scholarly journals Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myofibrils

2000 ◽  
Vol 346 (2) ◽  
pp. 393-402 ◽  
Author(s):  
Gerry A. SMITH ◽  
Henry B. F. DIXON ◽  
Heide L. KIRSCHENLOHR ◽  
Andrew A. GRACE ◽  
James C. METCALFE ◽  
...  
Keyword(s):  
Experimental Data ◽  
Dissociation Constant ◽  
Dissociation Rate ◽  
Troponin C ◽  
Cardiac Contraction ◽  
Myofibrillar Atpase ◽  
Dissociation Rate Constants ◽  
Contraction And Relaxation ◽  
Ferret Heart

The measurement of cardiac Ca2+ transients using spectroscopic Ca2+ indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca2+ buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca2+ dissociation rate constants and which would account for the observed effects of 19F-NMR indicators on the cardiac Ca2+ transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca2+ dependency of myofibril activation in cardiac myocytes is mediated by a single Ca2+-binding site on troponin C. A model based on 1:1 Ca2+ binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca2+ binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca2+ binding, giving 58 and 2.0 μM respectively. In addition to reproducing our experimental data on the concentration of free Ca2+ ions in the cytoplasm ([Ca2+]i), the resulting Ca2+ and ATP affinities given by fitting of the model also provided good predictions of the Ca2+ dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca2+ mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca2+ indicators. The new model was used to estimate the extent of perturbation of the Ca2+ transient caused by different concentrations of indicators. As little as 10 μM of a Ca2+ indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca2+]i and 30 μM will cause approx. 50% reduction in the peak-systolic [Ca2+]i in a heart paced at 1.0 Hz.

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo

2004 ◽  
Vol 286 (6) ◽  
pp. H2089-H2095 ◽  
Author(s):  
Brian B. Roman ◽  
Paul H. Goldspink ◽  
Elyse Spaite ◽  
Dalia Urboniene ◽  
Ron McKinney ◽  
...  
Keyword(s):  
Troponin I ◽  
Cardiac Contraction ◽  
Phosphorylation Sites ◽  
Developed Pressure ◽  
Contraction And Relaxation ◽  
Intracellular Targets ◽  
Camp Levels

Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-ε, may slow the rates of cardiac contraction and relaxation (+dP/d t and −dP/d t). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for Ser43 and Ser45 and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater −dP/d t than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/d t increased by 33% and −dP/d t increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ.

scholarly journals Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.

1980 ◽  
Vol 84 (1) ◽  
pp. 141-150 ◽  
Author(s):  
L G Bergen ◽  
G G Borisy
Keyword(s):  
Electron Microscope ◽  
Rate Constants ◽  
Dissociation Constants ◽  
Dissociation Rate ◽  
Association Constants ◽  
Directional Growth ◽  
Microtubule Protein ◽  
Electron Microscope Analysis ◽  
Dissociation Rate Constants

Microtubules are polar structures, and this polarity is reflected in their biased directional growth. Following a convention established previously (G. G. Borisy, 1978, J. Mol. Biol. 124:565--570), we define the plus (+) and minus (-) ends of a microtubule as those equivalent in structural orientation to the distal and proximal ends, respectively, of the A subfiber of flagellar outer doublets. Rates of elongation were obtained for both ends using flagellar axonemes as seeds and porcine brain microtubule protein as subunits. Since the two ends of a flagellar seed are distinguishable morphologically, elongation of each end may be analyzed separately. By plotting rates of elongation at various concentrations of subunit protein, we have determined the association and dissociation rate constants for the plus and minus ends. Under our conditions at 30 degrees C, the association constants were 7.2 X 10(6) M-1 s-1 and 2.25 X 10(6) M-1 s-1 for the plus and minus ends, respectively, and the dissociation constants were 17 s-1 and 7 s-1. From these values and Wegner's equations (1976, J. Mol. Biol. 108:139--150), we identified the plus end of the microtubule as its head and calculated "s," the head-to-tail polymerization parameter. Surprisingly small values (s = 0.07 +/- 0.02) were found. The validity of models of mitosis based upon head-to-tail polymerization (Margolis et al., 1978, Nature (Lond.) 272:450--452) are discussed in light of a small value for s.

Calculated R.R.K.M. unimolecular dissociation rate constants for hydrazine

1969 ◽  
Vol 47 (14) ◽  
pp. 2593-2599 ◽  
Author(s):  
D. W. Setser ◽  
W. C. Richardson
Keyword(s):  
Experimental Data ◽  
Lower Bounds ◽  
Rate Constants ◽  
Chemical Activation ◽  
Dissociation Rate ◽  
Upper And Lower Bounds ◽  
Unimolecular Dissociation ◽  
Complex Models ◽  
Dissociation Rate Constants ◽  
Activated Complex

Unimolecular rate constants for hydrazine dissociation by thermal and chemical activation have been calculated according to the R.R.K.M. theory. The two activated complex models used in the calculations represent plausible upper and lower bounds to the rate constants. The calculations are mainly directed toward establishing expected decomposition to stablilization ratios of N2H4 produced by combination of NH2 radicals; however, a general comparison to available experimental data for hydrazine dissociation is made.

Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein βγ subunits

2002 ◽  
Vol 362 (3) ◽  
pp. 725-731 ◽  
Author(s):  
Sonja KRUGMANN ◽  
Matthew A. COOPER ◽  
Dudley H. WILLIAMS ◽  
Phillip T. HAWKINS ◽  
Len R. STEPHENS
Keyword(s):  
Plasma Membrane ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Lipid Vesicles ◽  
Dissociation Rate ◽  
Lipid Monolayers ◽  
Dissociation Rate Constants

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein βγ subunits (Gβγs). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P2, and the Gβγs are localized at the plasma membrane. We have addressed the mechanism by which Gβγs regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gβγs incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gβγs. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gβγs in vitro. We conclude that Gβγs activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

scholarly journals NAD(P)H: FMN-oxidoreductase functioning under macromolecular crowding: in vitro modeling

2019 ◽  
Vol 486 (4) ◽  
pp. 500-503
Author(s):  
A. E. Govorun ◽  
E. N. Esimbekova ◽  
V. A. Kratasyuk
Keyword(s):  
Rate Constants ◽  
Vibrio Fischeri ◽  
Macromolecular Crowding ◽  
Dissociation Rate ◽  
Common Belief ◽  
Native Conformation ◽  
The Common ◽  
Dissociation Rate Constants

The functioning of Vibrio fischeri NAD(P)H: FMN-oxidoreductase (Red) under conditions of macromolecular crowding (MMC) modeled in vitro by adding biopolymers (starch and gelatin) was studied. The dissociation rate constants and the activation energies of dissociation of Red to the subunits were calculated; the process of denaturation of Red was analyzed. It was shown that the functioning of Red both under conditions of MMC and diluted solutions is the same. The result refutes the common belief that due to MMC the stabilization of enzymes’ native conformation occurs in vivo when compared to in vitro.

scholarly journals Does protein binding modulate the effect of angiotensin II receptor antagonists?

2001 ◽  
Vol 2 (1_suppl) ◽  
pp. S54-S58 ◽  
Author(s):  
Marc P Maillard ◽  
Catherine Centeno ◽  
Åsa Frostell-Karlsson ◽  
Hans R Brunner ◽  
Michel Burnier
Keyword(s):  
Angiotensin Ii ◽  
Protein Binding ◽  
Plasma Proteins ◽  
Dissociation Rate ◽  
At1 Receptor ◽  
Ang Ii ◽  
Receptor Antagonists ◽  
At1 Receptor Antagonists ◽  
Dissociation Rate Constants

Introduction Angiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%). With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists. Methods A radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II) antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin. Results The in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists. Conclusion Although the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

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