The objective is to understand the physical-chemical significance of the simple observation that all biological """"""""active"""""""" transport systems are ion transport systems, and its implication that ion activation is a central biochemical mechanism. The most direct line of experiments toward this objective would be mass incorporation of a purified ion pump into or onto a bilayer lipid membrane, so that the kinetics of initial charge transfer can be observed and related to known chemical events. The transport protein chosen is the plasma membrane H+-ATPase of fungal membranes, which may have the simplest reaction chemistry of all ATP-driven ion pumps: It transfers one proton across the membrane for each ATP molecule split, without identifiable coupling to any other ion; and it does so as an Mr = 100,000 protein of the E1-E2 class. Bilayer techniques for active transport systems will be used. Complementary experiments, designed for both support and backup, will be conducted to measure pump electrical transients in lipid vesicles. Both the bilayer and the vesicle experiments will use photoreleasable """"""""caged"""""""" ATP. Two related, but less direct, lines of experiments will be pursued which have grown out of extensive previous work in this laboratory. One of these is to generalize the techniques for direct electrical-kinetic study of active transport systems in situ, as developed for the large-celled species Neurospora, to other less convenient microorganisms. The model cell chosen is the yeast Saccharomyces, whose protoplasts will be fused into 10-12 um diameter cells and then studied by whole-cell patch recording. The plasma membrane proton pump and/or several cotransport systems will be examiend. The final line of experiments proposed is to carry out a detailed steady-state kinetic study of the proton-coupled glucose transport system in Neurospora using current-voltage techniques supplemented with isotope flux measurements. The data will be analyzed by means of recent general kinetic theoretical models (however, avoiding the assumption of equilibrium binding) in order to specify the order of binding and release of substrates and driver ions, as well as the apparent binding constants.
