Fluorescence Titration and Fluorescence Stopped-Flow Studies on Skeletal Muscle Troponin Labeled with Fluorescent Reagent

1982 ◽  
Vol 92 (4) ◽  
pp. 1141-1149 ◽  
Author(s):  
Takayoshi IIO ◽  
Hiroshi KONDO
Keyword(s):  
Skeletal Muscle ◽  
Stopped Flow ◽  
Fluorescence Titration

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle

1986 ◽  
Vol 229 (1254) ◽  
pp. 85-95 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Binding Affinity ◽  
Kinetic Studies ◽  
Rabbit Skeletal Muscle ◽  
Fluorescent Label ◽  
Weakly Bound ◽  
Thin Filaments ◽  
Fluorescence Titration ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

We have used actin labelled in Cys–374 with N -(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin–troponin–tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin–tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin–subfragment 1 complex from a state in which actin is weakly bound ( K a = 5.9 x 10 4 M -1 ) to a more tightly bound complex ( K a = 1.7 x 10 7 M -1 ) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin–tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.

scholarly journals The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site

1980 ◽  
Vol 189 (3) ◽  
pp. 561-567 ◽  
Author(s):  
D Roberts ◽  
G L Kellett
Keyword(s):  
Skeletal Muscle ◽  
Cyclic Amp ◽  
Rate Constant ◽  
Catalytic Site ◽  
Stopped Flow ◽  
Diffusion Controlled ◽  
Inhibitory Site ◽  
Kinetics Of ◽  
Step Mechanism ◽  
Effector Binding

1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.

scholarly journals Reduced uterine perfusion pressure decreases functional capillary density in skeletal muscle

2015 ◽  
Vol 309 (12) ◽  
pp. H2002-H2007 ◽  
Author(s):  
Graham M. Fraser ◽  
Jude S. Morton ◽  
Sydney M. Schmidt ◽  
Stephane Bourque ◽  
Sandra T. Davidge ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Continuous Flow ◽  
Perfusion Pressure ◽  
Capillary Density ◽  
Functional Capillary Density ◽  
Stopped Flow ◽  
Sprague Dawley ◽  
Capillary Perfusion ◽  
Pregnant State ◽  
Uterine Perfusion

The purpose of this study was to examine the functional and structural capillary density in the reduced uterine perfusion pressure (RUPP) model, which when performed during pregnancy is an established animal model of preeclampsia. We hypothesized that the RUPP model would be associated with capillary rarefaction and impaired capillary perfusion, which would be more pronounced in the pregnant state. Female Sprague-Dawley rats ( n = 32) were randomized to nonpregnancy (Nonpregnant) or breeding (Pregnant) at 12 wk of age and again to RUPP or SHAM surgeries on gestational day (GD) 14 (or equivalent age in nonpregnant rats). On GD 20 (or equivalent), capillary structure and perfusion of the extensor digitorum longus were imaged using digital intravital video microscopy. Functional videos were analyzed by a blinded observer to measure capillary density, expressed as capillaries per millimeter intersecting three staggered reference lines (200 μm). Flow was scored as the percentage of capillaries having 1) continuous, 2) intermittent, or 3) stopped flow. Total capillary density was not different between groups. There was a main effect of RUPP surgery resulting in decreased continuous flow vessels ( P < 0.01) and increased stopped flow ( P < 0.01), which was driven by differences between pregnant animals (Continuous flow: pregnant SHAM 80.1 ± 7.8% vs. pregnant RUPP 67.8 ± 11.2%, P < 0.05) (Stopped flow: pregnant SHAM 8.7 ± 3.2% vs. pregnant RUPP 17.9 ± 5.7%, P < 0.01). Our results demonstrate that the RUPP surgery is associated with a decrease in functional capillary density in skeletal muscle that is more pronounced in the pregnant state, which may contribute to the vascular pathophysiology observed in preeclampsia.

Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

1974 ◽  
Vol 32 ◽  
pp. 148-149
Author(s):  
D. E. Philpott ◽  
A. Takahashi
Keyword(s):  
Skeletal Muscle ◽  
Endothelial Cells ◽  
Salivary Gland ◽  
Carotid Artery ◽  
Basement Membrane ◽  
Coronary Vessels ◽  
Ruthenium Red ◽  
E Coli ◽  
Old Rats ◽  
The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

Ultra-Rapid Freezing of Isolated Skeletal Muscle Fibers

1977 ◽  
Vol 35 ◽  
pp. 576-577
Author(s):  
Joachim R. Sommer ◽  
Nancy R. Wallace
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Granular Material ◽  
Nuclear Envelope ◽  
Intracellular Calcium ◽  
Ruthenium Red ◽  
Threshold Concentration ◽  
Rapid Freezing ◽  
Frog Skeletal Muscle ◽  
Electrical Isolation

After Howell (1) had shown that ruthenium red treatment of fixed frog skeletal muscle caused collapse of the intermediate cisternae of the sarcoplasmic reticulum (SR), forming a pentalaminate structure by obi iterating the SR lumen, we demonstrated that the phenomenon involves the entire SR including the nuclear envelope and that it also occurs after treatment with other cations, including calcium (2,3,4).From these observations we have formulated a hypothesis which states that intracellular calcium taken up by the SR at the end of contraction causes the M rete to collapse at a certain threshold concentration as the first step in a subsequent centrifugal zippering of the free SR toward the junctional SR (JSR). This would cause a) bulk transport of SR contents, such as calcium and granular material (4) into the JSR and, b) electrical isolation of the free SR from the JSR.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

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