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.

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 Effects of actin filament cross-linking and filament length on actin-myosin interaction.

1985 ◽  
Vol 101 (5) ◽  
pp. 1850-1857 ◽  
Author(s):  
T R Coleman ◽  
M S Mooseker
Keyword(s):  
Atpase Activity ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Filament Length ◽  
Maximum Enhancement ◽  
Actomyosin Atpase ◽  
Cross Linker

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration-dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2-25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.

Effect of taxol on the interaction of tubulin with myosin filaments

1984 ◽  
Vol 62 (9) ◽  
pp. 878-884 ◽  
Author(s):  
Toshihiro Fujii ◽  
Tatsuo Suzuki ◽  
Akira Hachimori ◽  
Michiyo Fujii ◽  
Yoshiyuki Kondo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Atpase Activity ◽  
Cross Section ◽  
Molar Ratio ◽  
Tubulin Polymerization ◽  
Myosin Filaments ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The interaction between polymerized tubulin from porcine brain and myosin from rabbit skeletal muscle was examined. The addition of myosin to the solution of tubulin polymerized by taxol resulted in a remarkable increase in turbidity within a few minutes at 37 °C, and a dense and stable precipitate was formed. The maximal molar ratio of tubulin bound to myosin was calculated to be about 4, while the value was about 2 when 6S tubulin was used. Both podophyllotoxin and colchicine suppressed the taxol-dependent increase of the binding of tubulin to myosin, but only when they were preincubated with tubulin prior to addition of taxol. 6S tubulin inhibited with aetin-activated Mg2+-ATPase activity of myosin, and polymerized tubulin inhibited the Mg-ATPase more than 6S tubulin. Dense precipitates of tubulin and myosin were observed by thin-section electron microscopy. Microtubules were observed to be entangled in myosin filaments and single microtubules were occasionally surrounded by five myosin filaments in a cross section, similar to actin–myosin arrays in muscle. After incubation of tubulin with myosin, taxol was able to induce tubulin polymerization in the same way as it polymerized microtubules in the absence of myosin.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

scholarly journals A model for the uptake and release of Ca2+ by sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 739-749 ◽  
Author(s):  
G W Gould ◽  
J M McWhirter ◽  
J M East ◽  
A G Lee
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
External Medium ◽  
Spontaneous Release ◽  
Calculated Concentration ◽  
Effects Of Ph ◽  
Hydrolysis Of ◽  
Uptake And Release

On addition of ATP to vesicles derived from the sarcoplasmic reticulum (SR) of skeletal muscle, Ca2+ is accumulated from the external medium. Following uptake, spontaneous release of Ca2+ occurs in the presence or in the absence of ATP. These processes of Ca2+ uptake and release were simulated by using the models derived for ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227; Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730] and for Ca2+ release from passively loaded vesicles [McWhirter, Gould, East & Lee (1987) Biochem. J. 245, 713-722]. The simulations are consistent with measurements of the effects of pH, K+, Ca2+ and Mg2+ on uptake and release of Ca2+. The increase in maximal Ca2+ accumulation observed in the presence of maleate is explained in terms of complexing of Ca2+ and maleate within the SR. The calculated concentration of ADP generated by hydrolysis of ATP has a large effect on the simulations. The effects of an ATP-regenerating system on the measured Ca2+ uptake is explained in terms of both removal of ADP and precipitation of Ca3(PO4)2 within the vesicles. It is concluded that both the process of Ca2+ uptake and the process of Ca2+ release seen with SR vesicles can be interpreted quantitatively in terms solely of the properties of the Ca2+ + Mg2+-activated ATPase.

Effects of prolonged low frequency stimulation on skeletal muscle sarcoplasmic reticulum

1995 ◽  
Vol 73 (8) ◽  
pp. 1154-1164 ◽  
Author(s):  
E. R. Chin ◽  
H. J Green ◽  
F. Grange ◽  
J. Dossett-Mercer ◽  
P. J. O'Brien
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Metabolic Response ◽  
Contractile Activity ◽  
Creatine Phosphate ◽  
Energy Status ◽  
Muscle Lactate ◽  
Muscle Energy

The role of prolonged electrical stimulation on sarcoplasmic reticulum (SR) Ca2+sequestration measured in vitro and muscle energy status in fast white and red skeletal muscle was investigated. Fatigue was induced by 90 min intermittent 10-Hz stimulation of rat gastrocnemius muscle, which led to reductions (p < 0.05) in ATP, creatine phosphate, and glycogen of 16, 55, and 49%, respectively, compared with non-stimulated muscle. Stimulation also resulted in increases (p < 0.05) in muscle lactate, creatine, Pi, total ADP, total AMP, IMP, and inosine. Calculated free ADP (ADPf) and free AMP (AMPf) were elevated 3- and 15-fold, respectively. No differences were found in the metabolic response between tissues obtained from the white (WG) and red (RG) regions of the gastrocnemius. No significant reductions in SR Ca2+ATPase activity were observed in homogenate (HOM) or a crude SR fraction (CM) from WG or RG muscle following exercise. Maximum Ca2+uptake in HOM and CM preparations was similar in control (C) and stimulated (St) muscles. However, Ca2+uptake at 400 nM free Ca2+was significantly reduced in CM from RG (0.108 ± 0.04 to 0.076 ± 0.02 μmol∙mg−1protein∙min−1in RG–C and RG–St, respectively). Collectively, these data suggest that reductions in muscle energy status are dissociated from changes in SR Ca2+ATPase activity in vitro but are related to Ca2+uptake at physiological free [Ca2+] in fractionated SR from highly oxidative muscle. Dissociation of SR Ca2+ATPase activity from Ca2+uptake may reflect differences in the mechanisms evaluated by these techniques.Key words: sarcoplasmic reticulum, contractile activity, Ca2+sequestration, energy status, red and white gastrocnemius.

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.

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