Active transport of inorganic phosphate and magnesium ions by beef heart mitochondria.

In 19491 our laboratory described the binding of inorganic phosphate by a mitochondrial preparation of rabbit kidney. This binding was shown to be linked to electron transfer since reagents that were found to inhibit this process exerted a comparable effect on the binding Although the phosphate taken up by the mitochondrion estimated as inorganic phosphate, nonetheless, some of the properties of the accumulated phosphate argued in favor of a form of phosphate other than inorganic phosphate (hence the designation of "gel phosphate"). This binding phenomenon was subsequently studied by Crane and Lipmann.2 The possible relation of the phosphate binding system to the phenomenon of oxidative phosphorylation was the central theme of Crane and Lipmann's investigation and later of the investigations of Pressman,3 but no simple relation could be found. Among others, Bartley and Davies4 and Amoore5 considered phosphate binding by mitochondria from the standpoint of both active and passive transport. In the present communication, we shall present evidence that the binding of inorganic phosphate by intact beef heart mitochondria is a process coupled to electron transfer and that this translocation of phosphate (our description of the binding phenomenon) is a process that shares one or more intermediate steps with that of oxidative phosphorylation but differs in respect to the final step(s). Electron transfer, indeed, energizes both coupled phosphorylation and coupled translocation but these are alternative (and possibly competitive) expressions of the same underlying coupling mechanism. Under the conditions and in presence of the additions specified in the legend for Table 1, massive accumulation of inorganic phosphate by the mitochondrion is readily demonstrable. As much as 1 ,umole of phosphate can be bound per mg of mitochondrial protein under optimal conditions. From the estimate6 of the number of mitochondria per mg of protein (9 X 109) and from the computed volume of the mitochondrion of beef heart muscle (0.2 ,3) and the fluid volume of the mitochondrion (0.1 3), it can be calculated that the final concentration of phosphate in the mitochondrion would be about IM when 1 4mole is taken up per mg of protein. Our own determinations of the total water content of the packed pellets which underlie the calculations are in close agreement with those of Green and Oda.6 The binding of phosphate is completely inhibited by the classical inhibitors of electron transfer tested (antimycin A, cyanide, and azide), by some reagents that uncouple phosphorylation (2,4-dinitrophenol and dicoumarol), but not by oligomycin-a highly effective inhibitor of coupled phosphorylation. The concentration levels at which these respective inhibitors achieved complete inhibition of binding in no case exceeded the levels normally used for complete inhibition of electron transfer. The binding requires: (1) a suitable substrate (there is little if any specificity in this regard); (2) Mg++; and (3) an electron acceptor (02). These are absolute requirements as shown by the data of Table 1. In the presence of ADP, phosphate