Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.     

Calcium-binding sites in rat myocardial sarcolemma.

Myocardial sarcolemmal preparations have the ability to bind Ca²⁺; this binding shows saturation kinetics, has a pH optimum of 7.4 to 7.8, and is stimulated by ATP. Scatchard analysis reveals both high- and low-affinity binding sites. Both classes are inhibited by ruthenium red, whereas only the high-affinity sites are affected by lanthanum and hydroxylamine. Electrophoresis in the presence of sodium dodecyl sulfate suggests that the high-affinity sites are associated with a protein peak of molecular weight of about 100,000. Enzyme treatment of the membranes suggested that proteins accounted for most of the ⁴⁵Ca binding with phospholipids and sialic acids residues playing a secondary role. Interaction with different function group reagents indicated that the carboxyl residues were necessary for the calcium binding, whereas thiol, amino, and sulfhydryl groups were unimportant.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Analogues of the Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Antagonist Ned-19 Indicate Two Binding Sites on the NAADP Receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca²⁺-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca²⁺ release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca²⁺ release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca²⁺ release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca²⁺ release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.     

Effect of Mg2+ and spermine on the kinetics of Ca2+ transport in rat-liver mitochondria

Plots relating the initial rate of mitochondrial Ca²⁺ transport to the Ca²⁺ concentration (kinetic plots) have a hyperbolic shape in a Ca²⁺ concentration range of 2.5–100 µM as measured in sucrose or KCl media. In the presence of Mg²⁺ or a polyamine spermine, which both are competitive inhibitors of Ca²⁺ binding to low affinity sites at the membrane surface, the shape of the plots becomes sigmoidal. At higher concentrations of these agents linear kinetic plots are obtained as measured in a sucrose medium. In a KCl medium the sigmoidality of the kinetic plots is enhanced by an increase in the Mg²⁺ or spermine concentration. It is suggested that Mg²⁺ and spermine affect the kinetics of Ca²⁺ transport by interfering with Ca²⁺ binding to low affinity sites of the membrane surface and that the binding of Ca²⁺ to these sites is the first step of the mitochondrial Ca²⁺ transport.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Multivalent cations and ligand affinity of the type 1 insulin-like growth factor receptor on P2A2-LISN muscle cells

Mouse P₂A₂-LISN myoblasts are transfected cells that overexpress the human type 1 insulin-like growth factor (IGF) receptor. Because the type 1 IGF receptor is the major binding site for both IGF-I and IGF-II, this cell line is an excellent model to determine the effect of multivalent cations on ligand binding specifically to this type of receptor. Competitive binding assays were performed to characterize IGF binding and Scatchard analysis to quantify affinity (K_a). ¹²⁵I-IGF-I, ¹²⁵I-IGF-II, and ¹²⁵I-R³-IGF-I bind only to the type 1 IGF receptor on these cells. Zn²⁺ increased binding of the three ligands to the type 1 IGF receptor by 17 to 35%. Cd²⁺ significantly increased binding of ¹²⁵I-IGF-I, although by only 8%. La³⁺ and Cr³⁺ did not effect binding. Au³⁺ decreased IGF binding by approximately 56%. Scatchard analysis produced nonlinear concave-down plots yielding binding constants for high and low affinity sites. Zn²⁺ increased the strength of only the high affinity sites. Au³⁺ decreased the affinity of both high and low affinity sites. Zn²⁺ increased binding with a half-maximal effect between 40 μM and 60 μM. Half-maximal dose of Au³⁺ was > 130 μM. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) and only these cations were found to affect receptor binding indicating similar mechanisms of action. Thus, multivalent cations may alter IGF binding to cell surface receptors ultimately controlling growth. Physiologically this may be especially important for the growth promoting effects of Zn²⁺. J. Cell. Physiol. 176:392–401, 1998. © 1998 Wiley-Liss, Inc.     

Analysis of a calcium ion binding system composed of two different sites

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca⁺⁺ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca⁺⁺ binding sites and without such solute were titrated with Ca⁺⁺ using Mx as an indicator of free Ca⁺⁺ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca⁺⁺; the association constants of the high affinity site and low affinity site are 4.3 × 10⁵ M^-1 and 9 × 10³ M^-1, respectively. The advantages and limitations of this methodology are discussed.     

125I-Ifenprodil: Synthesis and Characterization of Binding to a Polyamine-Sensitive Site in Cerebral Cortical Membranes

Abstract: The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by ¹²⁵I-ifenprodil, a noncompetitive NMDA receptor antagonist, are described. ¹²⁵I-ifenprodil was synthesized using Na¹²⁵I in the presence of chloramine-T and purified by paper chromatography. Binding of the ¹²⁵I-ligand was optimal at pH 7.7 in 5 mM Tris · HCl buffer. Equilibrium binding of ¹²⁵I-ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 μM). Zn²⁺, Ca²⁺, and Mg²⁺ inhibited specific binding of ¹²⁵I-ifenprodil in a concentration-dependent manner, with IC₅₀ values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (K_D) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single-binding-site model (Hill coefficient = 0.95), with a density of binding sites (B_max) of 141 pmol/mg of protein. Binding of ¹²⁵I-ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3-diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine-sensitive binding of ¹²⁵I-ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that σ/antitussive agents partially inhibited specific binding, but inclusion of the σ drug GBR 12909 had little effect on the binding of ¹²⁵I-ifenprodil, suggesting this site was not involved. The binding site labeled by ¹²⁵I-ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the σ site.     

Calcium binding to placental plasma membranes as measured by rate of diffusion in a flow dialysis system

The Ca²⁺-binding properties of placental plasma membranes were studied using a flow dialysis system.Ca²⁺-binding was not detectable at pH 4.0, but increased at higher pH values to a maximum binding at pH 11.0.Two types of Ca²⁺-binding sites were identified: high-affinity sites with dissociation constant K_s = 3.1 · 10^{−5 M} and a capacity of 26 nmoles per mg protein; low-affinity sites with K_s = 1.1 · 10^{−3 M} and a capacity of 266 nmoles per mg protein.The affinities of Mg²⁺ and Sr²⁺ for the high-affinity sites were 10-fold lower than that of Ca²⁺, and for the low-affinity sites were 4- and 8-fold lower, respectively.The placental plasma membranes contain sites for Ca²⁺ with capacity, specificity and affinity within the range reported for other membranes involved in an active transport of Ca²⁺ (mitochondria, sarcoplasmic reticulum, cardiac microsomes). The presence of high-affinity Ca²⁺ sites as well as Ca²⁺-ATPase implicates these membranes in Ca²⁺ transport from the maternal to the fetal circulation.     

An increase of calcium/manganese binding sites in cell ghosts associated with the acquisition of aggregative competence in Dictyostelium discoideum.

In Dictyostelium discoideum, the formation of multicellular aggregates represents the first morphogenetic event that leads ultimately to the construction of fruiting bodies. The altered adhesive properties of the cells can be demonstrated in ghosts derived from them which consist of largely intact membranes containing a few empty vesicles and exploded mitochondria but lacking nuclei, RNA, soluble cytoplasm and ATP [4]. A cofactor requirement for the aggregation of the ghosts can be satisfied by the following divalent cations: Ca²⁺, Mn²⁺, Zn²⁺ and Cu²⁺. In this paper it is shown that associated with the acquisition of aggregative competence is a 15–20-fold increase in the ghosts of sites capable of binding either Ca²⁺ or Mn²⁺ with relatively high affinity.     

Ca-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. , 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 (Ca²⁺/monomer). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.     

Phospholamban stoichiometry in canine cardiac muscle sarcoplasmic reticulum

Treatment of cardiac sarcoplasmic reticulum with the crosslinking reagent dithiobis (succinimidyl propionate) in the presence of¹²⁵I-calmodulin, resulted in the formation of a 40,000-dalton affinity labeled component, consisting of a 11, phospholamban:¹²⁵I-calmodulin complex. In parallel experiments, sarcoplasmic reticulum was phosphorylated in the presence of calmodulin and [-³²P]ATP, and then treated with the crosslinking reagent to produce an affinity labeled component consisting of a 11, calmodulin:³²P-phospholamban complex. These experiments permitted determination of the amount of¹²⁵I and³²P incorporated into the 40,000-dalton complexes, as well as the amount of³²P incorporated into the 23,000-dalton form of phospholamban. If 1 mol of Ca²⁺-dependent ATPase phosphoprotein represents 1 mol of 100,000-dalton Ca²⁺-dependent ATPase monomer, then there are 4.88±1.33 mol Ca²⁺-dependent ATPase/mol of phospholamba. If there are 2 mol of Ca²⁺-dependent ATPase phosphoprotein/mol of 100,000-dalton Ca²⁺-dependent ATPase monomer, then there are 9.76±2.66 mol Ca²⁺-dependent ATPase/mol phospholamban.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Transformation of (Ca + Mg)ATPase of calmodulin-depleted erythrocyte membranes into a high Ca-affinity form by Ca

Specific activity and Ca²⁺-affinity of (Ca²⁺+Mg²⁺)ATPase of calmodulin-depleted ghosts progressively increase during preincubation with 0.1–2 mM Ca²⁺. Concomitantly, the increment in ATPase activity caused by calmodulin and the binding of calmodulin to ghosts decrease. The effects of calcium ions are abolished by the addition of calmodulin. ATP protects the enzyme from a Ca²⁺-dependent decrease of the maximum activity but does not seem to influence the Ca²⁺-dependent transformation of the low Ca²⁺-affinity enzyme into a high Ca²⁺-affinity form.     

Transformation of (Ca2+ + Mg2+)ATPase of calmodulin-depleted erythrocyte membranes into a high Ca2+-affinity form by Ca2+

Specific activity and Ca²⁺-affinity of (Ca²⁺+Mg²⁺)ATPase of calmodulin-depleted ghosts progressively increase during preincubation with 0.1–2 mM Ca²⁺. Concomitantly, the increment in ATPase activity caused by calmodulin and the binding of calmodulin to ghosts decrease. The effects of calcium ions are abolished by the addition of calmodulin. ATP protects the enzyme from a Ca²⁺-dependent decrease of the maximum activity but does not seem to influence the Ca²⁺-dependent transformation of the low Ca²⁺-affinity enzyme into a high Ca²⁺-affinity form.     

Ca2+-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle

Previous studies showed that Ca²⁺ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. $\text{256}$, 10203–10207). Initial equilibrium dialysis measurements of Ca²⁺ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca²⁺ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 ($\text{Ca}{}^{\mathrm{2+}}\text{monomer}$). K_a1 and K_a2 values of 4.2 μM and 12.1 μM, respectively, were estimated by extrapolation of apparent constants to infinite dilution of protein. K_a3 (51.3 μM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca²⁺ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.     

Effects of calmodulin on the (Ca2+ + Mg2+)ATPase partially purified from erythrocyte membranes.

The stimulation of the (Ca²⁺ + Mg²⁺)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583). The (Ca²⁺ + Mg²⁺)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound ¹²⁵I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca²⁺ + Mg²⁺)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.     

Identification of the coated vesicle proteins that bind calmodulin

The constituent proteins of coated vesicles responsible for binding calmodulin were identified by photoaffinity labeling with the reagent azido-¹²⁵I-calmodulin. Three protein complexes with apparent molecular weights of 130,000, 93,000 and 52,000 were labeled. Specificity was demonstrated by the dependence of labeling on Ca²⁺, and by its reduction in the presence of unlabeled calmodulin or Stelazine. Urea-soluble components of coated vesicles and material isolated by Sepharose CL4B chromatography formed a 52,000 MW labeled complex. Subtracting an apparent molecular weight of calmodulin of 20,000 from the weights of the covalently labeled complexes, the coated vesicle proteins that bind calmodulin are 110,000, 73,000 and 32,000 MW. The 32,000 MW protein is thought to participate in the coat structure but the other two are most likely associated with the vesicle.     

Apparent “negative cooperativity” kinetics in the absence of a nonlinear Scatchard plot of thyrotropin-receptor interaction in a human thyroid adenoma

A human thyroid adenoma (benign nodule) was identified which exhibited a linear Scatchard plot of ¹²⁵I-TSH binding, characteristic of a single class of binding site with high affinity (K_d = 0.5±0.1 nM) and low binding capacity (0.8±0.2 pmol/mg protein). In contrast, Scatchard analysis of binding to adjacent normal thyroid was nonlinear, suggesting the presence of high and low-affinity binding sites with K_d's of 0.4±0.2 and of 27.9±11.0 nM and capacities of 0.7±0.3 and 1.8±1.0 pmol/mg protein, respectively. Dissociation of bound ¹²⁵I-TSH from membranes of both adenoma and normal tissue revealed identical enhancement of dissociation in the presence of excess native hormone, thought to be evidence for the “negative cooperativity” model of hormone-receptor interaction. Furthermore, adenylate cyclase from both tissues was equally responsive to TSH. Thus, a thyroid adenoma which contains TSH-responsive adenylate cyclase still exhibited enhanced dissociation by native hormone, even though Scatchard analysis yielded a single, non-cooperative class of binding sites. This suggests that enhanced dissociation of bound hormone does not provide a demonstration of negatively-cooperative site-site interaction. Furthermore, nonlinear Scatchard plots, typical of TSH binding in normal thyroid, represent two classes of binding sites, of which the high affinity type is responsible for stimulation of adenylate cyclase.     

Further characterization of the in vitro binding of phytochrome to a membrane fraction enriched for mitochondria

This study employs ¹²⁵I-labeled phytochrome (¹²⁵I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. The binding of ¹²⁵I-Pfr to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of ¹²⁵I-Pr. However, ¹²⁵I-Pfr and ¹²⁵I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum ¹²⁵I-Pfr binding to membranes from light-exposed plants occurs within 30 seconds and is optimized in a reaction buffer containing 5 millimolar MgCl₂ at pH 6.8. Scatchard plots of the binding data for Pfr indicate a single high-affinity site with an affinity constant of 1.79 × 10¹¹ per molar. When optimal binding conditions are used, over 20% of the ¹²⁵I-P added is bound and a stoichiometry of about 100 molecules per mitochondrion is attained. When the specificity of binding is tested using competition experiments with a 15-fold excess of unlabeled phytochrome, ¹²⁵I-Pfr shows no specific binding to rat liver mitochondria.