Lee and 

 Eiler (1951) under the conditions described by those authors. At 

 low temperatures, 2-7°, only the y-phosphorus of adenosine or 

 guanosine triphosphate is removed. This can be estimated as 

 inorganic phosphate by the modified Berenblum and Chain 

 technique, and counted for radioactivity in the same solution 

 (Heald, 1956^). 



Analysis of Phosphates Insoluble in Acid Extractants 



The writer's experience here has been with the separation of 

 phosphates in the non-phospholipid fraction. Methods of analysis 

 of phospholipids have been described by Sperry (1955), Lovern 

 (1955) and Lees (1957). Details of the separation of phospholipids 

 into groups by chromatography upon silicic acid, and methods of 

 analysis of some of the fractions obtained have been described by 

 Hanahan et al. (1957), Dittmer et al (1955), Lea and Rhodes 

 (1953, 1954), and Rhodes and Lea (1957), and the limitations of 

 such methods have been noted (Barron and Hanahan, 1958). 

 Analysis by preliminary separation into groups, alkaline hydrolysis 

 and subsequent separation of the glyceride fractions by paper 

 chromatography (Dawson, 1954) has been extensively used to 

 study the metabolism of radioactive phosphate in cerebral lipids. 



