Low Strength Buffer Research Articles

Adsorption behaviors of neutral and ionizable compounds on hybrid stationary phases in the absence (BEH-C18) and the presence (CSH-C18) of immobile surface charges

The adsorption behaviors of a neutral (caffeine) and a positively charged compound (nortriptylinium) are investigated on two RPLC/hybrid stationary phases, eluted with a low ionic strength buffer (phosphate buffer, WSpH 2.63, I=10mM). The first phase, bridge ethylene hybrid (BEH), is neutral at all pHs whereas the second, charged surface hybrid (CSH), contains a protonated ligand at WWpH <7. The band profiles of these two compounds eluted by mixture of acetonitrile and water were recorded under overloaded conditions. The adsorption isotherms of the neutral compound on both columns were well accounted for by a heterogeneous Linear-Langmuir (LL) model, which has an adsorption–desorption equilibrium constants about twice larger for caffeine on the CSH than on the BEH, due to charge–dipole interactions. In contrast, at low loadings (0.3 and 1.2μL, C=30g/L), the adsorption isotherm of the charged compound can be accounted for by a homogeneous electrostatically modified Langmuir (EML) or by a heterogeneous bi-EML implicit isotherms onto the CSH and BEH adsorbent, respectively. Electrostatic repulsions definitely account the lesser retention of the ionizable compound on CSH than on BEH. This is explained by the surface potential of CSH-C18 at 20mV and by the subsequent decrease of the equilibrium constant of weak adsorption sites (C18 environment) and removal of the strong adsorption sites (accessible silanols). At the highest sample loadings (5 and 20μL, C=30g/L), the EML and the bi-EML isotherms failed because some adsorbate–adsorbate interactions take place when bulk concentrations exceed 0.2g/L. The experimental data were then successively fitted to an empirical heterogeneous Langmuir–Moreau (LM) explicit isotherm. The best saturation capacities of the empirical heterogeneous LM isotherm is consistent with the manufacturer's estimate of the surface concentration of residual silanols onto the BEH- and CSH-C18 endcapped adsorbent (<0.1μmol/m2).

Potassium and ionic strength effects on the conformational and thermal stability of two aldehyde dehydrogenases reveal structural and functional roles of K⁺-binding sites.

Many aldehyde dehydrogenases (ALDHs) have potential potassium-binding sites of as yet unknown structural or functional roles. To explore possible K+-specific effects, we performed comparative structural studies on the tetrameric betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (PaBADH) and on the dimeric BADH from spinach (SoBADH), whose activities are K+-dependent and K+-independent, respectively, although both enzymes contain potassium-binding sites. Size exclusion chromatography, dynamic light scattering, far- and near-UV circular dichroism, and extrinsic fluorescence results indicated that in the absence of K+ ions and at very low ionic strength, PaBADH remained tetrameric but its tertiary structure was significantly altered, accounting for its inactivation, whereas SoBADH formed tetramers that maintained the native tertiary structure. The recovery of PaBADH native tertiary-structure was hyperbolically dependent on KCl concentration, indicating potassium-specific structuring effects probably arising from binding to a central-cavity site present in PaBADH but not in SoBADH. K+ ions stabilized the native structure of both enzymes against thermal denaturation more than did tetraethylammonium (TEA+) ions. This indicated specific effects of potassium on both enzymes, particularly on PaBADH whose apparent T m values showed hyperbolical dependence on potassium concentration, similar to that observed with the tertiary structure changes. Interestingly, we also found that thermal denaturation of both enzymes performed in low ionic-strength buffers led to formation of heat-resistant, inactive soluble aggregates that retain 80% secondary structure, have increased β-sheet content and bind thioflavin T. These structured aggregates underwent further thermal-induced aggregation and precipitation when the concentrations of KCl or TEACl were raised. Given that PaBADH and SoBADH belong to different ALDH families and differ not only in amino acid composition but also in association state and surface electrostatic potential, the formation of this kind of β-sheet pre-fibrillar aggregates, not described before for any ALDH enzyme, appear to be a property of the ALDH fold.

Proteomic Analysis of Duck Fatty Liver during Post-Mortem Storage Related to the Variability of Fat Loss during Cooking of “Foie Gras”

Fat loss during cooking of duck "foie gras" is the main problem for both manufacturers and consumers. Despite the efforts of the processing industry to control fat loss, the variability of fatty liver cooking yields remains high and uncontrolled. To understand the biochemical effects of postslaughter processing on fat loss during cooking, this study characterizes for the first time the protein expression of fatty liver during chilling using a proteomic approach. For this purpose the proteins were separated according to their solubility: the protein fraction soluble in a buffer of low ionic strength (S) and the protein fraction insoluble in the same buffer (IS). Two-dimensional electrophoresis was used to analyze the S fraction and mass spectrometry for the identification of spots of interest. This analysis revealed 36 (21 identified proteins) and 34 (26 identified proteins) spots of interests in the low-fat-loss and high-fat-loss groups, respectively. The expression of proteins was lower after chilling, which revealed a suppressive effect of chilling on biological processes. The shot-gun strategy was used to analyze the IS fraction, with the identification of all the proteins by mass spectrometry. This allowed identification of 554 and 562 proteins in the low-fat-loss and high-fat-loss groups, respectively. Among these proteins, only the proteins that were up-regulated in the high-fat-loss group were significant (p value = 3.17 × 10(-3)) and corresponded to protein from the cytoskeleton and its associated proteins. Taken together, these results suggest that the variability of technological yield observed in processing plants could be explained by different aging states of fatty livers during chilling, most likely associated with different proteolytic patterns.

Evaluation of Amino Acid O-Phosphoserine as Ligand for the Capture of Immunoglubulin G from Human Serum

The amino acid ortho-phosphoserine (OPS) immobilized on agarose gel was evaluated as a ligand for adsorption of polyclonal human immunoglobulin G (IgG) from human serum in the presence of low ionic strength buffers. Screening of buffer systems showed sodium phosphate as the buffer that exhibited higher IgG purity values. Through breakthrough curve analysis for agarose-OPS (feeding of 31.93 mg of total protein per mL of gel), a purification factor of 5.4 with an IgG purity of 89 % was obtained (based on IgG, IgM, IgA, HSA, and Trf nephelometric analysis). IgG adsorption equilibrium studies showed that these data followed the Langmuir-Freundlich model, with cooperativity parameter (n) equal to 1.74, indicating the presence of positive cooperativity, probably due to multipoint interactions. The maximum IgG binding capacity was 24.2 mg mL(-1), near the value for the bioaffinity ligand protein A. The agarose-OPS adsorbent provides an attractive alternative for capturing of IgG from human serum.

Substrate-selective and Calcium-independent Activation of CaMKII by α-Actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

Enhancement of Pressure-Induced Hemolysis by Aquaporin-1 Inhibitors in Human Erythrocytes

Abstract Pressure-induced hemolysis reflects sensitively membrane perturbations in erythrocytes. The functions of transporters and channels are inhibited by sodium p-chloromercuribenzoate (pCMB). So, we tested the effects of pCMB on hemolysis of human erythrocytes at 200 MPa. Pressure-induced hemolysis was enhanced by pCMB, but restored by dithiothreitol. Such effects of pCMB were also observed in N-ethylmaleimide-pretreated erythrocytes. Similar hemolytic properties were observed by aquaporin-1 (AQP1) inhibitors such Hg2+ and Au3+, but not by inhibitors of Na+–K+ pump, Gardos channels, and glucose transporter. In fact, the results of atomic absorption spectrometry showed the binding of mercury to AQP1. From the 1H spin–spin relaxation time of water, we found that pressure-induced hemolysis was enhanced upon inhibition of water transport. Flow cytometric analysis demonstrated the enhancement of hemolysis by suppression of fragmentation under pressure. Detachment of cytoskeletal proteins from the membrane by low ionic strength buffer was unaffected by pCMB. Moreover, diameters of vesicles released from intact and pCMB-treated erythrocytes at 200 MPa were 438 and 584 nm, respectively. In both vesicles, spectrin contents were almost the same. Taken together, these data suggest that pCMB bound to AQP1 inhibits water transport and enhances the hemolysis due to the suppression of fragmentation under pressure, but not affect membrane–cytoskeleton interactions.

Properties of β-thioglucoside hydrolases (TGG1 and TGG2) from leaves of Arabidopsis thaliana

Myrosinases (β-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen- and sulfur-containing secondary metabolites, to give a chemically unstable intermediate, glucose and sulfate. This catalysis initiates a chemical defense in crucifer plants as a response to the tissue-damaging activities of herbivores and pathogens. To characterize the individual and collective biochemical properties of the myrosinase enzymes found in the aerial tissues of Arabidopsis thaliana, we purified TGG1 and TGG2, which share 73% amino acid identity, individually from T-DNA insertion lines of Arabidopsis using lectin affinity and anion exchange chromatography. Electrophoresis under denaturing conditions and the mobility of nondenatured TGG1 and TGG2 protein on gel filtration chromatography indicated that the native proteins exist as dimers of 150 and 126kDa, respectively.Despite their relatively similar kinetic parameters, both enzymes had distinct physicochemical properties such as extractability in low ionic strength buffer and electrophoretic mobility following deglycosylation treatment. Deglycosylation under nondenaturing conditions had limited effects on TGG1 and no effect on TGG2 activity. Both enzymes functioned across a broad range of temperatures (up to 60°C) and pH values (5–10). These results demonstrate that myrosinases have the ability to function in environments like the digestive tract of insect herbivores that are significantly different from the environment in a damaged plant.

Continuous microfluidic DNA and protein trapping and concentration by balancing transverse electrokinetic forces

Sample pre-concentration can be a critical element to improve sensitivity of integrated microchip assays. In this work a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields to assess the ability to concentrate a sample prior to other enzymatic modifications or capillary electrophoretic separations. Employing a pressure-driven flow to perfuse the microchannel, negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main daughter channel. Experimental results demonstrated that, depending of the buffer selection, different DNA migration and accumulation dynamics were seen. Charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility, high electrophoretic mobility, high ionic strength buffer or migrated towards an equilibrium position within the channel in a high electroosmotic mobility, high electrophoretic mobility, low ionic strength buffer. The various migration behaviours are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the bounding walls. Under continuous flow conditions, DNA samples were concentrated several-fold by balancing these transverse electrokinetic forces. The electrokinetic trapping technique presented here is a simple technique which could be expanded to concentrate or separate other analytes as a preconditioning step for downstream processes.

Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE) activity. The enzyme showed optimum activity at alkaline pH (8.5) and 45oC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5) inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5) inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45oC; it only lost 5% of its activity after incubation at 45oC for 40 minutes. The Michaelis-Menten constant (Km) for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.

The Effect of Tension on Closely Spaced Nicks in Naked DNA Molecules

Cells always protect their genomic integrity from environmental stresses and harmful chemicals produced during metabolism. Double-stranded breaks(DSB) in DNA caused by factors such as free radicals or ionizing radiation play an important role in triggering DNA repair pathways and apoptosis. These DSBs are often formed by closely spaced nicks. Although methods exist to quantify single nicks on plasmids, the detection of multiple nicks or clustered nicks on DNA molecules is not so straight forward. By stretching single DNA molecules in low ionic strength buffers, sections containing clustered nicks denature locally and induce DSB. The detection of closely spaced nicks on double-stranded (ds) DNA is done by applying tension on DNA molecules with dual-beam optical tweezers and notating the time at which the DNA breaks in relation to the starting time (sustaining time). The effects of nicks caused by the sample preparation process and oxidative agents in the buffer were investigated. DNA sustaining time under low force (4∼6 pN) is around 50 minutes in low salt buffer. Higher forces (14∼16 pN) cause the sustaining time to shift toward a shorter time frame (∼30 minutes). The sustaining time of DNA molecules prepulled to a predetermined maximum force (14∼16, 24∼26, or 34∼36 pN) also show shifts toward shorter time.

The Origins of Cross-Bridges in Active and Rigor Insect Flight Muscle are not as Predicted from Acto-S1 and X-Ray Crystallography

In fast-frozen, actively contracting insect flight muscle (IFM) fibers we noted a surprising azimuthal slew in the myosin lever arms that was wholly unexpected from the currently available crystal structures of isolated myosin S1 and which indicates the crystal structures may not reflect the normal in situ constraints within the sarcomere where myosin cross-bridges are restricted to originate from a well defined zone on the thick filament [Wu et al., 2010, PLoS-ONE, 5(9): e12643]. However, previous studies did not reveal the S2 domains, which most accurately define the myosin head origin. Here, we have used electron tomography (ET) of Lethocerus IFM fibers in rigor in which the filament lattice has been swollen in low ionic strength buffer to view the S2 origins of rigor “lead bridges”. These rigor lead bridges bind to the same region of the thin filament as myosin heads of active muscle so their origins should reflect the same thick filament positions as active heads. We examined 80 nm thick transverse sections cut with a vibrating knife to minimize section compression and shearing artifacts, and imaged with < 60 e-/A2 total exposure during each tilt series to minimize radiation-induced section thinning. This analysis shows two different distributions of lead bridge origins depending on the presence of the rear bridges. However, the origins are consistent with target zone accessibility of strong binding myosin heads being limited to two successive 14.5 nm crowns. Subvolume averages of both thick filaments as well as actin filaments are being pursued with the goal of reassembling a region of the filament lattice using high S/N averages. Supported by NIGMS and NIAMSD.

The sclerostin-bone protein interactome

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

Fatty acid binding and oxidation kinetics for wild type P450(BM3) (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k (cat). The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.

Verification of a decrease in the rigidity of the phage λ DNA polymeric chain in low ionic strength aqueous solutions by testing the polymer-polymer interlink interactions

Changes in the rigidity of the polymetric chain of phage lambda double-strand DNA have been studied by laser correlation spectroscopy. It was shown that, as the ionic strength increases, the effect of the screening of the hydrodynamic interaction of the links of the polymeric chain specific for polymeric coils arises in a DNA solution. It is assumed that the screening occurs when the threshold of the overlapping of DNA coils is achieved. The overlapping of coils is the result of a previously observed significant rise of DNA coil size from abnormally small DNA coils in low ionic strength buffers (about 10(-2) M Na+ or less) to maximum possible large coils in the 5SSC and 5SSC-like buffers. Further analysis of the far interlink interactions in linear lambda phage DNA coils in similar buffers at pH 7 and 4 confirms the earlier proposal about the role of H+ ions in the appearance of abnormally small DNA coils. The abnormal decrease in the DNA coil size in low ionic strength buffers is not a specific feature of lambda phage DNA only.

Fc domain mediated self-association of an IgG1 monoclonal antibody under a low ionic strength condition

Recently, we reported that IgG1 monoclonal antibody A (MAb A) underwent liquid-liquid phase separation and separated into light and heavy phases under a low ionic strength condition. The liquid-liquid phase separation was induced due to self-association of MAb A in the heavy phase when the initial concentration of MAb A was between the two critical concentrations [Nishi et al., Pharm. Res., 27, 1348-1360 (2010)]. Here, we determined the interaction site of MAb A by using proteolytic Fab and Fc fragments of MAb A. The mean hydrodynamic diameter of the Fc fragment increased in a low ionic strength buffer, and furthermore the SPR measurement detected interactions of the Fc fragment with both whole MAb A and the Fc fragment, whereas the Fab fragment interacted with neither whole MAb A nor the Fc fragment. No binding was detected under an isotonic ionic strength condition. Zeta potential of MAb A was significant positive below pH 5.5 and negative above pH 6.5. Between pH 5.5 and 6.5 where the phase separation is significantly induced, MAb A had only a small positive or negative net charge. The isothermal titration calorimetry dilution method revealed that dissociation of MAb A accompanied endothermic heat changes, suggesting that intermolecular interactions among MAb A molecules were attributed to the enthalpically driven process. These results suggest that liquid-liquid phase separation of MAb A is mediated by a weak electrostatic intermolecular interaction among MAb A molecules mainly at Fc portions.

Integrated Glass Microdevice for Nucleic Acid Purification, Loop-Mediated Isothermal Amplification, and Online Detection

A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h.

Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme.

Adenosine 5'-phosphoramidate (NH₂-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH₂-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH₂-pA. The enzyme functions as a homodimer (2 x 15,800 Da). At the optimum pH of 7.0, the K(m) for NH₂-pA was 0.5 µM and k(cat) 0.8 s⁻¹ (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms.

Rennet-induced aggregation of milk containing homogenized fat globules. Effect of interacting and non-interacting fat globules observed using diffusing wave spectroscopy

The dynamics of the interactions between casein micelles and oil droplets during rennet-induced gelation were described using rheology and diffusing wave spectroscopy. Two different colloidal states were obtained by preparing WPI-stabilized oil droplets in either milk serum or low ionic strength buffer: flocculated and non-flocculated. These two colloidal states strongly affected the diffusion of the non-interacting droplets in the gel and the elasticity of the protein network. In homogenized milk, the droplets interact with the protein network and play a structuring role, causing an increased elastic modulus. However, after displacement of the protein from the interface using Tween 20, the interpretation of the rheological data would lead to incorrect conclusions without light scattering measurements. This work demonstrated that an in-depth understanding of the colloidal properties of the oil droplets is necessary to fine-tune the interactions between these particles and the casein matrix in dairy gels.

Electron Tomography of Thick Sections of Insect Flight Muscle

Insect flight muscle (IFM) is a good model system within which to visualize actin-myosin interactions due to its highly ordered lattice of actin and myosin filaments. Lethocerus flight muscle is perhaps the best ordered muscle in nature. Electron tomography (ET) of Lethocerus IFM has recently resulted in a model for the weak to strong transition that incorporates large azimuthal changes in the position of the lever arm compared to that predicted from crystal structures of myosin subfragment 1 in both the nucleotide free and transition states (Wu et al. PLoS-ONE, Sept. 2010). Those studies did not visualize the S2 domain in either the raw tomogram or in subvolume averages which would clarify the crossbridge origin. Here we have used ET of IFM fibers in rigor in which the filament lattice has been swollen in low ionic strength buffer to view where S2 emerges from the thick filament backbone as a test of the weak to strong transition. Previous ET on myac layers (single filament layers containing alternating myosin and actin filaments) of these same swollen rigor fibers revealed the S2 domain with clarity. In the present work, we are examining 80 nm thick transverse and longitudinal sections of swollen rigor IFM fibers in order to visualize all of the crossbridges originating from each 14.5 nm crown on the thick filament, but especially the so-called lead bridges, which bind the thin filament within the same target zone of isometric contraction. Class averages of both thick filaments as well as myac layers are being pursued. The thick filaments show subfilaments in the backbone and many of the myac layer raw repeat subvolumes show S2. Progress on this study will be presented. Supported by NIGMS and NIAMSD.

Proteomic analysis of semimembranosus and biceps femoris muscles from Bayonne dry-cured ham

The aim of the study was to delineate and compare the proteomic maps of two muscles of dry-cured ham: the biceps femoris and the semimembranosus. For this purpose, we used two-dimensional electrophoresis on a subcellular muscle fraction: insoluble protein in low ionic strength buffer. After protein identification by MALDI–TOF mass spectrometry and bioinformatic analyses, we found differences in expression levels in the two muscles. Seventy-three proteins or fragments were differentially expressed: 43 were over-represented in semimembranosus and 30 in biceps femoris. Although the study was performed on the insoluble protein fraction in low strength ionic buffer, protein and fragment identifications by mass spectrometry showed that most of the proteins were involved in energy metabolism. The differences observed between the two muscles can be explained by the differences in salt and moisture content in the course of dry-cured ham processing.