scholarly journals The mechanism of inhibition of the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde

1996 ◽  
Vol 317 (2) ◽  
pp. 439-445 ◽  
Author(s):  
Yamin M. KHAN ◽  
Anthony P. STARLING ◽  
J. Malcolm EAST ◽  
Anthony G. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Order Rate Constant ◽  
Molar Ratio ◽  
Slow Step ◽  
Phosphoryl Transfer ◽  
Back Reaction ◽  
Mechanism Of Inhibition ◽  
Fluorescence Change ◽  
Similar Conformation

Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ratio of label to ATPase. The affinity of the ATPase for Ca2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissociation of Ca2+ from the Ca2+-bound ATPase is also unaffected and Mg2+ increases the rate of dissociation, as for the unlabelled ATPase. Effects of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ binding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The rate of phosphorylation is independent of the concentration of ATP above 25 μM ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction between phosphorylated ATPase and ADP is little affected, suggesting that the slow step in phosphorylation could be the slow conformation step before phosphoryl transfer. The rate of dephosphorylation of the phosphorylated ATPase is also decreased, suggesting that a similar conformation change could be involved in the dephosphorylation step. The rate of the Ca2+ transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predominantly in a Ca2+-free, phosphorylated form at steady state in the presence of ATP.

Sarcolipin uncouples hydrolysis of ATP from accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum

2002 ◽  
Vol 361 (2) ◽  
pp. 277-286 ◽  
Author(s):  
Wendy S. SMITH ◽  
Robert BROADBRIDGE ◽  
J. Malcolm EAST ◽  
Anthony G. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Fatty Acyl ◽  
Molar Ratio ◽  
Fluorescence Emission Spectra ◽  
Α Helix ◽  
Hydrolysis Of

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane α-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C22 or C24) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca2+-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca2+ by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca2+ in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca2+ can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca2+ mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

scholarly journals Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin

1992 ◽  
Vol 283 (2) ◽  
pp. 525-529 ◽  
Author(s):  
M Wictome ◽  
I Henderson ◽  
A G Lee ◽  
J M East
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Dissociation Constant ◽  
State Level ◽  
Calcium Pump ◽  
Molar Ratio ◽  
Steady State Level ◽  
Mechanism Of Inhibition ◽  
Nanomolar Range

The steady-state ATPase activity of sarcoplasmic-reticulum (Ca(2+)-Mg2+)-ATPase is inhibited by thapsigargin at a molar ratio of 1:1, with a dissociation constant for thapsigargin estimated to be in the sub-nanomolar range. In the presence of thapsigargin, only a single Ca2+ ion binds to the ATPase. Similarly, addition of thapsigargin to the ATPase incubated in the presence of Ca2+ results in the release of one of the two originally bound Ca2+ ions. As monitored by the fluorescence of nitrobenzo-2-oxa-1,3-diazole-labelled ATPase, thapsigargin appears to shift the transition between E1 and E2 conformations towards E2. Addition of thapsigargin prevents phosphorylation of the ATPase by P(i) and results in a very low steady-state level of phosphorylation of the ATPase by ATP, as observed previously for nonylphenol.

scholarly journals Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with the cross-linker o-phthalaldehyde

1996 ◽  
Vol 317 (2) ◽  
pp. 433-437 ◽  
Author(s):  
Yamin M. KHAN ◽  
Matthew WICTOME ◽  
Malcolm EAST ◽  
Anthony G. J. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Fluorescence Emission ◽  
Molar Ratio ◽  
Cross Linking ◽  
Fluorescence Emission Spectrum ◽  
Cross Link ◽  
Subsequent Reaction ◽  
Cross Linker

The Ca2+-ATPase in the sarcoplasmic reticulum of skeletal muscle reacts with o-phthalaldehyde (OPA) to form a fluorescent isoindole product. The stoichiometry of labelling of the ATPase is 9 nmol of isoindole/mg of ATPase, corresponding to a 1:1 molar ratio of isoindole:ATPase. There is no evidence for any intermolecular cross-linking. Isoindole formation is faster in the presence of methylamine, but the stoichiometry of labelling is unchanged, whereas in the presence of 2-mercaptoethanol the level of labelling is much higher. It is concluded that OPA reacts with a single Cys residue (defining the specificity of the reaction) in a fast step, subsequent reaction with a Lys residue to form the isoindole being rate-controlling. Labelling the ATPase with OPA in the absence of methylamine leads to total loss of ATPase activity, whereas in the presence of methylamine, the decrease in ATPase activity on reaction is small. We conclude that the loss of ATPase activity probably follows from formation of the intramolecular cross-link rather than from the initial modification of the Cys residue. Reaction with OPA is not affected by the presence of ATP, ADP or Ca2+, so that the reactive Cys is not part of a ligand-binding site. The fluorescence emission spectrum of the labelled ATPase indicates a hydrophobic environment for the isoindole ring.

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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