Single frog rods consisting of the outer segment and the ellipsoid were investigated by the whole-cell patch-clamp technique. When the recording pipette was filled with a simple intracellular medium containing potassium as the principal cation, a slow increase in dark voltage (hyperpolarization) associated with a decay of the photoresponses was observed. The hyperpolarization started at a dark voltage of −27 ± 8 mV, followed an exponential course, and leveled out at −52 ± 6 mV. The time constant was proportional to the access resistance of the preparations. With a pipette medium containing a 0.5 or 1.0 μM cGMP, the initial dark voltage was shifted to more positive values and the tendency of hyperpolarization was clearly attenuated. Similar results were obtained with 1 mM GTP. The effects of GDP and of ATP were less significant. In experiments with 1 mM GTP plus 1 mM ATP, the dark voltage behaved as in experiments with only GTP. The stabilizing action of GTP was amplified by EGTA so that with 1 mM GTP plus 1 mM free EGTA the dark voltage was stable at a level of −15 mV. It is concluded that the preparations lose intracellular components such as cGMP and GTP by diffusion into the recording pipette and that the losses are prevented or reduced when the pipette medium contains these nucleotides in nearly physiological concentrations. For the internal transmitter cGMP, the results suggest that its free concentration does not exceed 1 μM.

Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tumt) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tumt·GDP complex (KGDP = 1.0 ± 0.1 μM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tumt was used to measure the dissociation constant of the EF-Tumt·GTP complex (KGTP = 18 ± 9 μM). The analysis of these data required information on the dissociation constant of the EF-Tumt·mantGDP complex (KmGDP = 2.0 ± 0.5 μM), which was measured by equilibrium dialysis. Both KGDP and KGTP for EF-Tumt are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tumt·GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tumt. These values are in agreement with a simple equilibrium binding interaction between EF-Tumt and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tumt·GDP complex.