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1.
Scallop sarcoplasmic reticulum (SR), visualized in situ by freeze-fracture and deep-etching, is characterized by long tubes displaying crystalline arrays of Ca2+-ATPase dimer ribbons, resembling those observed in isolated SR vesicles. The orderly arrangement of the Ca2+-ATPase molecules is well preserved in muscle bundles permeabilized with saponin. Treatment with saponin, however, is not needed to isolate SR vesicles displaying a crystalline surface structure. Omission of ATP from the isolation procedure of SR vesicles does not alter the dimeric organization of the Ca2+-ATPase, although the overall appearance of the tubes seems to be affected: the edges of the vesicles are scalloped and the individual Ca2+-ATPase molecules are not clearly defined. The effect of Ca2+ on isolated scallop SR vesicles was investigated by correlating the enzymatic activity and calcium-binding properties of the Ca2+-ATPase with the surface structure of the vesicles, as revealed by electron microscopy. The dimeric organization of the membrane is preserved at Ca2+ concentrations where the Ca2+ binds to the high affinity sites (half-maximum saturation at pCa approximately 7.0 with a Hill coefficient of 2.1) and the Ca2+-ATPase is activated (half-maximum activation at pCa approximately 6.8 with a Hill coefficient of 1.84). Higher Ca2+ concentrations disrupt the crystalline surface array of the SR tubes, both in the presence and absence of ATP. We discuss here whether the Ca2+-ATPase dimer identified as a structural unit of the SR membrane represents the Ca2+ pump in the membrane. 相似文献
2.
Previously, we showed that incubation of the scallop sarcoplasmic reticulum (SR) with EGTA at above 37 degrees C resulted in the uncoupling of ATP hydrolysis with Ca2+ transport [Nagata et al. (1996) J. Biochem. 119, 1100-1105]. We have extended this study by comparing the kinetic behavior of Ca2+ release and binding to the uncoupled SR with that of intact scallop or rabbit SR. The change in the Ca2+ concentration in the reaction medium, as determined as the absorption of APIII, was followed using a stopped flow system. Intact scallop SR was preincubated with Ca2+ in the presence of a Ca2+ ionophore, A23187, and then ATP was added to initiate the reaction. The Ca2+ level in the medium increased to the maximum level in several seconds, and then slowly decreased to the initial low level. The rising and subsequent slow decay phases could be related to the dissociation and reassociation of Ca2+ with the Ca-ATPase, respectively. When uncoupled scallop SR vesicles were preincubated with CaCl2 in the absence of A23187 and then the reaction was initiated by the addition of ATP, a remarkable amount of Ca2+ was released from the SR vesicles into the cytosolic solution, whereas, with intact scallop or rabbit SR, only a sharp decrease in the Ca2+ level was observed. Based on these findings, we concluded that the heat treatment of scallop SR in EGTA may alter the conformation of the Ca-ATPase, thereby causing Ca2+ to be released from the enzyme, during the catalytic cycle, at the cytoplasmic surface, but not at the lumenal surface of SR vesicles. 相似文献
3.
Methods for preparing native scallop sarcoplasmic reticulum vesicles, largely purified membranous scallop sarcoplasmic reticulum Ca2+-ATPase, and nonionic detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase are described. The effect of a range of polyoxyethylene-based detergents on the solubilized Ca2+-ATPase was tested. Decaethylene glycol dodecyl ether (C12E10) supported the highest levels of activity, although C12E8 and C12E9 were more routinely used. Arrhenius plots of Ca2+-ATPase activity, where the assays were carried out with the same pH at all temperatures (7.4), showed a region of nonlinearity at 10 degrees C. A very similar plot was obtained when no compensation was made for pH variation with temperature. Both the break in the Arrhenius plot and the activation energies for the scallop sarcoplasmic reticulum above and below the break were very similar to those found for lobster sarcoplasmic reticulum (Madeira, V. M. C., Antunes-Madeira, M. C., and Carvalho, A. R. (1974) Biochem. Biophys. Res. Commun. 65, 997-1003). The Arrhenius plot of the scallop Ca2+-ATPase in C12E8 no longer showed the nonlinearity at 10-12 degrees C seen with the native sarcoplasmic reticulum, but instead a break now appeared at 20-21 degrees C. This is close to the Arrhenius break temperature of rabbit Ca2+-ATPase in C12E8 and of a perturbation in C12E8 (Dean, W. L. (1982) Biophys. J. 37, 56-57). 相似文献
4.
M Abe Y Shirakata D Sato K Konishi T Watanabe J Nakamura 《Journal of biochemistry》1992,112(6):822-827
Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year. 相似文献
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6.
A Martonosi 《Biochimica et biophysica acta》1968,150(4):694-704
7.
Ca2+ efflux was studied in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In experimental conditions in which the Ca2+ pump is reversed, the rate of Ca2? efflux varies with the ADP, orthophosphate and Mg2+ concentrations of the assay medium and is inhibited by Na+. 相似文献
8.
The three-dimensional structure of scallop sarcoplasmic reticulum membranes has been determined from electron micrographs of two classes of stain-filled tubules by helical reconstruction methods. These structures are characterized by dimer ribbons of Ca2+-ATPase molecules running diagonally around the tube wall. Deep right-handed grooves separate the ribbons. The elongated, curved units of the dimer (approximately 95 A long in the radial direction; 60 to 70 A axially, and about 30 A wide) are displaced axially by approximately 34 A and are connected at their outer ends by a bridge running nearly parallel to the tube axis. The monomers make a second contact at their inner ends. Adjacent units with the same orientation form a strong contact that is responsible for the ribbon appearance. Comparison of tubules of different diameter shows that one set of connections between the dimer ribbons is conserved: the inner ends of axially displaced dimers appear to make contact along a left-handed path almost perpendicular to the major grooves. The lipid bilayer cannot be clearly identified. The two-dimensional map obtained from flattened tubules is consistent with the three-dimensional reconstruction in showing dimer ribbons connected by a weak contact across the grooves, strongly resembling the inter-dimer bond observed in three dimensions. The two-dimensional map shows a 2-fold axis relating units of the dimer, but the three-dimensional tubes show a slight axial polarity that may arise from the presence of proteins other than the Ca2+-ATPase. 相似文献
9.
Philip Palade Christine Dettbarn Donald Brunder Philip Stein Gary Hals 《Journal of bioenergetics and biomembranes》1989,21(2):295-320
Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca2+ release are here tentatively classified as activators of a Ca2+-induced Ca2+ release channel preferentially localized in SR terminal or as likely activators of other Ca2+ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca2+ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca2+ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (sarcoballs) designed to make the pharmacological study of SR Ca2+ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered. 相似文献
10.
Characterization of sarcoplasmic reticulum from skeletal muscle 总被引:11,自引:0,他引:11
11.
C J Limas 《Biochemical and biophysical research communications》1980,95(2):541-546
Phosphatidate (PA) inhibits calcium accumulation by cardiac sarcoplasmic reticulum (SR) and enhances its Ca++ ATPase activity. These effects seem to be related to a phosphatidate-induced increase in the calcium permeability of the SR membrane with resultant calcium release. The amount of calcium released by phosphatidate is dependent both on the calcium concentration outside the SR vesicles and the internal calcium concentration. The ionophoric effects of phosphatidate on the sarcoplasmic membrane provide a novel pathway for controlling Ca++ transport in the cardiac cell. 相似文献
12.
A two-dimensional projection map was computed of the Ca2+-ATPase molecules in sarcoplasmic reticulum, isolated from rabbit skeletal muscle. Crystalline arrays of Ca2+-ATPase molecules were formed by incubating the membrane vesicles with phospholipase A2 and dialysing against Tris/HCl buffer. Ca2+-ATPase molecules appear as quasi-triangular blobs in the projection map and seem to form dimers. The projection map seems to indicate an enzyme conformation somewhat similar to vanadate-induced crystals but different from lanthanide-induced crystals of Ca2*-ATPase. 相似文献
13.
Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes 总被引:10,自引:0,他引:10
A transition in the temperature dependences of Ca2+ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca2+ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca2+ accumulation and ATPase activity are uncoupled, and Ca2+ accumulation is inhibited, while ATPase activity and passive Ca2+ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change. 相似文献
14.
Summary Ca2+-induced Ca2+ release at the terminal cisternae of skeletal sarcoplasmic reticulum was demonstrated using heavy sarcoplasmic reticulum vesicles. Ca2+ release was observed at 10 m Ca2+ in the presence of 1.25mm free Mg2+ and was sensitive to low concentrations of ruthenium red and was partially inhibited by valinomycin. These results suggest that the Ca2+-induced Ca2+ release is electrogenic and that an inside negative membrane potential created by the Ca2+ flux opens a second channel that releases Ca2+. Results in support of this formulation were obtained by applying a Cl– gradient or K+ gradient to sarcoplasmic reticulum vesicles to initiate Ca2+ release. Based on experiments the following hypothesis for the excitation-contraction coupling of skeletal muscle was formulated. On excitation, small amounts of Ca2+ enter from the transverse tubule and interact with a Ca2+ receptor at the terminal cisternae and cause Ca2+ release (Ca2+-induced Ca2+ release). This Ca2+ flux generates an inside negative membrane potential which opens voltage-gated Ca2+ channels (membrane potential-dependent Ca2+ release) in amounts sufficient for contraction. 相似文献
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16.
Summary The structural consequences of clamping the transepithelial potential difference across the toad's urinary bladder have been examined. Reducing the potential to zero (short-circuiting) produced no apparent changes in the morphology of any of the four cell types which comprise the epithelium. Computer assisted, morphometric analysis of quick frozen specimens revealed no measurable difference in granular cell volume between open- and short-circuited preparations. However, when the open-circuit potential was quantitatively reversed (serosa negative with respect to mucosa), some of the preparations showed a marked increase in granular cell volume. To examine this more systematically twelve preparations were voltage-clamped at 50 mV (serosa negative); eight of the twelve revealed prominent granular cell swelling relative to control, short-circuited preparations. Only in this group of eight had the external circuit current fallen substantially during the clamping interval. Mitochondria-rich cells were not affected detectably. Application of the diuretic amiloride prior to clamping at reversed potential prevented granular cell swelling in every case. Goblet cells which were often affected by the –50 mV clamp were not protected by the diuretic. Granular cell swelling thus appeared to be dependent on sodium entry at the mucosal surface. We also observed that, after voltage reversal, the apical tight junctions of the bladders were blistered as they are with hypertonic mucosal media. This blistering was associated with an increase in passive ionic permeability and was not prevented by application of amiloride. This finding is consistent with the evidence that the junction is a complex barrier with asymetric, and hence, rectifying properties for intrinsic ionic conductance as well as hydraulic permeability. These findings, together with others from the literature, lead to the conclusion that the granular cells constitute the principal, if not sole, elements for active sodium transport across toad urinary bladder and that they swell when sodium entry exceeds the transport capacity of the pump at the basal-lateral surface. 相似文献
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18.
S T Ohnishi 《Journal of biochemistry》1979,86(4):1147-1150
A new method is introduced which allows the study of calcium-induced calcium release from fragmented sarcoplasmic reticulum. Results obtained with this method are in agreement with those obtained by previous investigators using skinned muscle fiber. It was also found that anesthetic drugs and alcohol increased the calcium- and caffeine-induced calcium release from the sarcoplasmic reticulum. 相似文献
19.
Rapid Ca2+ release from the sarcoplasmic reticulum (SR) can be triggered by either binding of heavy metals to a sulfhydryl (SH) group or by catalyzing the oxidation of endogenous groups to a disulfide. Ca2+ release has been monitored directly using isolated vesicle preparations or indirectly by monitoring phasic contractions in a skinned fiber preparation. SH oxidation triggered by addition of Cu2+ /mercaptans, phthalocyanine dyes, reactive disulfides, and various anthraquinones appears to involve a direct interaction with the Ca2+ release protein from the SR. A model is presented in which reversible oxidation and reduction of endogenous SH groups results in the opening and closing of the Ca2+ release channel from the SR.Abbreviations SR
sarcoplasmic reticulum
- SH
sulfhydryl
- T-tubule
transverse tubule
- 2,2-DTDP
2,2-dithiodipyridine
- 4,4-DTDP
4,4-dithiodipyridine
- DTT
dithiothreitol 相似文献
20.
In this article the morphology of sarcoplasmic reticulum, classification of Ca(2+)-ATPase (SERCA) isoenzymes presented in this membrane system, as well as their topology will be reviewed. The focus is on the structure and interactions of Ca(2+)-ATPase determined by electron and X-ray crystallography, lamellar X-ray and neutron diffraction analysis of the profile structure of Ca(2+)-ATPase in sarcoplasmic reticulum multilayers. In addition, targeting of the Ca(2+)-ATPase to the sarcoplasmic reticulum is discussed. 相似文献