Interaction of α-catulin with dystrobrevin contributes to integrity of dystrophin complex in muscle

The dystrophin complex is a multimolecular membrane-associated protein complex whose defects underlie many forms of muscular dystrophy. The dystrophin complex is postulated to function as a structural element that stabilizes the cell membrane by linking the contractile apparatus to the extracellular matrix. A better understanding of how this complex is organized and localized will improve our knowledge of the pathogenic mechanisms of diseases that involve the dystrophin complex. In a Caenorhabditis elegans genetic study, we demonstrate that CTN-1/α-catulin, a cytoskeletal protein, physically interacts with DYB-1/α-dystrobrevin (a component of the dystrophin complex) and that this interaction is critical for the localization of the dystrophin complex near dense bodies, structures analogous to mammalian costameres. We further show that in mouse α-catulin is localized at the sarcolemma and neuromuscular junctions and interacts with α-dystrobrevin and that the level of α-catulin is reduced in α-dystrobrevin-deficient mouse muscle. Intriguingly, in the skeletal muscle of mdx mice lacking dystrophin, we discover that the expression of α-catulin is increased, suggesting a compensatory role of α-catulin in dystrophic muscle. Together, our study demonstrates that the interaction between α-catulin and α-dystrobrevin is evolutionarily conserved in C. elegans and mammalian muscles and strongly suggests that this interaction contributes to the integrity of the dystrophin complex.     

A comparison of two methods for fitting the integrated Michaelis–Menten equation (Short Communication)

The methods of Atkins & Nimmo (1973) and Fernley (1974) for fitting the integrated Michaelis-Menten equation were compared by using the same sets of simulated experimental data. The method of Fernley (1974) is to be preferred because it gives precise and unbiased estimates of the Michaelis-Menten parameters over a wide range of substrate concentrations. However, the estimates may not be symmetrically distributed, especially at low substrate concentrations.     

The effect of surfactant structure on ribonuclease A–surfactant interactions (Short Communication)

The enzymic activity of ribonuclease A was measured in the presence of several surfactants at pH7.2. Cationic surfactants with trimethylammonium and pyridinium head groups do not deactivate or denature the enzyme, whereas n-dodecylamine hydrochloride, like the anionic surfactant sodium n-dodecyl sulphate, deactivates and denatures ribonuclease A.     

The detection of the messenger ribonucleic acid for the α-lactalbumin of guinea-pig milk (Short Communication)

RNA was extracted from the polyribosomes isolated from the mammary glands of a lactating guinea pig and injected into Xenopus oocytes. On incubation the oocytes effected the biosynthesis of alpha-lactalbumin.     

Fluorescence studies of protein–sterol relationships in human plasma lipoproteins (Short Communication)

Cholesta-5,7,9(11)-trien-3beta-ol and its oleate ester were incorporated into human low-density lipoprotein and reconstituted high-density lipoprotein. The unesterified sterol was more efficient than its ester in quenching tryptophan fluorescence, especially in low-density lipoprotein. The results, which indicate that in such lipoproteins unesterified sterols are more closely associated with peptide than are esterified sterols, are used to assess possible structures for the lipoproteins.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

M?ssbauer effect in the `super-reduced' form of the high-potential iron–sulphur protein from Chromatium (Short Communication)

Mössbauer-effect studies of the super-reduced form of Chromatium high-potential iron–sulphur protein indicate that the iron atoms are in a similar valency state to those in reduced ferredoxin from Clostridium pasteurianum, with possibly some inequivalence between the iron atoms within the four-iron centre. Mössbauer spectroscopy also shows magnetic differences between the four-iron centres in the two proteins.     

Nitrogenase of Klebsiella pneumoniae: evidence for an adenosine triphosphate-induced association of the iron–sulphur protein (Short Communication)

The effect of various nucleotides on the Fe-containing component of nitrogenase of Klebsiella pneumoniae was investigated by ultracentrifugation and thiol-group reactivity towards 5,5'-dithiobis-(2-nitrobenzoate). In the absence of Na(2)S(2)O(4), ATP and ADP produced changes in sedimentation behaviour and thiol-group reactivity consistent with association of the protein.     

Polyprenyl pyrophosphate–p-hydroxybenzoate polyprenyltransferase activity in mitochondria of broad-bean seeds and yeast (Short Communication)

Cell-free homogenates prepared from broad-bean seeds and yeast cells are capable of synthesizing 4-carboxy-2-polyprenylphenols from p-hydroxybenzoate and either isopentenyl pyrophosphate or protein-bound polyprenyl pyrophosphates (produced by incubating a Micrococcus lysodeikticus extract with isopentenyl pyrophosphate). The mitochondria contained all the polyprenyl pyrophosphate-p-hydroxybenzoate polyprenyltransferase activity; however, unlike the homogenates they could not synthesize a side chain from isopentenyl pyrophosphate and had to be provided with protein-bound polyprenyl pyrophosphates.     

Increased liver l-serine–pyruvate aminotransferase activity under gluconeogenic conditions (Short Communication)

Rat liver l-serine-pyruvate aminotransferase activity exceeds markedly the normal adult value (a) in the neonatal period, (b) after glucagon injection and (c) after alloxan injection, observations that reinforce the suggestion from comparative findings that the aminotransferase has a role in gluconeogenesis. Some findings, however, argue in favour of l-serine dehydratase as the enzyme of gluconeogenesis from l-serine.     

The protein–polysaccharide complex of bovine nasal cartilage. Studies on the protein core

1. Chondromucoprotein from bovine nasal cartilage was purified by cetylpyridinium chloride or by bismuth nitrate in acetone. 2. Amino acid compositions of crude and purified preparations were compared and few differences were found, in spite of the decrease in protein content on purification. 3. Amino acid analysis of bismuth-purified material revealed the existence of four groups of amino acids. Within each group, the amino acids were present in approximately equimolar concentrations. 4. Amino end-group assay on the same material showed six alpha-DNP derivatives. 5. A molecular weight of 6.3x10(5) for the protein-polysaccharide complex was calculated from the latter analysis.     

The effect of systematic error on the accuracy of Michaelis constants and maximum velocities estimated by using the integrated Michaelis–Menten equation (Short Communication)

Systematic errors in initial substrate concentration (s₀), product concentration and reaction time give much larger errors in the Michaelis–Menten parameters unless s₀ is treated as an unknown parameter. These errors are difficult to detect because the fitted curve deviates little from the data. The effect of non-enzymic reaction is also examined.     

Interaction of ATP with a macromolecular translocation inhibitor of the nuclear binding of “activated” receptor-glucocorticoid complex

Incubation of 1–5 mM ATP with nuclei and partially purified “activated” receptor-[³H]triamcinolone acetonide complex from rat liver cytosol had no significant effect on association of the activated complex with the nuclei. However, when the nuclear uptake was reduced by the macromolecular translocation inhibitor in the rat liver cytosol, addition of 5 mM ATP restored the uptake to the level without inhibitor. ADP and AMP as well as other nucleotides tested could not overcome the inhibitory effect of macromolecular inhibitor.     

The phenomenological model of muscle contraction with a controller to simulate the excitation–contraction (E–C) coupling

We previously proposed a systematic motor model for muscle with two parallel Maxwell elements and a force generator P. The motor model showed the non-linear behavior of a muscle, such as the force–velocity relation and the force depression and enhancement, by using weight functions. Our newly proposed muscle model is based on the molecular mechanism of myosin cross-bridges. We assume that each parallel Maxwell element represents the mechanical properties of weak and strong binding of the myosin head to actin. Furthermore, we introduce a controller to simulate the excitation–contraction coupling of the muscle. The new muscle model satisfies all the properties obtained in our previous model and reduces the wasted energy of the viscous component to less than 5% of the total energy. The controller enables us to simulate contractions of slow and fast twitch muscles, which are driven by an artificial action potential or a processing electromyography signal despite their same mechanical components. The maximum velocities are calculated to be 3.4L₀ m/s for the fast twitch muscle model and 2.5L₀ m/s for the slow twitch muscle model, where L₀ is the initial length of the muscle model.     

Synthesis of Morin–Zinc(II) Complex and its Interaction with Serum Albumin

A morin–zinc(II) complex (MZ) was synthesized and its interaction with bovine serum albumin (BSA) were studied by molecular spectroscopy including fluorescence emission spectra, UV-visible spectra, circular dichroism (CD) spectra, three-dimensional fluorescence spectra, and synchronous fluorescence spectra. The interaction mechanism of BSA and MZ was discussed by fluorescence quenching method and Förster non-radiation energy transfer theory. The thermodynamic parameters ΔH ^θ, ΔG ^θ, ΔS ^θ at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond forces played the most important role in the reaction. The fluorescence probe experiment showed that the binding site of MZ is in subdomain IIA of BSA and the distance between BSA and MZ is 3.17 nm at normal body temperature. The conformation changes of BSA in presence of MZ were investigated by CD spectra and three-dimensional fluorescence spectra.     

γ-Hydroxybutyric acid binding sites: Interaction with the GABA-benzodiazepine-picrotoxin receptor complex

The effect of three compounds known to allosterically modulate binding to the GABA/benzodiazepine/picrotoxin receptor complex on 4-hydroxy-2,3 [³H]butyric acid (GHB) binding was investigated. Pentobarbital, pentylenetetrazole, and picrotoxin enhanced [³H]GHB binding in a dose dependent fashion. Pentobarbital enhanced 4-hydroxy-2,3 [³H]butyric acid binding was associated with an increase in B_max while pentylenetetrazole and picrotoxin altered the affinity of GHB for its binding site producing a decrease in K_d. These findings suggest that the GHB and GABA receptor complex may share certain moieties in common.     

Structural and some medicinal characteristics of the copper(II)–hydroxychloroquine complex

In the first phase of this study, the binding of hydroxychloroquine to the copper(II) cation is examined using liquid chromatography–mass spectrometry (LC–MS), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), Fourier transform-ion cyclotron resonance spectrometry (FT-ICR) and nuclear magnetic resonance (¹H and ¹³C NMR) in one and two dimensions. The data suggest the metal–ligand complex is a polarity adaptive molecule. In the second phase of the study, the complexes activity is tested against the National Cancer Institute’s 60 cell line panel. Its anti-cancer activity is compared to quinine, Cu(II)–quinine and hydroxychloroquine. It serves as a base line for future anti-cancer complexes in which hydroxychloroquine is utilized for its ability to impact cell autophagy.     

Interaction of α-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT)

The human Na⁺/multivitamin transporter (hSMVT) has been suggested to transport α-lipoic acid (LA), a potent antioxidant and anti-inflammatory agent used in therapeutic applications, e.g. in the treatment of diabetic neuropathy and Alzheimer disease. However, the molecular basis of the cellular delivery of LA and in particular the stereospecificity of the transport process are not well understood. Here, we expressed recombinant hSMVT in Pichia pastoris and used affinity chromatography to purify the detergent-solubilized protein followed by reconstitution of hSMVT in lipid bilayers. Using a combined approach encompassing radiolabeled LA transport and equilibrium binding studies in conjunction with the stabilized R-(+)- and S-(−)-enantiomers and the R,S-(+/−) racemic mixture of LA or lipoamide, we identified the biologically active form of LA, R-LA, to be the physiological substrate of hSMVT. Interaction of R-LA with hSMVT is strictly dependent on Na⁺. Under equilibrium conditions, hSMVT can simultaneously bind ∼2 molecules of R-LA in a biphasic binding isotherm with dissociation constants (K_d) of 0.9 and 7.4 μm. Transport of R-LA in the oocyte and reconstituted system is exclusively dependent on Na⁺ and exhibits an affinity of ∼3 μm. Measuring transport with known amounts of protein in proteoliposomes containing hSMVT in outside-out orientation yielded a catalytic turnover number (k_cat) of about 1 s⁻¹, a value that is well in agreement with other Na⁺-coupled transporters. Our data suggest that hSMVT-mediated transport is highly specific for R-LA at our tested concentration range, a finding with wide ramifications for the use of LA in therapeutic applications.     

Interactions of quinone with the iron-sulfur protein of the bc(1) complex: is the mechanism spring-loaded?

Since available structures of native bc(1) complexes show a vacant Q(o)-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme-substrate complex at the Q(o)-site. This is formed by binding of both ubiquinol (QH(2)) and the dissociated oxidized iron-sulfur protein (ISP(ox)). A binding constant of approximately 14 can be estimated from the displacement of E(m) or pK for quinone or ISP(ox), respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme-product complex is also formed at the Q(o)-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in E(m) of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in E(m) reflects a binding constant of approximately 4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Q(o)-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states.