14N1H and 2H1H cross-relaxation in hydrated proteins.

The frequency dependence of the proton spin-lattice relaxation time T1 of solid hydrated bovine serum albumin and alpha-chymotrypsin has been measured over 4.5 decades in the range 10(4) to 3 X 10(8) Hz mainly by the aid of the field-cycling technique. The comparison between H2O- and D2O-hydrated samples permitted the distinction of exchangeable and unexchangeable protons. In all cases the 14N1H cross-relaxation dips due mainly to the amide groups have been observed. In addition, in the case of the deuterium exchanged proteins a 2H1H quadrupole dip appears. The amide groups act as relaxation sinks due to the coupling of the amide proton to 14N and adjacent protons. Outside of the dip regions the proton-proton coupling dominates. The fluctuations of the 14N1H and 1H1H interactions are of a different type. The unexchangeable protons show a T1 dispersion outside of the quadrupole dip regions given by the exceptional power law T1 approximately v0.75 +/- 0.05. It is shown that apart from structural information of the 14N spectra, 14N1H cross-relaxation spectroscopy permits the determination of correlation times in the range 10(-7) s less than tau less than 10(-4)S.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine

Summary Methods are described which demonstrate the use of unidirectional influx of¹⁴C-tetraphenylphosphonium (¹⁴C-TPP⁺) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential () and Na⁺-dependent sugar influx was determined for these cells at various Na⁺ and -methylglucoside (-MG) concentrations. The results show a high degree of dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of . No transinhibition by intracellular sugar (40mm) can be detected. Sugar influx in the absence of Na⁺ is insensitive to 1.3mm phlorizin and independent of . The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na⁺ binding is the potential-dependent step (Na⁺ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.     

The effects of ionophores on the fluorescence of the cation 3,3''-dipropyloxadicarbocyanine in the presence of pigeon erythrocytes, erythrocyte ''ghosts'' or liposomes.

1. Pigeon erythrocytes, resealed lysed erythrocytes or liposomes derived from erythrocyte lipids were suspended in solutions containing up to 2 micrometer-3,3'-dipropyloxadicarbocyanine iodide. Gramicidin, valinomycin, nigericin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or combinations of these, were used to induce electrical diffusion potentials dependent on Na+, K+ or protons. In each instance hyperpolarization of the cell membrane lowered the fluorescence of the cell suspension, a process that was completed in about 1 min. Subsequent depolarization caused an increase in fluorescence. 2. Quenching of the fluorescence of the cell suspension appeared to be due to the reversible binding of the dye to the cells. Much larger amounts of dye were bound, both to the intact and to the resealed erythrocytes, than would be expected if partitioning of the dye cation followed the Nernst equation. The dependence of the binding on the extracellular dye concentration was studied in the presence and absence of valinomycin. The results were consistent with the suggestion of Sims, Waggoner, Wang & Hoffman [(1974) Biochemistry 13, 3315-3330] that the dye was bound at both membrane surfaces and that, at low dye concentrations, hyperpolarizing the cells promoted dye binding at the inner membrane surface. 3. The applications of the technique are limited by the circumstance that the direct effect of the electric field on the uptake of the dye into the cells is amplified by a binding process that may be affected by other physiological variables.     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

Inhibition of fructose-1,6-bisphosphatase by a new class of allosteric effectors

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.