685 



William L. Ogren and David W. Krogmann 



Thus non aqueous chloroplasts, when supplemented with appropriate lipids, 

 will carry out the Hill reaction at rates that are comparable to those 

 observed with chloroplasts prepared in the conventional aqueous media. 



While data are available on the pyridine nucleotide content of whole 

 leaves and of chloroplasts isolated in aqueous media, a more realistic 

 appraisal of water soluble coenzymes in chloroplasts might be obtained by 

 measurements with chloroplasts isolated in non aqueous medium. In 1954, 

 Anderson and Vennesland had reported values of the TPN and DPN content 

 of spinach leaves . Recently Das and Crane published a value for the 

 TPN content of chloroplasts isolated in isotonic sucrose medium . 

 Using the method of Das and Crane which involves chromatographic 

 separation of the coenzymes and direct enzymatic analysis , we have 

 confirmed both these reports and the data are presented in Table II. 



Table II 



Spinach Pyridine Nucleotide Distribution 



mjxmoles per |jimole chlorophyll 

 DPN TPN 



Whole leaves 17.4 5.6 



Chloroplasts prepared in 



aqueous 0. 4 M sucrose 0.033 0.089 



Chloroplasts prepared in 



non aqueous media 8. 87 5. 06 



It is apparent that a very small fraction of pyridine nucleotide in the leaf 

 appears in the chloroplasts. Data are also presented in this table for the 

 TPN and DPN content of chloroplasts prepared in non aqueous media. This 

 preparation contains approximately half of the total leaf pyridine 

 nucleotides. While most of the leaf TPN is found in the chloroplast, it is 

 not the predonninant fornn of pyridine nucleotide coenzyme in this fraction. 

 This is disquieting in view of the assumptions that TPN is the biosynthetic 

 coenzyme and chloroplasts are biosynthetic organelles. 



To exploit the non aqueous chloroplast preparation further, 

 measurements were made of the inorganic phosphate, ADP and ATP levels 

 in the whole leaf and in chloroplasts prepared in non aqueous solvents. The 

 data, obtained by the usual colorimetric measurement of phosphate and 

 enzymatic measurements of ADP and ATP, are given in Table III. 



