CLUES FROM ASSOCIATED EVENTS 



tern catalysing electron translocation; an adenosine triphospha- 

 tase system catalysing a reversible translocation of OH - ions 

 from inorganic phosphate; and a charge-impermeable mem- 

 brane in which the oxido-reduction and adenosine triphosphatase 

 systems are orientated in opposition, so that the movement of 

 electrons across the membrane during oxido-reduction must push 

 a corresponding number of OH~ ions from inorganic phosphate, 

 thus generating phosphorylium for donation to adenosine diphos- 

 phate. If this hypothesis were correct, one might reasonably sup- 

 pose that uncouplers of oxidative phosphorylation could act by 

 making the membrane permeable to charge. 



It has been discovered, by means of a simple titration tech- 

 nique, that the normally very low rate at which protons diffuse 

 through the membranes of washed suspensions of Micrococcus 

 lysodeikticus (Gilby and Few, 1958) and of rat-liver mitochon- 

 dria isolated as described by Myers and Slater (1957) is in- 

 creased manyfold by the addition of dicoumarol, 2,4-dinitro- 

 phenoxide, azide, and other uncouplers of oxidative phosphoryl- 

 ation. At low concentration of uncouplers of the dinitrophenol 

 type, the rate of conduction of protons across the membrane 

 is proportional to the amount of uncoupler added. The concen- 

 trations of the agents which give nearly complete uncoupling 

 (e.g., for the mitochondria: 20 MM-discoumarol; 100 /*M-dinitro- 

 phenoxide; 2 mAf-azide; for the bacteria: 10 times the concen- 

 trations of the same agents) have been found to catalyse the 

 passage of protons through the membranes of resting mitochon- 

 dria or bacteria at rates equivalent to the rates of passage of 

 the electrons through the respective oxido-reduction systems 

 during uncoupled glutamate oxidation. 



These observations are entirely consistent with the chemios- 

 motic coupling hvpothesis which they were designed to test. 

 They also imply that the dinitrophenol type of uncoupling agent 

 owes its activity to two factors (a) solubility in a nonpolar re- 

 gion in the membrane of the phosphorylation system (Shaw, 

 Lannon and Tapley, 1959), and (b) the capacity for conducting 

 protons across this region. The latter capacity can possibly be 

 ascribed most simply to the presence of two or more alternative 

 weakly acidic groups between which the proton-bonding elec- 

 tron can pass by way of the 7r-orbitals present in all uncouplers 

 of this type. 



He also wrote (1961b): 



In the exact sciences, cause and effect are no more than events 

 linked in sequence. Biochemists now generally accept the idea 

 that metabolism is the cause of membrane transport. The under- 

 lying thesis of the hypothesis put forward here is that if the 

 processes that we call metabolism and transport represent events 



85 



