A note on the distribution of phosphorus in photophosphorylating spinach chloroplast preparations.

The distribution of phosphorus compounds in various leaf components has been examined in some detail, particularly for tobacco, where interest has centered in part on the study of nucleic acids, especially in relation to virus infection (2-7). The method adopted for determining phosphorus distribution in the present study was based on the procedure of Ogur and Rosen (8), and on the results of others applying similar methods to the study of chloroplasts (2-5). In addition to inorganic phosphate (Pi) and total phosphate, four fractions were differentiated, i.e. acid-soluble P, lipide P, nucleic acid P, and residual P (presumably protein-bound). No attempt was made to differentiate RNA-P from DNA-P, except for a few enzymatic degradation studies described in the following section. Procedure for Determining Phosphorus Distribution-A sample containing 1.5 to 2.0 mg. of chlorophyll was diluted to 3 ml., and 2 ml. of ice-cold 0.5 N perchloric acid were added. After 10 minutes at 0’ the precipitate was centrifuged down and the supernatant poured off. The extraction with cold perchloric acid was repeated, and the combined extracts were analyzed for inorganic phosphate and total phosphate by the method of Fiske and SubbaRow (9). The precipitate from the cold perchloric acid extraction was suspended in a 1:4 water-ethanol mixture. The supernatant obtained after centrifugation was poured off and retained. The precipitate was extracted twice with 7-ml. portions of a 3 : 1 mixture of ethanol-ethyl ether by gentle boiling in a water bath for 3 minutes. The total phosphorus content of the combined supernatants, determined after solvent evaporation, was called phospholipide phosphorus. The residue from the previous treatment was extracted twice with 5-ml. portions of 0.5 N perchloric acid for 20 minutes at 70”, and the total phosphorus content of the extracts was determined. This was called nucleic acid phosphorus. The phosphorus content of the residue from the hot perchloric acid extraction was designated “protein” phosphorus. This accounted for only a