Measurement of interprotein distances in the acto-subfragment 1 rigor complex

Using enzymatic labeling, we have conjugated the fluorescence probe dansylcadaverine (DNC) to Gln-41 of rabbit skeletal muscle actin with the intention of utilizing the dansyl chromophore as a donor in fluorescence resonance energy transfer (FRET) distance measurements. The fluorescence decay of DNC-actin was found to consist of two decay constants (8.23 and 21.2 ns) that were associated with two different but partially overlapping spectra of the dye. Three different chemical points on myosin subfragment 1 (S1) were labeled with suitable acceptors: reactive thiol 1 (SH1) and Cys-136 on LC3 were modified with tetramethylrhodamine 5- (and 6-) iodoacetamide (ITMR); Lys-83 (RLR) was derivatized with trinitrobenzenesulfonate. In the rigor complex of the two labeled proteins, fluorescence resonance energy transfer took place, the efficiency of which was 10.9, 9.28, and 3.73% for the transfer from Gln-41 to SH1, Cys-136 (LC3), and RLR, respectively. The limits of the F?rster critical distance for each pair were obtained from the analysis of the polarization spectra of the donor and of the acceptors. The kappa 2(2/3) distances from actin Gln-41 to the three points on S1 were 63, 66, and greater than 37 A for SH1, Cys-136 (LC3), and RLR, respectively.     

Epigenetic aspects of HP1 exchange kinetics in apoptotic chromatin

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (∼40%) recovery of GFP-HP1α and GFP-HP1β after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic.     

Oxygen consumption characteristics of porcine hepatocytes

Oxygen uptake rate (OUR) of hepatocytes is an important parameter for the design of bioartificial liver assist (BAL) devices. Porcine hepatocytes were cultured in a specially constructed measurement chamber with an incorporated mixing system and a Clark polarographic oxygen electrode. Signal noise associated with conventional Clark electrode implementations was circumvented by the combination of real time digital numerical averaging and subsequent finite impulse response (FIR) spectral filtering. Additional software allowed for the automated generation of cellular oxygen consumption coefficients, namely, Vmax and K0.5, adding a high degree of objectivity to parameter determination. Optimization of the above numerical techniques identified a 0.1 Hz/200 data point sample size and a 0.004 Hz cutoff frequency as ideal parameters. Vmax values obtained for porcine hepatocytes during the first two weeks of culture showed a maximal consumption of 0.9 nmole/sec/10(6) cells occurring on Day 4 post seeding, and a gradual decrease to 0.31 nmole/sec/10(6) cells by Day 15. K0.5 values increased from 2 mm Hg on Day 2 to 8 mm Hg by Day 8, with gradual subsequent decrease to 4 mm Hg by Day 15. The Vmax and K0.5 values measured for porcine cells were higher than maximal values for rat hepatocytes (Vmax: 0.43 nmole/sec/10(6) cells, K0.5: 5.6 mmHg) and thus may necessitate significantly altered BAL device design conditions to ensure no oxygen limitations. Finally, these results highlight the need for species specific characterization of cellular function for optimal BAL device implementations.     

Analysis of proton exchange kinetics with time-dependent exchange rate

Mass spectrometry is used to probe the kinetics of hydrogen–deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange. The data are described by a power-like function which is based on a time-dependence of the exchange rate. Experimental data ranging over many time scales is considered and accurate fits of a power-like function are obtained. Results of fittings show correlation between faster hydrogen–deuterium exchange and increase of pH. Furthermore a model is presented that discriminates between easily exchangeable hydrogens (located in close proximity to the protein surface) and those protected from the exchange (located in the protein interior). A possible interpretation of the model and its biological significance are discussed.     

Reactions of 1-N6-ethenoadenosine nucleotides with myosin subfragment 1 and acto-subfragment 1 of skeletal and smooth muscle

The fluorescent nucleotides epsilon ADP and epsilon ATP were used to study the binding and hydrolysis mechanisms of subfragment 1 (S-1) and acto-subfragment 1 from striated and smooth muscle. The quenching of the enhanced fluorescence emission of bound nucleotide by acrylamide analyzed either by the Stern-Volmer method or by fluorescence lifetime measurements showed the presence of two bound nucleotide states for 1-N6-ethenoadenosine triphosphate (epsilon ATP), 1-N6-ethenoadenosine diphosphate (epsilon ADP), and epsilon ADP-vanadate complexes with S-1. The equilibrium constant relating the two bound nucleotide states was close to unity. Transient kinetic studies showed two first-order transitions with rate constants of approximately 500 and 100 s-1 for both epsilon ATP and epsilon ADP and striated muscle S-1 and 300 and 30 s-1, respectively, for smooth muscle S-1. The hydrolysis of [gamma-32P] epsilon ATP yielded a transient phase of small amplitude (less than 0.2 mol/site) with a rate constant of 5-10 s-1. Consequently, the hydrolysis of the substrate is a step in the mechanism which is distinct from the two conformational changes induced by the binding of epsilon ATP. An essentially symmetric reaction mechanism is proposed in which two structural changes accompany substrate binding and the reversal of these steps occurs in product release. epsilon ATP dissociates acto-S-1 as effectively as ATP. For smooth muscle acto-S-1, dissociation proceeds in two steps, each accompanied by enhancement of fluorescence emission. A symmetric reaction scheme is proposed for the acto-S-1 epsilon ATPase cycle. The very similar kinetic properties of the reactions of epsilon ATP and ATP with S-1 and acto-S-1 suggest that two ATP intermediate states also occur in the ATPase reaction mechanism.     

Positive inotropic effects of low dATP/ATP ratios on mechanics and kinetics of porcine cardiac muscle

Substitution of 2'-deoxy ATP (dATP) for ATP as substrate for actomyosin results in significant enhancement of in vitro parameters of cardiac contraction. To determine the minimal ratio of dATP/ATP (constant total NTP) that significantly enhances cardiac contractility and obtain greater understanding of how dATP substitution results in contractile enhancement, we varied dATP/ATP ratio in porcine cardiac muscle preparations. At maximum Ca(2+) (pCa 4.5), isometric force increased linearly with dATP/ATP ratio, but at submaximal Ca(2+) (pCa 5.5) this relationship was nonlinear, with the nonlinearity evident at 2-20% dATP; force increased significantly with only 10% of substrate as dATP. The rate of tension redevelopment (k(TR)) increased with dATP at all Ca(2+) levels. k(TR) increased linearly with dATP/ATP ratio at pCa 4.5 and 5.5. Unregulated actin-activated Mg-NTPase rates and actin sliding speed linearly increased with the dATP/ATP ratio (p < 0.01 at 10% dATP). Together these data suggest cardiac contractility is enhanced when only 10% of the contractile substrate is dATP. Our results imply that relatively small (but supraphysiological) levels of dATP increase the number of strongly attached, force-producing actomyosin cross-bridges, resulting in an increase in overall contractility through both thin filament activation and kinetic shortening of the actomyosin cross-bridge cycle.     

Increased cross-bridge cycling kinetics after exchange of C-terminal truncated troponin I in skinned rat cardiac muscle

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions ( approximately 50%) in maximum force and cooperative activation as well as increases ( approximately 50%) in myofilament Ca(2+) sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca(2+) sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca(2+) sensitivity, we measured Ca(2+) binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca(2+) binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca(2+) binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.     

Effect of nucleotide structure on cardiac myosin subfragment 1 transient kinetics

Transient kinetic data of the hydrolysis of several nucleotides (TTP, CTP, UTP, GTP) by cardiac myosin subfragment 1 (S1) were analyzed to obtain values for the equilibrium constant for nucleotide binding and rate constants for the S1-nucleotide isomerization and the subsequent nucleotide hydrolysis as well as the magnitudes of the relative fluorescence enhancements of the myosin that occur upon isomerization and hydrolysis. These data are compared with data from a previous study with ATP. Nucleotide binding is found to be relatively insensitive to nucleotide ring structure, being affected most by the group at position C6. Isomerization and hydrolysis are more sensitive to nucleotide structure, being inhibited by the presence of a bulky group at position C2. Kinetic parameters decrease as follows: for binding, GTP greater than UTP approximately TTP greater than ATP greater than CTP; for isomerization, ATP greater than UTP approximately TTP approximately CTP greater than GTP; for hydrolysis, ATP greater than TTP greater than CTP approximately UTP greater than GTP. Fluorescence enhancements appear to be most dependent upon the relative values of the individual rate constants.     

Bovine cardiac myosin subfragment 1. Transient kinetics of ATP hydrolysis

The kinetics of binding and hydrolysis of ATP by bovine cardiac myosin subfragment 1 has been reinvestigated. More than 90% of the total fluorescence amplitude associated with ATP hydrolysis occurs with an apparent second-order rate constant of 8.1 X 10(5) M-1 S-1 and a limiting rate constant of approximately 140 S-1 (100 mM KCl, 50 mM 1,3-bis-[tris(hydroxymethyl)methylamino]-propane, 10 mM MgCl2, pH 7.0, 20 degrees C); the remaining 10% occurs more slowly (approximately 1 S-1). The observed rate constants are independent of subfragment 1 concentration under pseudo first-order conditions for ATP with respect to protein. The fraction of protein which hydrolyzes ATP rapidly is not a function of the nucleotide or protein concentration and appears to be constant irrespective of ionic strength or temperature within the range studied (50-100 mM KCl, pH 7.0, 15-20 degrees C). These data are compared to that obtained previously using subfragment 1 prepared by a different method which showed ATP-dependent aggregation of two protein species.     

Immunochemical analysis of porcine cardiac C-protein

C-protein has been isolated from pig heart and its immunochemical properties studied. It is extracted with myosin, and separated from the myosin on a DEAE-Sephadex column. The amount of C-protein recovered from crude myosin is approx. 3.5%. The molecular weight of C-protein is 150,000. Anti-C-protein serum reacts with crude myosin and purified C-protein but not with purified myosin in immunodiffusion plates. Cardiac C-protein does not react with anti-skeletal white muscle C-protein serum. Immunoblotting experiments show that anti-cardiac C-protein serum reacts with a Mr = 150,000 component in myofibrils or crude myosin. C-protein is located in the A-band, except the M-line region, of the myofibrils. These results indicate that C-protein is an intrinsic component of the thick filaments in pig heart myofibrils.     

Oxygen radicals in cardiac surgery

Most cardiac surgical procedures require the use of prolonged induced myocardial ischemia. Experimental models of global myocardial ischemia which mimic cardiac surgical techniques have been developed to investigate the possibility of oxygen free radical development during prolonged myocardial ischemia or upon reperfusion. In such experiments, various free radical scavenging agents, including superoxide dismutase, catalase, and mannitol, have been shown to improve the tolerance of the heart to protracted global ischemia. Use of these agents has improved cardiac functional recovery and has attenuated the biochemical and structural changes which occur due to prolonged ischemia and reflow. In a recently developed porcine experimental model, the effects of preexisting regional myocardial ischemia with superimposed global ischemia and reperfusion have been studied, with free radical scavenging agents administered in an attempt to reduce myocardial infarction and improve regional functional recovery. In most such studies completed to date, free radical scavenging agents have resulted in better myocardial preservation, suggesting, at least indirectly, that there may be an oxygen free radical-mediated component of the ischemia-reperfusion injury seen in such models. Techniques for directly measuring myocardial oxygen free radical levels may allow for early clarification of the development of such toxic species in the clinical cardiac surgical setting.     

The kinetics of transdermal ethanol exchange.

The kinetics of ethanol transport from the blood to the skin surface are incompletely understood. We present a mathematical model to predict the transient exchange of ethanol across the skin while it is being absorbed from the gut and eliminated from the body. The model simulates the behavior of a commercial device that is used to estimate the blood alcohol concentration (BAC). During the elimination phase, the stratum corneum of the skin has a higher ethanol concentration than the blood. We studied the effect of varying the maximum BAC and the absorption rate from the gut on the relationship between BAC and equivalent concentration in the gas phase above the skin. The results showed that the ethanol concentration in the gas compartment always took longer to reach its maximum, had a lower maximum, and had a slower apparent elimination rate than the BAC. These effects increased as the maximum BAC increased. Our model's predictions are consistent with experimental data from the literature. We performed a sensitivity analysis (using Latin hypercube sampling) to identify and rank the importance of parameters. The analysis showed that outputs were sensitive to solubility and diffusivity within the stratum corneum, to stratum corneum thickness, and to the volume of gas in the sampling chamber above the skin. We conclude that ethanol transport through the skin is primarily governed by the washin and washout of ethanol through the stratum corneum. The dynamics can be highly variable from subject to subject because of variability in the physical properties of the stratum corneum.     

Tritium exchange kinetics of yeast ribosomal subunits

Tritium exchange kinetics of 60 S and 40 S ribosomal subunits from Saccharomyces cerevisiae were studied using a rapid centrifugal, ultrafiltration procedure. This assay used commercially available disposable columns and microconcentrators. The tritium-labeled ribosome was separated from the tritiated solvent using a prepacked gel-filtration column. The labeled ribosome was applied to a microconcentrator and the exchange-out kinetics of the ribosome was measured by centrifugation of the ribosome solution and measurement of the amount of radioactivity present in the filtrate. One major advantage of this method is its simplicity and rapidity. With this method, the tritium exchange-out behavior of 60 S and 40 S ribosomal subunits and of subunits during reassociation were determined. The two subunits exhibited different exchange-out rates. Both subunits consisted of multiple classes of exchangeable protons. Considerable conformational changes in both subunits were evident during subunit reassociation, as additions of equal molar quantities of unlabeled 40 S subunits to labeled 60 S subunits caused an immediate increase in the exchange rate. Similarly, an increase in the exchange rate in the small subunits upon addition of unlabeled 60 S subunits was observed.     

Oxygen exchange in leaves in the light

Photosynthetic O₂ production and photorespiratory O₂ uptake were measured using isotopic techniques, in the C₃ species Hirschfeldia incana Lowe., Helianthus annuus L., and Phaseolus vulgaris L. At high CO₂ and normal O₂, O₂ production increased linearly with light intensity. At low O₂ or low CO₂, O₂ production was suppressed, indicating that increased concentrations of both O₂ and CO₂ can stimulate O₂ production. At the CO₂ compensation point, O₂ uptake equaled O₂ production over a wide range of O₂ concentrations. O₂ uptake increased with light intensity and O₂ concentration. At low light intensities, O₂ uptake was suppressed by increased CO₂ concentrations so that O₂ uptake at 1,000 microliters per liter CO₂ was 28 to 35% of the uptake at the CO₂ compensation point. At high light intensities, O₂ uptake was stimulated by low concentrations of CO₂ and suppressed by higher concentrations of CO₂. O₂ uptake at high light intensity and 1000 microliters per liter CO₂ was 75% or more of the rate of O₂ uptake at the compensation point. The response of O₂ uptake to light intensity extrapolated to zero in darkness, suggesting that O₂ uptake via dark respiration may be suppressed in the light. The response of O₂ uptake to O₂ concentration saturated at about 30% O₂ in high light and at a lower O₂ concentration in low light. O₂ uptake was also observed with the C₄ plant Amaranthus edulis; the rate of uptake at the CO₂ compensation point was 20% of that observed at the same light intensity with the C₃ species, and this rate was not influenced by the CO₂ concentration. The results are discussed and interpreted in terms of the ribulose-1,5-bisphosphate oxygenase reaction, the associated metabolism of the photorespiratory pathway, and direct photosynthetic reduction of O₂.     

Native connectin from porcine cardiac muscle

Native connectin was isolated from porcine cardiac muscle using the method developed for the preparation of native connectin from chicken breast muscle (Kimura et al. (1984) J. Biochem. 96, 1947-1950). It was not necessary to keep cardiac muscle at 0 degrees C before preparation: the proteolysis of alpha-connectin to beta-connectin proceeded during the preparation of myofibrils. Cardiac connectin showed almost the same properties as those of skeletal muscle connectin: mobility in SDS gel electrophoresis, filamentous structure under an electron microscope, circular dichroism spectra, UV absorption spectra, and amino acid composition. Porcine cardiac connectin cross-reacted with antiserum against chicken breast muscle connectin as revealed by an immunoblot method. Immunoelectron microscopical observations revealed an abundance of connectin antigenic sites around the A-I junction area of cardiac myofibrils. Cardiac connectin also interacted with myosin and actin filaments at low ionic strengths to form aggregates. The extent of interaction was somewhat weaker in the case of cardiac connectin than skeletal muscle connectin, regardless of the origin of myosin and actin (porcine cardiac and rabbit skeletal muscles). In conclusion, cardiac connectin is very similar, but not identical to skeletal muscle connectin.