Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation (original) (raw)
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23492 Orientation of Mg 2 +-ATPase in Transverse Tubul
2001
The orientation of the enzyme Mg"-ATPase (EC 3.6.1.3) in the transverse tubule (TT) membranes of skeletal muscle was investigated using highly purified chicken and rabbit TT vesicles. The percentage of sealed vesicles present in these preparations averaged 88 and 78%, respectively, as calculated from the detergent-induced increase in ouabain-sensitive (Na+,K+)ATPase activity, ATP-dependent ouabain binding, and lactate dehydrogenase activity (sarcoplasmic enzyme trapped in the TT vesicles). Sidedness of the sealed vesicles, estimated from latency of 5'-nucleotidase, acetylcholinesterase, and adenylate cyclase, was predominantly right-side out (69-76%, chicken TT and 62-70%, rabbit TT). In both chicken and rabbit native vesicles, high Mg2+-ATPase activity was detected by addition of ATP to the extravesicular medium; this activity was increased 14-12% by alamethicin pointing to the external ocalization of the active site. Furthermore, the enzymatic activity resulted partially ...
The presence of calcium stimulated adenosine triphosphatase (Ca2+,Mg2+-ATPase) activity in isolated transverse tubule (t-tubule) membranes is distinguished from magnesium adenosine triphosphatase (Mg2+-ATPase) activity on the basis of differing thermal stabilities. The Mg2+-ATPase is the major protein component of the t-tubule membrane, and it can be difficult to discriminate between the low levels of Ca 2÷ stimulated ATPase activity found in isolates of t-tubules compared to the much higher Mg2+-ATPase activity. Thermal analysis reveals different inactivation temperatures (T i) for the proteins responsible for ATP dependent calcium transport (T i = 49°C) and Mg2+-ATPase activity (T i = 57°C) in isolated t-tubule membranes. The differential scanning calorimetry profile of t-tubule membranes consists of three major components with transition temperatures (T m) of 51°C, 57°C and 63°C. Denaturation of the component with T m = 57°C correlates with inactivation of Mg2+-ATPase activity, and denaturation of the T m = 51°C component correlates with the inactivation of Ca2+,Mg2+-ATPase activity and calcium transport. The functions of the t-tubule membrane component or components that denature with T m = 63°C have yet to be identified. The lack of stimulation of calcium transport in isolated t-tubules by oxalate, the impermeability of isolated t-tubules to oxalate, and experiments performed on t-tubules with defined amounts of sarcoplasmic reticulum (SR) added suggest that contamination of the isolated t-tubules by SR is unlikely to account for the level of Ca2+,Mg2÷-ATPase activity detected. The presence of a Ca2+,Mg2+-ATPase in the t-tubule membrane would provide a mechanism that may be involved in the partial removal of calcium that is accumulated in the junctional space during muscle relaxation or calcium that is released from the terminal cisternae of sarcoplasmic reticulum during excitation-contraction coupling.
Uptake at Ca2+ mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1987
The (Ca2+ + MgZ+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Seplmdex G-50. The level of Ca 2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca 2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidyicholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles recoltstituted with dimyristoleoyl-or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca 2+ accumulated increased with increasing external K + concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca 2+ accumulation follows from an effect on the rate of Ca 2+ efflux mediated by the ATPase.
Effects of phospholipids on the function of the calcium-magnesium ATPase
Biochemistry, 1991
T h e ATPase activity for the (Ca2'-Mg2')-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [ (C 1 4 : I) P C ] than when it is reconstituted into dioleoylphosphatidylcholine [ (C l 8 : 1) P C ]. The rate of formation of phosphoenzyme on addition of A T P is slower for (C14:l)PC-ATPase than for the native ATPase or (C18:l)PC-ATPase. T h e reduction in rate of phosphoenzyme formation is attributed to a reduction
Characterization of transverse tubule membrane proteins: Tentative identification of the Mg-ATPase*1
Archives of Biochemistry and Biophysics, 1985
Vesiculated fragments of chicken skeletal muscle transverse tubule (TT) membranes were analyzed for their content of loosely associated and integral membrane proteins. Of particular interest was the identification of the magnesium-stimulated ATPase (Mg-ATPase), which is characteristically located in native isolated TT vesicles of chicken skeletal muscle [ R. A. Sabbadini and V. R. Okamoto (1983) Arch. B&hem, Biophys. 223, 10'7-1191. A number of the proteins found in vesicular TT preparations were found to be extractable by a mild Triton-X106 treatment and were identified as aldolase, enolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and pyruvate kinase. Approximately 60% of TT-associated protein was extracted with Triton, resulting in a twofold enrichment of the Mg-ATPase. Concommitantly, one core integral membrane protein possessing a M, of 102,000 was enriched, suggesting that it is responsible for the Mg-ATPase activity present in chicken skeletal muscle TT membranes.
Molecular and Cellular Biochemistry, 1988
Rat testicular microsomal membrane fraction contains both Mg+2-dependent and Mg+2-independent Ca +2-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg+2-independent Ca+2-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37 ~ and for latter one it is 40 ~ Among all the nuclestides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn +2, Ba +2 and Mn +2 have been found to inhibit Mg +2dependent Ca+2-ATPase activity whereas Mg+2-independent Ca+2-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadata, EDTA and EGTA. I50, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydral groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.
The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
(1) Media prepared with CDTA and low concentrations of Ca 2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na++ K +)-ATPase preparations are free of contaminant Mg 2 +. (2) In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. (3) In the absence of Mg 2+ the rate of phosphorylation in the presence of 1/~M Ca 2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca 2+ is increased to 5 mM. (4) The Kca for phosphorylation is 2/~M in the presence and 60/~M in the absence of Mg 2+. (5) Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca 2+ at the transport site and Mg 2+ at an activating site and that Ca 2+ replaces Mg 2+ at this site. (6) Under conditions in which it increases the rate of phosphorylation, Ca 2 + is without effect on the Ca2+-ATPase activity in the absence of Mg 2+ suggesting that to stimulate ATP hydrolysis Mg 2+ accelerates a reaction other than phosphorylation. (7) Activation of the EIP)E2P reaction by Mg 2+ is prevented by Ca 2+ after but not before the synthesis of EIP from E l and ATP, suggesting that Mg 2+ stabilizes E 1 in a state from which Mg 2+ cannot be removed by Ca 2+ and that Ca 2+ stabilizes EIP in a state insensitive to Mg 2+. (8) The response of the Ca2+-ATPase activity to Mg 2+ concentration is biphasic, activation with a KMg = 88 #M is followed by inhibition with a K i = 9.2 mM. Ca 2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg 2+, and Mg 2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca 2 + at the transport site of the Ca 2 +-ATPase.