The chemiosmotic theory proposes that in bacteria an inward movement of H+ is coupled to ATP synthesis by a proton-translocating ATPase (BFoF1), and one central problem is to understand how both electrical and chemical (pH) gradients drive ATP formation. A link between electrical and chemical events will be clarified by two studies. We will define the kinetic correspondence of membrane potential and pH gradient as driving forces for ATP formation by imposing artificial gradients and following ATP formation in both cells and vesicles of Streptococcus lactis; we will also initiate work also initiate work to drive ATP synthesis in vesicles using only an external electric field. In other work, we will examine some recently isolated mutants, presumed to have altered BFoF1-function on the basis of tests with intact cells. We will examine BFoF1 in these lines more directly, assessing the capacity to sustain an electrochemical H+ gradient under physiological conditions, and testing the ability to mediate ATP synthesis coupled to H+ movements in response to both electrical and chemical gradients. A coupling between H+ movements and anion transport will also be studied in S. lactis. In these cases we will give critical appraisal to the possibility of variable H+/lactate stoichoimetry, and direct special attention to characterization of phosphate exchange mediated by a newly discovered hexose phosphate transport system. This latter work will be extended to studies of hexose phsophate transport itself, with the goal of successful reconstitution of activity in the next three years. In parallel work, the focus on H+/anion symport will be maintained during selection of E. coli mutants that fail in the normal coupling between the proton and sugar phsophate movements. The goal of this work is an understanding of both the physiological and biochemical mechanisms that underly generation and utilization of ionic and electrical gradients across biomembranes, to clarify our view of how membrane transport reactions contribute to the normal physiological state.
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