1. The magnitude of the K+ gradient across the plasma membrane, which was in equilibrium with the membrane potential (E) of the tumour cells, was determined by the "null point" procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519--552] in which the fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. A mixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondria from interfering with the fluorescence signal. Transport functions at the plasmalemma were maintained under these conditions in the presence of glucose. 3. Physiological circumstances were found in which incubation with glycine or with glucose changed the "null point" value of E within the range--20mV to--100mV. The fluorescence intensity at the "null point" was an approximately linear function of E over that range. The procedure enabled E to be inferred form the fluorescence intensity in circumstances where titration to the "null point" was not feasible. 4. The rapid depolarization caused by l-methionine or glycine was shown in this way to have a maximum amplitude of about 60mV. A mathematical model of this process was devised. 5. The electrogenic Na+ pump hyperpolarized the cells up to about --80mV when the cellular and extracellular concentrations of K+ were roughly equal. 6. The observations show that the factors generating the membrane potential represent a major source of energy available for the transport of amino acids in this system.

1. The tumour cells were incubated at 37 degrees C in Ringer solutions containing glucose, 1 mM-methionine, various concentrations of Na+ and K+ and, in some instances, ouabain or valinomycin to lower the membrane potential generated by the Na+ pump. After about 30 min, when the system had reached a steady state, the ratio [extracellular Na+]/[cellular Na+] varied from about 0.6 to 3.2 with the ionic conditions. The membrane potential, determined by means of the fluorescent probe 3,3'-dipropyloxadicarbocyanine, also varied systemically from about zero to--55mV.2. the ratio [cellular methionine]/[extra-cellular methionine] varied from about 1 to 35 in these circumstances. The Na+ electrochemical gradient, measured in the same units, varied from about 1 to 30. Its magnitude in 46 assays was almost directly proportional to, though slightly smaller than, the methionine gradient. 3. A mathematical model was used to define the relation between these two gradients, which were not in equilibrium, owing to the presence of a leak pathway for the amino acid. On the assumption that the values of [cellular Na+] were correct, the methionine gradient was about 1.8 times larger than this version of the gradient hypothesis predicted.

1. Several methods of analysing progress curves of enzyme-catalysed reactions are discussed briefly in relation to their usefulness in a situation where a reaction product has a K(i) much lower than the K(m) for the substrate 2. A comparison is made of different methods of estimating initial rates in this situation. 3. The use of a computer curve-fitting routine capable of handling functions of more than one variable for the extraction of kinetic parameters from progress curves is described. 4. This method and that of fitting time as a polynomial in product concentration are applied to progress curves for the soluble mitochondrial adenosine triphosphatase and the results are compared with values obtained by more conventional methods.

1. Adenylyl imidodiphosphate is an inhibitor with high affinity for the soluble ATPase (adenosine triphosphatase) from mitochondria. 2. The reaction of the inhibitor with the ATPase is slow and estimates for the association and dissociation reaction rate constants are given. 3. The number of binding sites for the inhibitor appears to be doubled in the presence of 2,4-dinitrophenol. 4. Adenylyl imidodiphosphate is less effective as an inhibitor of the ATPase activity of this enzyme than of the inosine triphosphatase activity. It is also less effective on the ATPase of frozen-thawed or intact mitochondria and did not inhibit ADP-stimulated respiration by intact mitochondria.

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