The effect of physiologically occurring cations upon aequorin light emission. Determination of the binding constants.

1. The effect of K+, Na+, Mg2+ and pH upon the rate of aequorin utilization has been investigated in the presence of Ca2+. 2. The aequorin light emission in a medium simulating the in vivo cationic conditions for barnacle muscle fibres indicates that two Ca2+ are apparently involved in this process for free calcium concentrations higher than approx. 10(-5) M. However, for free calcium concentrations lower than 10(-6) M, the intensity of light emitted by aequorin shows a steeper dependency upon [Ca2+] than the square low relationship, indicating that a third Ca2+ should be involved in the process of aequorin light emission, as it has been previously predicted (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys. Acta. 396, 133-140). 3. The inhibitory effect of physiologically occurring cations upon the aequorin light emission can be explained by the cooperative action of two cations, competing with Ca2+ for the reactive sites on aequorin. 4. At a given concentration, Na2+ was found to have a stronger inhibitory effect upon the aequoring light emission than K+. 5. The experiments indicate a strong interaction between Na+ and K+ in this inhibitory process, since for a given total concentration of monovalent cations, a mixture containing both Na+ and K+ has a larger inhibitory effect on the aequorin light response than solutions containing either Na+ or K+ alone. 6. All other interactions between K+, Na+, H+ and Mg2+ appear to be weak. 7. The reaction schemes used for the explanation of these and other published results on aequorin (Moisescu, D.G., Ashley, C.C. and Campbell, A.K. (1975) Biochim. Biophys, Acta 396, 133-140 and Blinks, J.R. (1973) Eur. J. Cardiol. 1, 135-142) are described, and the 'absolute' binding constants of all physiologically occurring cations for aequorin have been determined. 8. Based on these parameters one can make accurate quantitative predictions for the aequoring light response under a variety of ionic conditions, and this suggests that it is possible to determine absolute free calcium concentrations providing that the ionic composition of the solutions is known, and that the relative rate of aequorin utilization is higher than 0.005.     

Proton NMR of aequorin. Structural changes concomitant with calcium-independent light emission

Aequorin, a Ca(II)-sensitive bioluminescent protein from jellyfish, emits light at 469 nm from an excited state of a substituted pyrazine (oxyluciferin) which results from the oxidation of a chromophore molecule that is noncovalently bound to the protein. The chromophore is oxidized when Ca(II) or other activating metal ions are bound by aequorin. In the absence of Ca(II), spontaneous emission of light, referred to as Ca(II)-independent light emission, occurs at a rate less than 10(-6) of that for Ca(II)-induced emission. Proton nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence were used to study structural changes of aequorin accompanying Ca(II)-independent light emission. Time course studies by 1H NMR and CD demonstrate that as a result of Ca(II)-independent light emission, aequorin progressively changes from a rigid, fully active form showing little segmental mobility to a practically unfolded, discharged (i.e., inactive) form in which a number of amino acid residues are significantly mobile. This slow discharged protein (SDP) is distinct in nature and conformation from aequorin which has been discharged by Ca(II), i.e., the blue fluorescent protein. The rate of Ca(II)-independent discharge of aequorin is substantially reduced in the presence of excess Mg(II); the time constant for inactivation at 5 degrees C is 30 days with no Mg(II) present and 70 days with Mg(II) present. The NMR spectra are nearly identical at a given stage of inactivation whether or not Mg(II) is present. Oxyluciferin remains bound to SDP. If it is removed, however, by column chromatography, the resulting apo-SDP partially refolds, and the segmental mobility acquired in the formation of SDP is significantly attenuated particularly for some of the aromatic amino acid residues.     

Photochemically-induced dynamic nuclear polarization proton-NMR of aequorin discharged by calcium-independent light emission

Photochemically-induced dynamic nuclear polarization was used to identify exposed amino-acid residues and to assign resonances in the 1H-NMR spectrum of Ca(II)-independent discharged (inactivated) aequorin. A previous nuclear magnetic resonance, circular dichroism and fluorescence study [Ray, B.D., Ho, S., Kemple, M. D., Prendergast, F. G. and Nageswara Rao, B.D. (1985) Biochemistry 24, 4280-4287] indicated that as the Ca(II)-activated bioluminescent protein aequorin from jellyfish spontaneously emits light in the absence of Ca(II), it changes from a rigid, fully active form to a discharged form in which a number of amino-acid residues are more mobile than in the native protein. Laser-photochemically-induced dynamic nuclear polarization experiments identified tryptophan and tyrosine residues, but not histidine residues, in Ca(II)-independent discharged aequorin to be accessible to the flavin dye used. These exposed residues are also among the mobile residues of the Ca(II)-independent discharged protein. Resonances of all the protons (including the alpha protons) of the accessible tryptophan and tyrosine residues were assigned with the aid of two-dimensional photochemically-induced dynamic nuclear polarization J-correlated spectroscopy. The oxidized chromophore, from which light is emitted in aequorin, was not accessible to the dye in the Ca(II)-independent discharged protein. No exposed residue was detected in the photochemically-induced dynamic nuclear polarization spectrum of Ca(II)-independent discharged aequorin from which the oxidized chromophore was removed, corroborating the previous finding that in this apo-discharged form the protein partially refolds and thereby loses some of the mobility acquired in the formation of the Ca(II)-independent discharged protein.     

Luminescence of peptide-bound terbium ions. Determination of binding constants

Luminescence of Tb3+ ions bound to a calmodulin fragment has been studied. It is shown that during their lifetime excited ions dissociate from the peptide. If concentration of free peptide is high enough they can be coordinated again. As a consequence, observed terbium luminescence lifetime and intensity depends not only on binding equilibrium, but also on concentration of free peptide molecules. In such a system terbium binding constant cannot be correctly determined by simple steady-state measurements of luminescence intensities. Instead, terbium luminescence decay curves measured at various peptide concentrations must be analysed. Such an analysis has been made for a fragment of the IIIrd calcium binding domain of rat testis calmodulin. Rate constant of terbium association and the equilibrium binding constant corresponding to the best fit of theoretical functions to experimental points have been determined.     

Determination of protein-ligand binding constants at equilibrium in biological samples.

Protein-ligand complexes can be separated functionally into two classes. "Specific" binding is characterized, in relative terms, by a high affinity for the ligand and a low binding capacity. "Non-specific" binding is characterized by a low affinity and a very large capacity. The calculation of equilibrium binding constants for any specific protein-ligand interaction requires the exact determination of the unbound ligand concentration and the specifically bound ligand concentration. These determinations usually require corrections for the contribution of non-specific binding. The use of two correction terms, kn and f, is proposed: kn is the product of the affinity constant k times the number of binding sites n of the non-specific components, while f is the fraction of the non-specific binding included in the experimental estimates of bound ligand. Several theoretical solutions using these terms are proposed for the calculation of specific binding constants. The practical choice of the correction factor may be different when the simultaneous measurement of the affinity constant and maximum number of binding sites, or when only the latter, is desired. In the case of complex binding systesm containing more than one specific component, the individual constants can be determined by non-graphical methods, using computer-aided iterative statistical calculations. A complete solution is given for a system containing two specific plus non-specific interactions and actual experiments are reported for steroid hormone-receptro complexes.     

The effect of alcohols on aequorin luminescence

The effect of alcohols from ethanol to octanol on aequorin luminescence was biphasic; enhancement of both peak light and total photon yield at low concentrations, and inhibition of these parameters at high concentrations. The potency of alcohols to exert these effects was in the same order as the oil/water partition coefficients of the alcohols. It is argued that neither of these effects is related to calcium binding by aequorin, since alcohols enhanced calcium-independent luminescence, and the inhibition of responses is not associated with a reduction in the rate of aequorin 'consumption' on binding of calcium.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

Determination of netropsin-DNA binding constants from footprinting data

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.     

Determination of the elastic constants of collagen by Brillouin light scattering

The high-frequency elastic properties of rat-tail tendon collagen have been investigated by means of Brillouin (inelastic) light scattering. Longitudinally and transversely polarised elastic waves of frequency about 10¹⁰ Hz have been observed propagating at various angles to the fibre axis of stretched, partially dried tendon. Assuming that the elastic properties of tendon are transversely isotropic, these measurements enable the five elastic constants for such a system to be determined. In particular the ratio of the Young's modulus for strain parallel to the axis to that for strain perpendicular to the axis ($\text{E}{}_{\mathrm{\parallel }}\text{E}{}_{\mathrm{\perp }}$) is found to be 1.43 and the ratio of the shear modulus to E_∥ is 0.28. In wet collagen only the longitudinal branch has been observed and in this case the ratio $\text{E}{}_{\mathrm{\parallel }}\text{E}{}_{\mathrm{\perp }}$ increases to 1.82. The absolute value for E_∥ in dry collagen is 11.9 GN m^?2 reducing to 5.1 GN m^?2 in wet collagen. An interpretation of these results in terms of the expected vibrations of the collagen molecular assembly is given. Possible applications to the determination of the mechanical properties of collagen composite materials such as bone are discussed as well as some measurements on silk and α- and β-keratins, which are fibrous proteins of different molecular conformation to collagen.     

The effect of a series of organic cations upon the plasmalemmal serotonin transporter, SERT

The aim of this work was to test the effect of a series of organic cations upon the activity of the plasma membrane serotonin transporter (SERT). The experiments were performed using the JAR cell line that constitutively expresses high levels of SERT, and rat intestine, whose mucosal epithelial cells also express SERT. Initial rates of (3)H-serotonin ((3)H-5HT; 200 nM) uptake were not changed by some of the organic cations tested (guanidine, N-methylnicotinamide, choline, atenolol, caffeine and theophylline), but were slightly (15-30%) inhibited by some other organic cations, at the highest concentrations tested (thiamine (3 mM), cimetidine (1 mM) and tetraethylammonium (3 mM)). On the other hand, some other organic cations reduced, in a concentration-dependent manner, uptake of (3)H-5HT by JAR cells (IC(50)s of 0.3, 1.3, 5.4, 89.3, 460 and 748 microM for quinidine, verapamil, propranolol, amiloride, nicotine and clonidine, respectively). Quinidine, clonidine and amiloride seem to be competitive inhibitors of (3)H-5HT uptake, whereas verapamil, nicotine and propranolol appear to be uncompetitive or non-competitive inhibitors. Moreover, quinidine, verapamil and propranolol trans-inhibited (3)H-5HT uptake, whereas clonidine, nicotine and amiloride were devoid of effect. Finally, these six organic cations were able to significantly increase the serosal-to-mucosal apparent permeability (P(app)) to (3)H-5HT of rat jejunum, ileum and colon. In conclusion, human and rat SERT-mediated transport is inhibited by several distinct organic cations, some of which are therapeutic agents or drugs of abuse. Knowledge on which organic cations interfere with SERT-mediated transport of 5HT will have major implications in tissues where 5HT plays important physiological roles (eg. central nervous system, intestine and placenta).     

The interaction of cations with lipopolysaccharide from Escherichia coli C as shown by measurement of binding constants and aggregation reactions.

Lipopolysaccharide from Escherichia coli C interacts with polyvalent cations at low ionic strength at more than one site. The first site has high affinity with a KD value of 10(-8) M for Ca2+ and even stronger binding for [(NH3)5CoNH2Co(NH3)5]5+ and La3+. The high-affinity site for the latter cations is beyond the sensitivity of the assay method. The second, low-affinity, site for bivalent cations has a Km of 10(-3) M, whereas, for tervalent and quinquevalent metal cations and spermine and hexacyclen (1,4,7,10,13,16-hexa-azacyclo-octadecane), this constant has a value of 10(-5) M. Binding of cations to the high-affinity site does not alter the aggregation state of the lipopolysaccharide, but combination with the low-affinity site gives particles twice the size of those of the sodium salt. Very high Ca2+ concentrations (30 mM) give particles eight times the size of those of the sodium salt.     

Determination of binding constants for N-acetyl-D-glucosamine oligomers with lysozyme

A method to determine intrinsic binding constants of lysozyme with substrate analogues such as N-acetyl-D-glucosamine dimer and trimer is proposed. The method is based on the competitive interaction of an anionic azo dye with substrate analogues for lysozyme. There are two binding sites for substrate analogues and dyes, respectively, on lysozyme. One binding mode of the substrate analogues to subsites D-F on lysozyme was non-competitive, and another binding mode to subsites A-C was competitive with the dye. From the binding constants obtained it is suggested that the binding of the substrate analogues to subsite D on lysozyme is weaker than the binding to the other subsites.     

The effect of monovalent cations upon 45Ca fluxes in frog sciatic nerve

Investigation of Ca fluxes in desheathed bundles of myelinated nerve of frog indicates an intracellular Ca concentration of 5 x 10(-4) mol . kg-1 (axoplasm) and an average transmembrane flux of 6 x 10(-8) mol. kg-1 . s-1 at an extracellular Ca concentration of 1 mM. Replacement of extracellular Na by isosmotic sucrose increases Ca influx threefold and decreases efflux by 50%. Similar, but significantly smaller, effects are observed when Tris or choline are substituted for Na. Li replaces Na without significant changes in Ca fluxes. The data demonstrate that Ca transmembrane fluxes in this preparation are sensitive to changes in the Na gradient. The observed flux changes, however, are too small to establish a Na-Ca exchange as the sole homeostatic mechanism for intracellular Ca. Moreover, as Li appears to serve as a good Na substitute and even Tris and choline interact with Ca flux, the exchange does not show the specificity described for squid axon.     

Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition

The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.