Martin Pring

A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4′-diisothiocyano stilbene-2,2′-disulfonic acid (DIDS) and its dihydro derivative (H2DIDS)

SummaryDIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid) and H2DIDS (4,4′-diisothiocyano-1,2-diphenyl ethane-2,2′-disulfonic acid) binding to the human red cell membrane proteins were studied as a function of concentration, temperature and time. Most binding sites were common to both. The common sites were in band 3 of SDS polyacrylamide gel electropherograms (Steck, 1974.J. Cell Biol.62∶1), an unidentified adjacent band, and glycophorin. Reversible and irreversible binding occurred; both inhibited sulfate equilibrium exchange. The time courses of irreversible binding to band 3 and total binding to the membrane as a whole were biphasic. About 20% of H2DIDS and >60% of DIDS binding were rapid, independent of temperature. Slow H2DIDS binding was monoexponential, activation enthalpy 23 kcal/mole. The stoichiometry of irreversible H2DIDS binding to band 3 was 1.1–1.2, concentration-dependent. Under the conditions studied (0–50 μm, hematocrit 10%, 5–37°C) binding to band 3 was a constant fraction of total binding, 0.7 for H2DIDS and 0.8 for DIDS.Inhibition was a linear function of total binding, binding to band 3, and therefore also to nonband 3 sites, with either inhibitor during both phases. H2DIDS inhibition was complete at 1.9×106 or 1.2×106 molecules/cell total and band 3 binding respectively. For DIDS the corresponding figures were 1.3×106 and 1.1×106.It is shown how reagents of mixed function can react with biphasic kinetics. Binding to multiple contiguous sites may exhibit concentration-dependent stoichiometry. Under such conditions a linear inhibition-binding relationship is neither a necessary nor a sufficient condition for the identification of transport sites.

Cellular uncoupling can unmask dispersion of action potential duration in ventricular myocardium. A computer modeling study.

Although slow conduction is a requirement for the preparation of sustained reentry, it alone is not sufficient for the initiation of reentry. Additionally, unidirectional block and recovery of excitability distal to the site of block must occur. Thus, a comprehensive description of the electrophysiological determinants of reentry must explain both slow conduction and unidirectional block. Although there is a growing body of research exploring the influence of axial resistivity and anisotropy on slow conduction, somewhat less is known about the relation of axial resistivity to spatial dispersion of action potential duration, a condition favorable to the development of unidirectional block. We hypothesized that when cells are well coupled, local differences in intrinsic action potential duration are not evident and that, as axial resistivity increases, local variation in action potential duration becomes manifest. We tested this hypothesis in a numerical model of electrical propagation in a grid of resistively coupled ionic current sources simulating a sheet of ventricular myocardium. Spatial dispersion of intrinsic action potential duration was simulated by varying the magnitude of the fully activated slow inward conductance in Beeler-Reuter membrane ionic kinetics. By then altering coupling resistance, we showed that dispersion of manifest action potential duration is masked in the setting of normal low-resistance cellular coupling and unmasked by increased axial resistance. When nonuniform anisotropy was simulated, dramatic pacing-site-dependent changes in both the pattern of activation and dispersion of action potential duration were noted. These findings may be important in understanding the mechanism of reentrant tachycardia initiation in the border zone of chronic, healed myocardial infarctions where evidence suggests that abnormal cellular coupling is the predominant electrophysiological derangement. In this study, we have shown, using a detailed ionic current-based model of cardiac electrical propagation, that changes in axial resistivity can modulate how spatial dispersion of intrinsic action potential duration is manifest.

Reactions of b-cytochromes with ATP and antimycin A in pigeon heart mitochondria.

In pigeon heart mitochondria antimycin A induces a red shift of less than 1 nm in the λmax of ferrocytochrome bK (Em7.0 + 40 mV; λmax 561 nm) but not of ferro-cytochrome bT (Em7.0 - 30 mV; λmax 565 nm). Antimycin A inhibits the ability of ATP to induce a measurable high-potential cytochrome bT. The stoichiometry of these interactions is one antimycin A per cytochrome bK or cytochrome bT. Antimycin A binding to mitochondrial membranes elicits no significant alteration of the oxidation-reduction midpoint potential of either cytochrome bK or T in the resting state. No spectrophotometric alterations were detected in either ferrocytochrome b on addition of ATP.

Voltage‐dependent calcium currents and cytosolic calcium in equine airway myocytes.

1. The relationship between voltage‐dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura‐2 AM‐loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/‐ 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium‐induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, (ii) increases in [Ca2+]i associated with I(Ca) were consistent with cytoplasmic buffering of calcium ions entering through voltage‐dependent calcium channels (VDCCs) only, (iii) there was a fixed instantaneous relationship between transmembrane calcium flux (J(Ca)) and the change in cytosolic free calcium concentration (delta [Ca2+]i) during I(Ca), (iv) caffeine (8 mM) triggered 8‐fold higher calcium transients than I(Ca), and (v) I(Ca) evoked following release of intracellular calcium by caffeine resulted in an equivalent delta[Ca2+]i‐J(Ca) relationship. 3. The time constant (T) for the decay in [Ca2+]i was 8.6 +/‐ 1.5 s (n = 8) for single steps and 8.6 +/‐ 1.1 s (n = 13) following multiple steps that increased [Ca2+]i to much higher levels. Following application of caffeine (8 mM), however, [Ca2+]i decay was enhanced (T = 2.0 +/‐ 0.2 s, n = 3). The rate of [Ca2+]i decay was not voltage dependent, was not decreased in the absence of extracellular Na+ ions, and no pump current was detected. 4. We conclude that under near physiological conditions, neither CICR nor Na(+)‐Ca2+ exchange play a substantial role in the regulation of I(Ca)‐induced increases in [Ca2+]i, and that, even following release of intracellular calcium by caffeine, Na(+)‐Ca2+ exchange does not play an appreciable role in the removal of calcium ions from the cytosol.

Band-3 protein-mediated anion conductance of the red cell membrane: Slippage vs ionic diffusion

The band 3 protein‐mediated, valinomycin‐induced KCl efflux continues to increase with increasing [KCl] when the Cl−/Cl− equilibrium exchange becomes saturated. This suggests the existence of a band 3‐mediated component of Cl− flux that contributes to the electrical conductance without being related to slippage; i.e., equilibration of the unloaded transport protein between the two membrane surfaces.

Chemical and Enzymatic Modification of Membrane Proteins and Anion Transport in Human Red Blood Cells

The paper is introduced by a review of the developments which lead to the suggestion of an involvement of the protein in band 3 (nomenclature of Steck, ref.2) in anion transport across the red cell membrane. Subsequently, it is shown that DIDS and its dihydroderivative H2DIDS, which both played an essential role in the reviewed work, not only combine with the protein in band 3 but also with other membrane constituents. At maximal inhibition 1.1–1.3 molecules of DIDS or H2DIDS are bound per molecule of protein in band 3. Combined treatment with 3h pDIDS and esternai chymotrypsin, pronase or papain demonstrates the existence of peptides in the protein in band 3 which differ with respect to their accessibility or susceptibility to proteolysis. Each enzyme affects the protein differently and produces different changes of anion transport. In contrast to external trypsin which has neither an effect on the protein in band 3 nor on anion transport, internal trypsin splits the protein in band 3 completely. Fragments of 58,000 and 48,000 Daltons remain attached to the membrane while other products of hydrolysis are released into the medium. Anion transport is partially inhibited but continues to exhibit the essential features seen in the intact cell. The described results are compatible with an involvement of some component of the protein in band 3 in anion transport. They show that additional evidence is required to provide more definitive proof of such involvement.

Pulsed nuclear magnetic resonance study of 39K in frog striated muscle.

Samples of 1 M KCl solution and 10 samples of intact frog striated muscle were studied at 4-7 degrees C and/or at 21-22 degrees C. Field inhomogeneity was minimized by using small sample volumes and by using a superconducting magnet designed specifically to provide highly homogeneous fields. In the present experiments, magnetic field inhomogeneity was measured to contribute less than 15% to the free induction decay observed for intracellular 39K. The signal-to-noise ratio of the measurements was enhanced by means of extensive time-averaging. The rates of nuclear relaxation for 39K in aqueous solution were 22 +/- 3 (mean +/- 95% confidence limits) s-1 at 4-7 degrees C and 15 +/- 2 s-1 at 21-22 degrees C. For intracellular 39K, (1/T2) was measured to be 327 +/- 22 s-1 and 229 +/- 10 s-1 at the lower and higher temperatures, respectively. The corresponding values for (1/T1) in the same muscle samples were 198 +/- 31 s-1 and 79 +/- 15 s-1 at 4-7 degrees C and at 21-22 degrees C, respectively. These results for 39K are similar to those previously obtained for intracellular 23Na. Since less than 1% of the intracellular 23Na has been estimated to be immobilized, fractional immobilization of intracellular 39K is also likely to be insubstantial.

The simulation and analysis by digital computer of biochemical systems in terms of kinetic models: I. The choice of integration method☆

Abstract Methods of simulation of biochemical experiments, by the numerical integration of the differential equations arising from models in terms of reaction networks, are discussed. Hand calculations are used to illustrate the type of integration method required. It is found that the optimum method depends both on the structure of the model and the current values of its parameters. A method which is optimum for the early stages of reaction is described. It is a suitable basis for a method which optimises itself as the integration proceeds, and this development is discussed.

The simulation and analysis by digital computer of biochemical systems in terms of kinetic models IV. Automatic derivation of enzymic rate laws

Abstract The application of the steady-state assumption to the enzyme species in kinetic models of biochemical systems is described. A set of rules for the derivation of the rate laws of complex enzymes under this assumption is available (King & Altman, 1956) . Two programmes, which carry out a symbolic derivation of this kind, and can be used as part of a generator system for simulation by this method, are described in detail. The limitations and advantages of this approach are discussed.

The simulation and analysis by digital computer of biochemical systems in terms of kinetic models: III. Generator programming

Abstract The desirability of the automatic translation of kinetic models, in terms of chemical reaction networks, into programmes for the simulation or fitting of biochemical experiments is pointed out. The general features necessary for a generator programme which writes a number of different integration programmes are shown. The basic operations of a Fortran programme to perform this are listed. Its advantages both for routine operations and development work are shown. This approach to simulation and analysis is compared with that of analogue computation.