Mn2+ transport in the unicellular cyanobacterium Anacystis nidulans

Mn²⁺ uptake by wild type of Anacystis nidulans IU 625 follows Michaelis-Menten type kinetics with apparent saturation at 27 μmol/l of Mn²⁺. Lineweaver-Burk plot of the data reveals a Km of 25 μmol/l and V_max of 1.2 nmol/mg protein · min. The uptake values at different pH (pH 4.0 to 9.0) exhibit a characteristic profile with an optimum at pH 6.0. The Mn²⁺ uptake rate in the dark is found to be similar to that in the light suggesting that energy for transport may not be directly derived from light reactions. The system seems to be specific for Mn²⁺ transport since simultaneous addition of Mg²⁺ partially inhibited the uptake in a competitive manner. Further, the alterations in Km and V_max values of mutants with respect to that of wild type strains suggest a genetic control of the transport system.     

Interaction of divalent metal ions with poly(riboinosinic acid)

Methods of differential UV and visible spectroscopy are used to study the interaction of Mg²⁺, Ca²⁺, Mn²⁺ and Cu²⁺ ions with four (4×I)‐ and single‐stranded poly I in solutions with 1 mol·l^–1 Na⁺. Up to concentrations of about 0.1 mol·l^–1 Mt²⁺, Mg²⁺ and Ca²⁺ ions do not bind to heteroatoms of hypoxanthine of 4×I. Cu²⁺ ions interact with N7 and/or N1 and possibly with O6 through the water molecule of the hydrate shell of the ion. It is likely that the interaction with O6 causes enolization of the hypoxanthine, N1 deprotonation and, as a result, this leads to the melting of the four‐stranded helix. In single‐stranded chains, Cu²⁺ ions induce the formation of compact particles which have the effective radii r_e ≈ 100 Å. The Mn²⁺‐induced differential spectra are similar to those observed in the presence of Cu²⁺ ions but in the case of Mn²⁺ they occur at concentrations about two orders of magnitude higher. In contrast to the positive cooperativity of the Cu²⁺ binding to bases of single‐stranded poly I, their interaction with 4×I has the negative cooperativity.     

Relationship between blood and myocardium manganese levels during manganese‐enhanced MRI (MEMRI) with T1 mapping in rats

Manganese ions (Mn²⁺) enter viable myocardial cells via voltage‐gated calcium channels. Because of its shortening of T₁ and its relatively long half‐life in cells, Mn²⁺ can serve as an intracellular molecular contrast agent to study indirect calcium influx into the myocardium. One major concern in using Mn²⁺ is its sensitivity over a limited range of concentrations employing T₁‐weighted images for visualization, which limits its potential in quantitative techniques. Therefore, this study assessed the implementation of a T₁ mapping method for cardiac manganese‐enhanced MRI to enable a quantitative estimate of the influx of Mn²⁺ over a wide range of concentrations in male Sprague‐Dawley rats. This MRI method was used to compare the relationship between T₁ changes in the heart as a function of myocardium and blood Mn²⁺ levels. Results showed a biphasic relationship between ΔR₁ and the total Mn²⁺ infusion dose. Nonlinear relationships were observed between the total Mn²⁺ infusion dose versus blood levels and left ventricular free wall ΔR₁. At low blood levels of Mn²⁺, there was proportionally less cardiac enhancement seen than at higher levels of blood Mn²⁺. We hypothesize that Mn²⁺ blood levels increase as a result of rate‐limiting excretion by the liver and kidneys at these higher Mn²⁺ doses. Copyright © 2010 John Wiley & Sons, Ltd.     

Enhancement of agonist binding to 5-HT1A receptors in rat brain membranes by millimolar Mn

Manganese in millimolar concentration caused increase in specific binding of [³H]8-OH-DPAT to rat hippocampal membranes up to 44% in comparison with experiments in the presence of Mg²⁺, while no significant differences were found in rat cortical membranes. Similar increase in high-affinity agonist binding sites by Mn²⁺ was found in displacement curves of 8-OH-DPAT, where antagonist [³H]WAY100635 was used as reporter ligand. The removal of bivalent ions with EDTA caused full loss of high-affinity binding of agonists, but not for antagonists. Therefore it was hypothesized, that the effect of Mn²⁺- and Mg²⁺-ions was modulated through their action on different G-proteins. Results showed that efficient coupling of G-protein and 5-HT_1A receptors is crucial to modify Mg²⁺ and Mn²⁺ effects, whereas Mn²⁺ is more potent stabilizer of agonist high-affinity binding, especially when GTPγS is present. Using Sf9 cells as model system, we have shown that G_i1 proteins are required to modulate Mn²⁺-dependent high-affinity agonist binding to 5-HT_1A receptors, but further studies are necessary to find the cofacors of Mn²⁺ modulation to signal transduction.     

Effects of varying the spacing within the D,D-35-E motif in the catalytic region of retroviral integrase

The catalytic domain of all retroviral integrases contains an Asp,Asp-35-Glu (D,D-35-E) motif with precisely 35 amino acids between the second aspartate and the glutamate. We have now made several mutations designed to alter the length or flexibility of a mobile loop within this 35-amino-acid spacer region in full-length Rous sarcoma virus integrase. Surprisingly, most of the mutants had enzymatic activity, including ones that shortened or lengthened the loop by up to 6 amino acids. Several size mutants exhibited the two biologically relevant activities of integrase in reactions with Mn²⁺, although they were inactive with Mg²⁺. No viruses containing integrase with an altered length, however, replicated in cell culture, and these viruses were blocked at the integration step. Thus, the conserved 35-amino-acid spacing is not absolutely required for enzymatic activity, but the correlation between infectivity and Mg²⁺-dependent activity supports magnesium as the metal cofactor used by integrase in vivo.     

Ca2+ bound to the high affinity divalent cation-binding site of actin enhances actophorin-induced depolymerization of muscle F-actin but inhibits actophorin-induced depolymerization of Acanthamoeba F-actin

Summary The cation tightly bound to actin, Mg²⁺ or Ca²⁺, affects the ability of actophorin to accelerate depolymerization of filaments and bind to monomers of actin prepared from rabbit skeletal muscle and Acanthamoeba castellanii. Actophorin interacted similarly with muscle and Acanthamoeba Mg²⁺-F-actin but depolymerized muscle Mg²⁺-F-actin more efficiently. Muscle Ca^2+-F-actin depolymerized about 5 times more rapidly than Mg²⁺-F-actin in the presence of actophorin but Acanthamoeba Ca^2+-F-actin was highly resistant to actophorin. Muscle actin subunits dissociated more rapidly than Acanthamoeba actin subunits from copolymers of muscle and Acanthamoeba Ca²⁺-actin upon addition of actophorin although Acanthamoeba actin dissociated much more rapidly from copolymers than from its homopolymer. The Kd of the 1:1 complex between actophorin and monomeric actin was somewhat lower for muscle Mg²⁺-ATP-G-actin than for both Acanthamoeba Mg²⁺-ATP-G-actin and muscle Ca²⁺-ATP-G-actin. The data for the interactions of actophorin with Acanthamoeba Ca²⁺-ATP-G-actin or muscle and amoeba Mg²⁺-and Ca²⁺-ADP-G-actin were incompatible with the formation of 1:1 actin:actophorin complexes and, thus, Kd values could not be calculated. While it may not be surprising that actophorin would interact differently with Mg²⁺-and Ca²⁺-actin, it is unexpected that the nature of the tightly bound cation would have such dramatically opposite effects on the ability of actophorin to depolymerize muscle and Acanthamoeba F-actin. Differential severing by actophorin, with Acanthamoeba Ca²⁺-actin being almost totally resistant, is sufficient to explain the results but other possibilities cannot be ruled out.     

Extracellular Mg2+ regulates activation of rat eag potassium channel

The rat homologue of Drosophila ether à gogo cDNA (rat eag) encodes voltage-activated potassium (K) channels with distinct activation properties. Using the Xenopus expression system, we examined the importance of extracellular Mg²⁺ on the activation of rat eag. Extracellular Mg²⁺ at physiological concentrations dramatically slowed the activation in a dose- and voltage-dependent manner. Other divalent cations exerted similar effects on the activation kinetics that correlated with their enthalpy of hydration. Lowering the external pH also resulted in a slowing of the activation. Protons competed with Mg²⁺ as the effect of Mg²⁺ was abolished at low pH. A kinetic model for rat eag activation was derived from the data indicating that all four channel subunits undergo a Mg²⁺-dependent conformational transition prior to final channel activation. The strong dependence of rat eag activation on both the resting potential and the extracellular Mg²⁺ concentration constitutes a system for fine-tuning K channel availability in neuronal cells. Received: 21 August 1995/Received after revision: 2 November 1995/Accepted: 29 November 1995     

Rod photoresponses in the absence of external sodium in retinae treated with phosphodiesterase inhibitors

Summary Exposure of isolated toad retinae to phosphodiesterase inhibitors induced changes in the ionic permeability of rod cells. Under similar conditions intracellularly recorded light responses were observed also in the absence of external Na⁺. Hyperpolarizing photoresponses in Na⁺-free media required the presence of divalent cations among which Mg²⁺, Mn²⁺ and Ba²⁺ were the most effective.Supported by a fellowship of the Scuola Normale Superiore, Pisa     

Effects of elevated magnesium and substrate on neuronal numbers and neurite outgrowth of neural stem/progenitor cells in vitro

Because a potential treatment for brain injuries could be elevating magnesium ions (Mg²⁺) intracerebrally, we characterized the effects of elevating external Mg²⁺ in cultures of neonatal murine brain-derived neural stem/progenitor cells (NSCs). Using a crystal violet assay, which avoids interference of Mg²⁺ in the assay, it was determined that substrate influenced Mg²⁺ effects on cell numbers. On uncoated plastic, elevating Mg²⁺ levels to between 2.5 and 10 mM above basal increased NSC numbers, and at higher concentrations numbers decreased to control or lower levels. Similar biphasic curves were observed with different plating densities, treatment durations and length of time in culture. When cells were plated on laminin-coated plastic, NSC numbers were higher even in basal medium and no further effects were observed with Mg²⁺. NSC differentiation into neurons was not altered by either substrate or Mg²⁺ supplementation. Some parameters of neurite outgrowth were increased by elevated Mg²⁺ when NSCs differentiated into neurons on uncoated plastic. Differentiation on laminin resulted in increased neurites even in basal medium and no further effects were seen when Mg²⁺ was elevated. This system can now be used to study the multiple mechanisms by which Mg²⁺ influences neuronal biology.     

A low pH form of thiamine pyrophosphatase from bovine brain

A preparation of thiamine pyrophosphatase from bovine brain exhibits an activity optimum at pH 6 and only sight activity is observed at pH 9–9.5. This low pH form of thiamine pyrophosphatase gives maximal activity with Mn²⁺. Other divalent cations (Mg²⁺, Ca²⁺ and Co²⁺ also satisfy the divalent cation requirement, but give loss activation than Mn²⁺. The low pH form of thiamine pyrophosphatase has not been previously observed independent of the high pH form. The data suggest that thiamine pyrophosphatase occurs in two separate enzyme forms in brain tissue.     

Intracellular Mg2+ inhibits the IP3-activated I K(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording I K(Ca)]

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP₃) can induce an outward Ca²⁺-activated K⁺ current [I _K(Ca)] some neural cells. We have investigated I _K(Ca) activated by intracellular injections of IP₃ in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg²⁺ by citrate. Theoretical calculations suggested free [Mg²⁺] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP₃-activated I _K(Ca) could be recorded when the free Mg²⁺ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised [Mg²⁺] blocks I _K(Ca). This appeared to be due to inhibition of the response to released Ca²⁺, since high [Mg²⁺] also blocked the response to intracellular injections of Ca²⁺ ions. Mean Mg²⁺ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP₃-induced I _K(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg²⁺.     

Effects of forskolin on crypt cells of rat distal colon

Na⁺-K⁺-2Cl^– cotransport activity has previously been shown to depend on both intracellular ATP and Mg²⁺, but the mechanisms remain unknown. Cotransport in avian erythrocytes can be stimulated by a variety of agents including cAMP and permeant serine/ threonine phosphatase inhibitors and is inhibited by prior depletion of either ATP with antimycin A, or Mg²⁺ by incubation in A23187 plus EDTA. However, when cells were first stimulated with cAMP rather than calyculin A then subjected to either depletion strategy, a differential effect was found. The phosphatase-inhibitor-treated cells were resistant to subsequent ATP or Mg²⁺ depletion while cAMP-treated cells were sensitive to both treatments. Parallel examination of protein phosphorylation confirmed that ATP or Mg²⁺ depletion leads to dephosphorylation of membrane proteins in cAMP-treated but not in calyculin-A-treated cells. These results suggest that, for cotransport, ATP and Mg²⁺ are required primarily to maintain the system in a phosphorylated state rather than as direct modulators. The relative effectiveness of okadaic acid (EC₅₀ 630 nM) and calyculin A (EC₅₀ 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably responsible for dephosphorylating the system. Cells stimulated by hypertonicity were also resistant to ATP or Mg²⁺ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

Sensitivity of RNA-synthesizing system of the lymphocytes of patients with malignant neoplasms to phytohemagglutinin and dexamethasone

Biosynthesis of RNA catalyzed by DNA-dependent RNA polymerase was demonstrated in a reconstructed system containing isolated lymphocyte nuclei, Mg²⁺ or Mn²⁺ salts, and ammonium sulfate, in the presence of four nucleoside triphosphates. Both Mg²⁺- and Mn²⁺-dependent forms of this enzyme were found in the nuclei of normal lymphocytes and of lymphocytes from patients with melanoma, lung carcinoma, and sarcoma. The activity of both forms of RNA polymerase in the nuclei of the patients' lymphocytes was higher than in normal analogs. The sensitivity of DNA-dependent RNA polymerase to dexamethasone and phytohemagglutinin was less marked in the nuclei from patients with lung carcinoma, melanoma, and sarcoma than in normal lymphocytes.Laboratory of Biochemistry of Tumors, All-Union Oncologic Center of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 449–452, April, 1977.     

Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis

Summary Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determinein situ the concentrations of Ca²⁺ and Mg²⁺ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2×10^–11 m Ca²⁺ and 2.7×10^–9 m Mg²⁺, the inexchangeably bound Mg²⁺ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca²⁺ bound to the I-band was not significantly above zero. The bound Mg²⁺ in the I-band was not exchangeable with Ca²⁺ even when the skinned fibres were exposed to 10mm Ca²⁺ solution. These results clearly indicate that Mg²⁺, rather than Ca²⁺, is the divalent cation bound to F-actin in the thin filamentsin situ. In the presence of 1mm Mg²⁺, the exchangeable Ca²⁺ bound to the I-band was increased as a function of the free Ca²⁺, while that in the A-band was not significantly changed with [Ca²⁺] up to 2 × 10^–5 m, and increased to approximately 0.8 mol Ca²⁺ per mol myosin at 10^–4 m Ca²⁺. At a saturating free Ca²⁺ in Tris-Cl solution, the bound Ca²⁺ content (2–3 mol Ca²⁺ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na⁺ enhanced Ca²⁺ binding to the level equivalent to 3–4 mol Ca²⁺ per mol troponin. The depressant effect of Tris on Ca²⁺ binding was greater in the absence of Mg²⁺. High concentrations of Tris also reduced the maximum tension induced by 10^–4 m Ca²⁺ buffered with 10mm EGTA. At 1.3×10^–7 m Ca²⁺, thought to be close to the cytoplasmic free Ca²⁺ in resting muscle, the I-band bound a significant amount of Ca²⁺: equivalent to about 1 mol Ca²⁺ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca²⁺ bound by the A-band at a free Ca²⁺ of 10^–4 m.     

Novel mutants of CHO cells resistant to adenosine analogs and containing biochemically altered form of adenosine kinase in cell extracts

Stable mutants which are approximately five- and eightfold resistant to an inosine analog, formycin B (Fom^r) have been selected in a single-step from Chinese hamster ovary cells at a frequency of approximately 10^–6. Cross-resistance studies with these mutants show that the Fom^r mutants exhibit increased resistance to all adenosine analogs (N- and C-nucleosides) examined and, in accordance with their cross-resistance pattern, the mutants exhibited decreased cellular uptake and phosphorylation of formycin B and various adenosine analogs. In cell hybrids formed with sensitive cells, the drug-resistant phenotype of these mutants behaved recessively. However, unlike mutants resistant to adenosine analogs that have been obtained previously, which contain no measurable activity of adenosine kinase (AK) in cell extracts, the two Fom^r mutants studied contained about 60 and 110% of the enzyme activity (compared to the parental cells) in their cell extracts. Biochemical studies with AK from the mutant cells show that in comparison to the wild-type enzyme, the mutant enzymes required much higher concentrations of the adenosine analog N⁷-(²-isopentenyl) formycin A for similar inhibition of [³H]adenosine phosphorylation. These results indicate that AK from the Fom^r mutants has lower affinity for phosphorylation of adenosine analogs in comparison to the enzyme from the parental cells. The genetic lesion in the Fom^r mutants may thus be directly affecting the structural gene for AK.     

Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation

Two effects of Mn²⁺ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence of absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a latch-like state, and the other involves direct Mn²⁺ activation of contractile proteins. Cells activated by Mn²⁺ in the presence of ATP and the absence of Ca²⁺, Mg²⁺ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn²⁺ contracting solution to prevent protein oxidation by Mn²⁺, the cells still contracted when exposed to Mn²⁺, but relaxed rapidly when the Mn²⁺ was removed. In the presence of DTT both the Mn²⁺ activation and the relaxation following removal of Mn²⁺ were more rapid than normal Ca²⁺-activated contractions and relaxations. The skinned fibers activated by Mn²⁺ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50mM) and then contracted again in response to Ca²⁺ and relaxed normally. The Mn²⁺ activation was not associated with myosin light chain phosphorylation, in contrast to Ca²⁺-activated contractions.A preliminary report of this work was given at the Biophysical Society Meeting, February 1987: Hoar PE, Kerrick WGL (1987) Mn²⁺ activates skinned smooth muscle cells directly without myosin light chain phosphorylation and by reversible oxidation. Biophys J 51:332a     

A genetic analysis of the role of calcineurin and calmodulin in Ca++-dependent improvement of NaCl tolerance of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae lacking activity of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin, show sensitivity to high concentrations of sodium that is partly reversed by the external supply of Ca²⁺. On long-time exposure to NaCl stress the mutants display an increased intracellular Na⁺/K⁺ ratio which is partially corrected by the addition of Ca²⁺, improving the sodium efflux of not only calcineurin-defective cells but also wild-type cells. We also demonstrate that the NaCl sensitivity of cmd mutants, expressing modified forms of calmodulin that do not bind Ca²⁺, is strongly reversed by the addition of Ca²⁺. This effect is highly dependent on calcineurin, since the NaCl tolerance of a cmd1-3 strain, carrying an additional mutation in calcineurin, is only slightly assisted by Ca²⁺. A striking characteristic of the loss of function of calcineurin is a several-fold increased content of intracellular Ca²⁺, localized mainly in subcellular compartment(s). If the compartmentalized Ca²⁺ pool is brought back to normal levels by an additional inactivating mutation of the vacuolar Ca²⁺-transporting ATPase, such double mutants do not significantly improve their tolerance to NaCl. Received: 15 August / 22 August 1996     

A monoclonal antibody that recognizes different conformational states of skeletal muscle troponin C and other calcium binding proteins

Summary Skeletal muscle troponin C contains four Ca²⁺-binding sites, two with a high affinity for Ca²⁺ that also bind Mg²⁺ competitively (Ca²⁺/Mg²⁺ sites) and two sites of lower affinity that are specific for Ca²⁺. We have characterized a monoclonal antibody (B₉D₉) that was produced against rabbit skeletal troponin C. The binding of this antibody to rabbit skeletal troponin C is sensitive to the binding of Ca²⁺. Increasing the Ca²⁺ concentration produces a decrease in the amount of antibody bound with a pK of approximately 6.9 which correlates with Ca²⁺ binding to the Ca²⁺/Mg²⁺ sites. Magnesium binding to rabbit skeletal troponin C had no effect on antibody binding. Thus the conformation of rabbit skeletal troponin C brought about by Ca²⁺ binding to these sites affects the antibody binding to its epitope. This epitope was unavailable for antibody binding in whole troponin. The antibody-binding site was localized in cyanogen bromide fragment CB9 of rabbit skeletal troponin C (residues 84–135). This antibody was also shown to cross-react with bovine cardiac troponin C., barnacle (Balanus nubilus) troponin C, bovine testis calmodulin and carp parvalbumin. In addition, the effect of Ca²⁺ on antibody binding seen with rabbit skeletal troponin C was also seen with bovine cardiac troponin C, and calmodulin. Thus these proteins appear to share a similar epitope and undergo similar structural changes. Wang and colleagues (1987) have presented evidence that rabbit skeletal troponin C at low pH has an elongated structure similar to that seen in the crystal structure and that at neutral pH its structure is more compact. We have found that in the absence of Ca²⁺ this antibody binds best to rabbit skeletal troponin C at low pH and its binding is reduced with increasingly alkaline pH. It is possible that the structural alterations brought about by changes in pH may also be responsible for the reduction in antibody binding. Since pH and Ca²⁺ have the same effect on antibody binding, this may mean that Ca²⁺ binding to the Ca²⁺/Mg²⁺ sites may also make rabbit skeletal troponin C more compact.