Calcium and magnesium ATPases of the spectrin fraction of human erythrocytes.

Using a rapid method of preparation, spectrin has been isolated from human erythrocytes and its ATPase activity investigated. The ATPase activity with calcium has two distinct components, one with optimal activity when calcium and ATP are of equal concentration (low-Ca-ATPase) and another which is activated above 1 mM CaCl2 and is maximal at 100 mM CaCl2. There is also a Mg-ATPase with maximal activity at 10 mM MgCl2. The high-Ca-ATPase of spectrin, but not the low-Ca-ATPase, is inhibited by magnesium, while the Mg-ATPase is inhibited by Ca in excess of ATP. None of these activities exhibits the calcium-stimulated magnesium-dependent activity characteristic of the red cell calcium pump.     

Transitions of serum albumin in patients with glomerulosclerosis 'in vivo' characterization by electrophoretic titration curves

HSA functions as a physiological transporter of solutes and small molecules that induce structural transitions 'in vitro'. Analysis of these transitions requires prior purification of HSA that could introduce bias due to conformational changes. We utilized electrophoretic titration curves to describe a neutral to acid (N-A) transition of HSA directly in sera of seven patients with active focal segmental glomerulosclerosis (FSGS). The divergent electrophoretic profile of HSA was characterized by a shift in the range of pHs between 4.5 and 7.5 with an average variation of free electrophoretic mobility corresponding to loss of 1 positive charge in the pKa protonation range of histidyl residues and should involve domain I of HSA. 'In-gel' determination by maleimide-PEO2-biotin of free SH 34 of domain I showed inaccessibility of the dye at this site in pathological HSA and alkylation with the same complex induced N-A transition in normal HSA. Potential binders of free imidazoles such as Ca++ and/or of SH 34 such as NO were excluded on the basis of direct titration and studies on binding stimulation. This is the first report describing a transition of HSA directly 'in vivo', and the utilization of electrophoretic titration curves was critical to this purpose. This transition appears to be specific to FSGS and is unrelated to the nephrotic syndrome, Ca++ and NO binding. Spectroscopic analysis will elucidate the structural implication.     

Structural changes in myosin during contraction and the state of ATP in the intact frog muscle.

The reactivity of myosin to [14C]-labeled N-ethylmaleimide ([14C]NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [14C]NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule. The reactivity of myosin to [14C]NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [14C]NEM into myosin and that upon the irreversible loss of ATP the rate increases. 31P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.     

Phosphorprotein intermediate in the Ca2+-dependent ATPase reaction of macrophage plasma membrane.

ATPase activity and phosphorylation by [gamma-32P] ATP of isolated plasma membrane of alveolar macorphages are stimulated in a parallel fashion by physiologic concentrations of Ca2+, with half-maximal activating effect of this ion at (3--7) X 10(-7) M. For various membrane preparations, a direct proportionality exists between Ca2+-dependent ATPase activity and amount of 32P incorporated. Labeling of membrane attains the steady-state level by 10 sec at 0 degrees C, and is rapidly reversed by adenosine diphosphate (ADP), K+ decreases the amount of membrane-bound 32P, mainly by enhancing the rate of dephosphorylation of the 32P-intermediate. Hydroxylamine causes a release of about 90% of 32P bound to the membrane, thus indicating that the 32P-intermediate contains an acyl-phosphate bond. When the labeled plasma membrane is solubilized and electrophoresed on acrylamide gels in the presence of sodium dodecyl sulphate, the radioactivity appears to be largely associated with a single protein fraction of 132,000 +/- 2,000 aarent molecular weight. These features of the macrophage Ca2+-ATPase suggest that the enzyme activity might be part of a surface-localized Ca1+-extrusion system, participating in the regulation of Ca2+-dependent activities of the macrophage.     

Alkaline ribonuclease associated with polyribosomes in fibroblasts of experimental granulation tissue.

Alkaline ribonuclease (RNase) from polyribosomes derived from experimental granulation tissue has been purified 1900-fold through affinity chromatography. The preparation was homogeneous in sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis with an estimated molecular weight of 15 000. Purified RNase was completely inhibited in the presence of divalent ions Mg2+(100 mM) and Ca2+(100 mM) but activated slightly with Na+(50 mM). The enzyme is an endonuclease and the best substrates were poly(U), mixed RNA from yeast, rRNA from granulation tissue and poly(C). The estimated apparent Km-values were 0.037, 0.064, 0.13 and 0.27 g1-1, respectively. In polyribosomes RNase occurred in both free and p-chloromercuribenzoate (pCMB)-liberated forms. The total activity was at the highest but the proportion of the free activity minimal in the granulation tissue during the maximal synthesis of collagen.     

High-performance ion-exchange chromatography of myosin using a DEAE-5PW column

High-performance ion-exchange chromatography of myosin using a DEAE-5PW packing was used to purify myosin from skeletal, cardiac and smooth muscle. This method produces high-speed resolution (30-min analysis) of myosin from contaminating myofibrillar proteins. The column has a high capacity for binding myosin (up to 1 g) and can be used for small-scale preparation of highly purified myosin. Gel analysis in the presence of sodium dodecyl sulfate showed recovery of myosin with very little contamination of other myofibrillar proteins. Myosin was also recovered from small biopsy samples (0.1 g) by a direct extraction technique with recovery of biological ATPase activity.     

Interaction between cytoplasmic (Ca2+--Mg2+) ATPase activator and the erythrocyte membrane.

Human red blood cells (RBC) contain a cytoplasmic, nonhemoglobin protein which activates the (Ca2+-Mg2+)ATPase of isolated RBC membranes. Results presented in this paper confirm that activation of (Ca2+-Mg2+)ATPase is associated with binding of the cytoplasmic activator to the membrane. Binding of the cytoplasmic activator is reversible and dependent on ionic strength and Ca2+. Cytoplasmic activator is sensitive to trypsin but is not degraded when intact RBC are exposed to trypsin. Cytoplasmic activator does not modify the (Ca2+-Mg2+)-ATPase of membranes from RBC exposed to activator prior to hemolysis. Thus, the activator is located in the cell and appears to act by binding to the inner membrane surface.     

Calcium-binding protein regucalcin stimulates the uptake of Ca2+ by rat liver mitochondria

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver mitochondria was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate mitochondrial 45Ca2+ accumulation. 45Ca2+ uptake was stimulated by the presence of regucalcin (1.0 and 2.0 microM). This stimulation was remarkable during 1.0 min after 45Ca2+ addition, while appreciable stimulation was no longer seen at 3 min. Regucalcin (2.0 microM)-induced stimulation of 45Ca2+ uptake was prevented by the presence of ruthenium red (1.0 microM) and lanthanum chloride (0.1 mM). Regucalcin (2.0 microM) did not increase the mitochondrial adenosine triphosphatase (ATPase) activity during 3.0 min after Ca2+ addition. Meanwhile, 45Ca2+, which accumulated in the mitochondria during 5.0 min after 45Ca2+ addition, was not released by the addition of regucalcin. Regucalcin may stimulate Ca2+ uptake in rat liver mitochondria independently of the energy.     

Substituent effects on the rate of formation of azomethine ylides. A computational investigation

The effect of substituents on the rate of conrotatory thermal cleavage of aziridine has been studied at the MP2(Full)/6-311++G(d,p)//MP2(Full)/6-31+G(d) level and also using SCS-MP2 methodology. While the parent compound has a high free energy of activation (194.6 kJ mol(-1)), this value could be drastically lowered by substituent effects. Anionic species were found to be particularly effective in increasing the calculated reaction rate. The potential utility of this approach in 1,3-dipolar cycloaddition is discussed.     

Development of impedimetric and optical calcium biosensor by using modified gold electrode with porcine S100A12 protein

We describe the development of a label free method to analyze the interactions between Ca(2+) and the porcine S100A12 protein immobilized on polyvinyl butyral (PVB). The modified gold electrodes were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and surface plasmon resonance (SPR) techniques. SEM analyses of PVB and PVB-S100A12 showed a heterogeneous distribution of PVB spherules on gold surface. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due the adsorption of PVB-S100A12, and confirm the existence of a positive response of the immobilized S100A12 to the presence of calcium ions. The biosensor exhibited a wide linear response to Ca(2+) concentrations ranging from 12.5 to 200mM. The PVB-S100A12 seems to be bound to the gold electrode surface by physical adsorption; we observed an increase of 1184.32m° in the SPR angle after the adsorption of the protein on the PVB surface (in an indication that 9.84ng of S100A12 are adsorbed per mm(2) of the Au-PVB electrode), followed by a further increase of 581.66m° after attachment of the Ca(2+) ions. In addition, no SPR response is obtained for non-specific ions. These studies might be useful as a platform for the design of new reusable and sensitive biosensing devices that could find use in the clinical applications.     

Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase.

EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 +/- 0.1 A is calculated, suggesting the sharing of a common ligand atomy by Mn2+ and T1+ on the ATPase. Addition of Pi increases this distance to 5.4 A consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the (Na+ + K+)-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATPase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.     

Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase.

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.     

Physico‐chemical study of sucrose and calcium ions interactions in alkaline aqueous solutions

Interactions between sucrose and calcium hydoxide have been the subject of our work. They were studied in dilute solutions (sucrose concentration <40% w/w, with a molar stoichiometric ratio Ca(OH)₂/sucrose <2) by several physico‐chemical methods, titration of free and bound calcium ions, ¹³C NMR spectroscopy, static light scattering and low shear viscosimetry. From these analyses we propose a polymer‐like complex formation with calcium ions bound to at least two sucrose molecules. Moreover, this polymer‐like aggregate can be described as a linear short chain at low Ca(OH)₂ and sucrose concentrations and as dense fractal particles at higher concentrations.     

DFT study of the mechanisms of in water Au(I)-catalyzed tandem [3,3]-rearrangement/Nazarov reaction/[1,2]-hydrogen shift of enynyl acetates: a proton-transport catalysis strategy in the water-catalyzed [1,2]-hydrogen shift

A computational study with the Becke3LYP density functional was carried out to elucidate the mechanisms of Au(I)-catalyzed reactions of enynyl acetates involving tandem [3,3]-rearrangement, Nazarov reaction, and [1,2]-hydrogen shift. Calculations indicate that the [3,3]-rearrangement is a two-step process with activation free energies below 10 kcal/mol for both steps. The following Nazarov-type 4pi electrocyclic ring-closure reaction of a Au-containing dienyl cation is also easy with an activation free energy of 3.2 kcal/mol in CH2Cl2. The final step in the catalytic cycle is a [1,2]-hydride shift, and this step is the rate-limiting step (with a computed activation free energy of 20.2 kcal/mol) when dry CH2Cl2 is used as the solvent. When this tandem reaction was conducted in wet CH2Cl2, the [1,2]-hydride shift step in dry solution turned to a very efficient water-catalyzed [1,2]-hydrogen shift mechanism with an activation free energy of 16.4 kcal/mol. Because of this, the tandem reaction of enynyl acetates was found to be faster in wet CH2Cl2 as compared to the reaction in dry CH2Cl2. Calculations show that a water-catalyzed [1,2]-hydrogen shift adopts a proton-transport catalysis strategy, in which the acetoxy group in the substrate is critical because it acts as either a proton acceptor when one water molecule is involved in catalysis or a proton-relay stabilizer when a water cluster is involved in catalysis. Water is found to act as a proton shuttle in the proton-transport catalysis strategy. Theoretical discovery of the role of the acetoxy group in the water-catalyzed [1,2]-hydrogen shift process suggests that a transition metal-catalyzed reaction involving a similar hydrogen shift step can be accelerated in water or on water with only a marginal effect, unless a proton-accepting group such as an acetoxy group, which can form a hydrogen bond network with water, is present around this reaction's active site.     

Interaction of hydroxyapatite with sodium chondroitin sulfate and calcium chondroitin sulfate in an aqueous phase

The suspension pH at a given concentration of hydroxyapatite (HAP) decreased with the concentration of calcium chondroitin-6-sulfate (CaChs), whereas it increased with the concentration of sodium chondroitin-6-sulfate (Na2Chs). The former effect is due to the increase in the concentration of H+ by ion exchange between H+ on the surface of HAP and Ca2+ of CaChs, and the latter is due to the protonation of phosphate ion (Pi) released from the surface of HAP. The absorbed amount of chondroitin-6-sulfate anion (Chs) by HAP was higher with CaChs than with Na2Chs over the concentration range examined. The equilibrium concentration of Pi decreased with increasing concentration of added CaChs because the concentration of free Ca2+, which dissociates from CaChs, regulates the free concentration of Pi through the restriction of the solubility product of HAP (Ksp). In contrast, that in the presence of Na2Chs increased with increasing concentration of added Na2Chs owing to the anion exchange between Chs and Pi of the HAP surface. The total concentration of Ca2+, which was released from HAP into the solution phase, increased after passing through a minimum with increasing concentration of added Na2Chs. That is, the concentration of Ca2+ free from Chs decreased with an increase in the concentration of released Pi owing to the restriction of the solubility product, whereas that of Ca2+ bound by Chs increased with increasing concentration of added Na2Chs through the ion exchange of Na+ with Ca2+. It was confirmed by the dialysis method that the value of Ksp was almost constant around 10, although HAP dissolves incongruently in the presence of Na2Chs.     

Alterations of transmembrane currents in frog atrial heart muscle induced by photoexcited gymnochrome A purified from the crinoid, Gymnochrinus richeri

The effects of gymnochrome A were tested on the electrical activity of the frog atrial heart muscle. Gymnochrome A (1-5 microM) did not alter the resting potential. Gymnochrome A (5 microM) slowed the initial depolarizing phase of the spontaneously beating action potential. Under voltage-clamp conditions gymnochrome A (5 microM) did not affect the electrical constant of the membrane and the kinetic parameters of the peak Na+ current (INa) recorded in the Ringer solution containing tetraethylammonium (2 mM) and Cd2+ (1 mM) but shifted the membrane potential at which the current both activated and reached its maximal value toward more negative membrane potentials. It did not alter the reversal potential for INa, indicating that the selectivity of the Na+ channels had not changed. These observations suggest that gymnochrome A binds to the membrane and shifts the activation of INa on the voltage axis by modifying the free negative fixed charges present at the membrane surface rather than by occupying a specific site on the Na+ channel. Photoexcited gymnochrome A transiently triggered an early outward current which lengthened the time-to-peak of INa and decreased its amplitude. In addition, photoexcited gymnochrome A blocked the background K+ current. This is, to our knowledge, the first time that such effects are reported on the cardiac muscle. These observations suggest that the photoexcitation of gymnochrome produces physico-chemical effects which lead to intracellular changes. Further experiments are required to determine their nature.     

Organic and inorganic di-cations for capillary silica coating and EOF modulation in CE: Example of application in PEG analysis

EOF measurements, by using 1,4-di-(4-aza-1-azonia-bicyclo[2.2.2]octane)butane diiodide, barium and strontium tetraborate as silica wall modifiers, are reported and, as an example of application, analysis of PEG (PEG 400-2000) polydisperse preparations in free solution CZE is shown. PEGs have been derivatized with phthalic anhydride so as to form singly or doubly charged derivatives with strong UV absorbance at 214 nm. Whereas separations in plain tetraborate buffer, pH 9.0, without any EOF control, did not lead to good resolution of all-size oligomers and suffered from long analysis times, excellent resolution of all oligomers up to 40 ethylene oxide (EO) units could be obtained under EOF control. Such EOF modulation was engendered by addition of 1 mM M7C4M7, a doubly charged organic cation able to stick tenaciously to the silica wall. Further modulation of EOF and silica surface modification could be achieved also by addition of inorganic cations, notably those of group II, whereas monovalent cations did not seem to affect much the EOF flux. Among the doubly charged cations investigated, Ca++, Mg++, Sr++ and Ba++, the latter did seem to offer best EOF control and reproducible runs. A judicious blend of M7C4M7 (0.33-1 mM range) with barium (10-20 mM range) allowed baseline resolution of all PEG oligomers investigated up to PEG 2000 and >40 EO units in length. In this last case, best results in terms of reproducibility and separation efficiency of the more heavy homologues were obtained using Li+ salt in small amounts.     

Temperature dependence of anisotropy decay and solvation dynamics of coumarin 153 in gamma-cyclodextrin aggregates

Effect of temperature on the fluorescence anisotropy decay and the ultraslow component of solvation dynamics of coumarin 153 (C153) in a gamma-cyclodextrin (gamma-CD) nanocavity are studied using a picosecond set up. The steady-state anisotropy (0.13 +/- 0.01) and residual anisotropy (0.14 +/- 0.01) in fluorescence anisotropy decay in an aqueous solution containing 7 microM C153 and 40 mM gamma-CD are found to be quite large. This indicates formation of large linear nanotube aggregates of gamma-CD linked by C153. It is estimated that >53 gamma-CD units are present in each aggregate. In these aggregates with rise in temperature, the average solvation time ((obs)) decreases markedly from 680 ps at 278 K to 160 ps at 318 K. The dynamic Stokes shift is found to decrease from 800 cm(-1) at 278 K to 250 cm(-1) at 318 K. The fraction of dynamic Stokes shift (f(d)) detected in a picosecond set up is calculated using the Fee-Maroncelli procedure. The corrected solvation time ((corr) = f(d)<(tau(s)>(obs)) displays an Arrhenius type temperature dependence. From the temperature variation, the activation energy and entropy of the solvation process are determined to be 12.5 kcal M(-1) and 28 cal M(-1) K(-1), respectively. The ultraslow component and its temperature dependence are ascribed to a dynamic exchange between bound and free water molecules.     

Effect of Oxygen Free Radicals on Myosin in Muscle Fibres. A DSC and EPR study

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural Distortion of Cycloalkynes Influences Cycloaddition Rates both by Strain and Interaction Energies

The reactivities of 2-butyne, cycloheptyne, cyclooctyne, and cyclononyne in the 1,3-dipolar cycloaddition reaction with methyl azide were evaluated through DFT calculations at the M06-2X/6-311++G(d)//M06-2X/6-31+G(d) level of theory. Computed activation free energies for the cycloadditions of cycloalkynes are 16.5–22.0 kcal mol⁻¹ lower in energy than that of the acyclic 2-butyne. The strained or predistorted nature of cycloalkynes is often solely used to rationalize this significant rate enhancement. Our distortion/interaction–activation strain analysis has been revealed that the degree of geometrical predistortion of the cycloalkyne ground-state geometries acts to enhance reactivity compared with that of acyclic alkynes through three distinct mechanisms, not only due to (i) a reduced strain or distortion energy, but also to (ii) a smaller HOMO–LUMO gap, and (iii) an enhanced orbital overlap, which both contribute to more stabilizing orbital interactions.