689 



Shigetoh Miyachi 



Acid soluble nucleotidic P compounds were adsorbed by charcoal 

 from the supernatant obtained by Procedure 1. The charcoal was separ- 

 ated by centrifugation and treated with H2SO4 (final cone. , 1 N) for 20 

 minutes at 100°. The amount of orthophosphate produced ( A20 P) was 

 assumed to be nucleotidic labile P. 



To the supernatant obtained by Procedure 1 was added a small quanti- 

 ty of carrier polyphosphate, and after adjusting the pH to 4. 0, poly-P"A" 

 was precipitated by the addition of Ba"*"^. The amount of poly-P"A" was 

 determined by assaying for orthophosphate liberated from the Ba -pre- 

 cipitate after hydrolysis with 1 NH2SO4 for 15 minutes at 100° (A15 P). 

 Preliminary experiments(^) showed that the acid soluble polyphosphate in 

 Chlorella cells was completely precipitated by this procedure. It has 

 been also shown that pyrophosphate is not precipitated by this procedure. 

 Poly -P"B" and poly -P"C" were determined by assaying A20 P in the super- 

 natant obtained by Procedure 111 and in the precipitate obtained by Pro- 

 cedure V, respectively. Poly -P"D" was separated from RNA -nucleo- 

 tides in the supernatant obtained by Procedure VI according to the char- 

 coal treatment: Charcoal was added to the supernatant, shaken vigor- 

 ously, and the RNA -nucleotides adsorbed on charcoal were removed by 

 centrifugation. Determination of poly-P"D" was done by assaying A15 P 

 in the nucleotide -free supernatant. Each polyphosphate, except poly- 

 phosphate "C", was purified and identified as long chained polyphosphate 

 (cf. 6,7). 



The charcoal separated as above was also treated with 1 NH2SO4 for 

 20 minutes at 100°. P^^ in the supernatant was a measure of acid-insolu- 

 ble nucleotidic labile P. Residual P^^ on the charcoal gave RNA - 

 nucleotide P^^ (RNA-P-^^). The RNA-P was also estimated from the 

 absorbancy at 260 mfj. in the RNA -fraction. 



Lip id -P was assayed by determining the amount of P in the super- 

 natant obtained in Procedure II. The supernatants obtained in Procedure 

 VII were regarded as the DNA fraction. The residue obtained in Pro- 

 cedure VII was regarded as the protein fraction. 



RESULTS 



Distribution of phosphorus in various compounds 



A typical result of analyses is reproduced in Fig. 1. The highest 

 P -content was shown in RNA-P and lipid -P. It should be mentioned, 

 however, that the procedures for the fractionation of the P-compounds 



