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1.
Ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat skeletal muscle by immunoferritin labeling of ultrathin frozen sections 总被引:9,自引:6,他引:3
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The ultrastructural localization of the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum in rat gracilis muscle was determined by indirect immunoferritin labeling of ultrathin frozen sections. Simultaneous visualization of ferritin particles and of adsorption- stained cellular membranes showed that the Ca2+ + Mg2+-ATPase was concentrated in the longitudinal sarcoplasmic reticulum and in the nonjunctional regions of the terminal cisternae membrane but was virtually absent from mitochondria, plasma membranes, transverse tubules, and junctional sarcoplasmic reticulum. Ferritin particles were found preponderantly on the cytoplasmic surface of the membrane, in agreement with published data showing an asymmetry of the Ca2+ + Mg2+- ATPase within the sarcoplasmic reticulum membrane. Comparison of the density of ferritin particles in fast and slow myofibers suggested that the density of the Ca2+ + Mg2+-ATPase in the sarcoplasmic reticulum membrane in a fast myofiber is approximately two times higher than in a slow myofiber. 相似文献
2.
M V Mikha?lova E Kh Safarian V P Nesterov 《Zhurnal evoliutsionno? biokhimii i fiziologii》1987,23(2):180-185
Using differential centrifugation in sucrose density gradient, from muscles of the frog fractions were obtained which contain fragments of sarcolemma, as well as membranes of T-system tubules and sarcoplasmic reticulum. In isolated membrane fractions, studies were made on the activity of cation-stimulated ATPases (Na+, K+-, Ca2+, Mg2+- and Mg2+-ATPases). Enzymic and electrophoretic analyses showed that the highest content of Mg2+-ATPases is typical of the fractions which are located on the surface of 35% sucrose. The data obtained indicate that Mg2+-ATPase is the enzyme which is specific for the membranes of T-system tubules in skeletal muscles of not only birds but amphibians as well. From cardiac muscle of the frog, membrane fraction was isolated which is similar (with respect to its predominant content of Mg2+-ATPase) to the membranes of T-system tubules. It is suggested that the presence of Mg2+-ATPase in these membranes is a common property of phasic striated muscle fibers in all mature vertebrate animals. 相似文献
3.
Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle 总被引:2,自引:0,他引:2
Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed. 相似文献
4.
A modified protocol for isolation of transverse tubules incorporated an extra stage of purification. The existence of an ATP-energized Ca2+ pump in transverse tubules isolated from rabbit skeletal muscle has been demonstrated. Isolated transverse tubules had a Ca-ATPase activity of 0.78 mu mol/min . mg; this was 300% in excess of that activity attributable to sarcoplasmic reticulum contamination. The distribution of part of the CaATPase activity and ATP-energized Ca2+ uptake coincided with the distribution of transverse tubules in isopycnic sucrose gradients loaded with mechanically disrupted triad junctions. Transverse tubules accumulated over 70 nmol of Ca2+/mg of protein; this uptake was abolished by the Ca2+ ionophore A23187. Neither digitoxin nor monensin inhibited Ca2+ uptake, indicating that Ca2+ accumulation did not occur through a sodium/calcium exchange. Conditions for half-maximal Ca2+ uptake were 5 micro M free Ca2+ and 10 micro M ATP. The Ca2+ pump of isolated transverse tubules was distinguished from the Ca2+ pump of sarcoplasmic reticulum and sarcolemma in that the transverse tubule Ca2+ pump: 1) was not enhanced by oxalate; 2) was not energized by acetyl phosphate, p-nitrophenyl phosphate, or 3-O-methylfluorescein phosphate; and 3) did not hydrolyze p-nitrophenyl phosphate or 3-O-methyl-fluorescein phosphate. Using Ca2+-dependent 3-O-methylfluorescein phosphatase as a marker for sarcoplasmic reticulum, the contamination of the transverse tubule preparation was calculated to be 6%. This agreed with a contamination level of 5% estimated by freeze-fracture electron microscopy. 相似文献
5.
The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases. 相似文献
6.
The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different. 相似文献
7.
K Ohlendieck F N Briggs K F Lee A W Wechsler K P Campbell 《European journal of biochemistry》1991,202(3):739-747
The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle. 相似文献
8.
Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel. 总被引:1,自引:0,他引:1
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The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites. 相似文献
9.
The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle 总被引:7,自引:3,他引:4
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《The Journal of cell biology》1993,120(4):969-980
The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T- tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma. 相似文献
10.
An enzymatically active Ca2+-stimulated ATPase has been isolated from the sarcolemmal sheets of human smooth muscle (myometrium). Ca2+-ATPase activity was quantitated in an assay medium which simulated the characteristic free ionic concentrations of the cytosol. New computer programs for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability constants, were used. In detergent-soluble form the enzyme has a high Ca2+-affinity expressed by an apparent Km (Ca2+) of 0.25 +/- 0.04 microM. The maximum specific activity (about 20 nmol of Pi/mg protein/min) was found in the micromolar domain of free-Ca2+ concentrations, the same levels required for normal maximal contractions in smooth muscle. The variation of free-Ca2+ concentration in the assay medium over 4 orders of magnitude (pCa 9 to pCa 5) resulted in a sigmoidal dependence of enzymatic activity, with a Hill coefficient of 1.4, which suggested the regulation of Ca2+-ATPase by allosteric effectors. The presence and the activator role of endogenous calmodulin in smooth muscle sarcolemma was proved by calmodulin-depletion experiments and by using suitable anticalmodulinic concentrations of trifluoperazine. The addition of exogenous calmodulin restored the enzyme activity. Apparently, the concentration of calmodulin in isolated smooth muscle sarcolemma is about 0.1% of sarcolemmal proteins, as deduced from the comparison of calmodulin-depletion and calmodulin-readdition experiments. Calmodulin increased significantly the enzyme Ca2+-affinity and Vmax (by a factor of about 10). At variance with the sarcoplasmic reticulum Ca2+-ATPase, the sarcolemmal Ca2+-ATPase is extremely sensitive to orthovanadate, half-maximal inhibition being observed at 0.8 microM vanadate. In conclusion, the Ca2+-ATPase isolated from smooth muscle sarcolemma appears very similar to the well-known Ca2+-pump ATPases of erythrocyte membrane, heart sarcolemma or axolemma. We suggest that this high-affinity Ca2+-ATPase represents the calmodulin-regulated Ca2+-extrusion pump of the smooth muscle sarcolemma. 相似文献
11.
Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle 总被引:16,自引:0,他引:16
M Rosemblatt C Hidalgo C Vergara N Ikemoto 《The Journal of biological chemistry》1981,256(15):8140-8148
A new method for isolating transverse tubule membranes from rabbit skeletal muscle has been developed. This procedure has the advantage of being mild, fast, and producing with good yields a purified membrane fraction. The transverse tubule membranes are purified by a discontinuous sucrose density centrifugation after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. Immunofluorescence staining of cryostat sections of rabbit psoas muscle with purified goat antibodies directed against the purified membranes shows that the reacting antigens are distributed at the boundary of the A and I bands of the myofibrils where transverse tubules are localized in mammalian muscle. The purified antibodies showed no cross-reactivity with sarcoplasmic reticulum, nor did they show any fluorescence staining of the muscle plasma membrane, indicating that the isolated membranes indeed originate from the transverse tubules. The transverse tubule fraction has a characteristic protein composition distinguishable from that of sarcoplasmic reticulum, a much higher cholesterol content than that of the crude microsomes, plasma membrane, and sarcoplasmic reticulum, and a phospholipid content about twice as high as that of sarcoplasmic reticulum and plasma membrane. The purified transverse tubule membrane has a distinct phospholipid composition with high contents of sphingomyelin and phosphatidylserine. A Mg2+-activated ATPase characteristic of the transverse tubule fraction undergoes a 20-30-fold increase in specific activity during purification. The levels of Ca2+-ATPase activity present in the purified transverse tubule fraction remain comparable to those of sarcoplasmic reticulum even after extensive removal of the latter. 相似文献
12.
Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion 总被引:6,自引:0,他引:6
Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals. 相似文献
13.
P Fernandez-Salguero F Henao J Laynez C Gutierrez-Merino 《Biochimica et biophysica acta》1990,1022(1):33-40
The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme. 相似文献
14.
The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase). 相似文献
15.
CARDIAC MUSCLE : A Comparative Study of Purkinje Fibers and Ventricular Fibers 总被引:30,自引:12,他引:18
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With light and electron microscopy a comparison has been made of the morphology of ventricular (V) and Purkinje (P) fibers of the hearts of guinea pig, rabbit, cat, dog, goat, and sheep. The criteria, previously established for the rabbit heart, that V fibers are distinguished from P fibers by the respective presence and absence of transverse tubules is shown to be true for all animals studied. No evidence was found of a permanent connection between the sarcoplasmic reticulum and the extracellular space. The sarcoplasmic reticulum (SR) of V fibers formed couplings with the sarcolemma of a transverse tubule (interior coupling) and with the peripheral sarcolemma (peripheral coupling), whereas in P fibers the SR formed only peripheral couplings. The forms of the couplings were identical. The significance, with respect to excitation-contraction coupling, of the difference in the form of the couplings in cardiac versus skeletal muscle is discussed together with the electrophysiological implications of the differing geometries of bundles of P fibers from different animals. 相似文献
16.
(Ca2+ + Mg2+)-dependent ATPase mRNA from smooth muscle sarcoplasmic reticulum differs from that in cardiac and fast skeletal muscles 总被引:6,自引:0,他引:6
We have investigated some characteristics of the sarcoplasmic reticulum (Ca2+ + Mg2+)-dependent ATPase (Ca2+-ATPase) mRNA from smooth muscle using specific cDNA probes isolated from a rat heart cDNA library. RNA blot analysis has shown that the Ca2+-ATPase mRNA expressed in smooth muscle is identical in size to the cardiac mRNA but differs from that of fast skeletal muscle. S1 nuclease mapping has moreover shown that the cardiac and smooth muscle isoforms possess different 3'-end sequences. These results indicate that a distinct sarcoplasmic reticulum Ca2+-ATPase mRNA is present in smooth muscle. 相似文献
17.
Is a guanine nucleotide-binding protein involved in excitation-contraction coupling in skeletal muscle? 总被引:2,自引:1,他引:1
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Plasma membrane depolarization causes skeletal muscle contraction by triggering Ca2+ release from an intracellular membrane network, the sarcoplasmic reticulum. A specialized portion of the sarcoplasmic reticulum, the terminal cisternae, is junctionally associated with sarcolemmal invaginations called the transverse tubules, but the mechanism by which the action potential at the level of the transverse tubules is coupled to Ca2+ release from the terminal cisternae is still mysterious. Here we show that: (i) GTP gamma S, a non-hydrolyzable analog of GTP, elicits isometric force development in skinned muscle fibre; (ii) GTP gamma S is unable to release CA2+ from isolated sarcoplasmic reticulum fractions; (iii) the threshold for tension development is shifted to higher GTP gamma S concentrations by pre-incubation with pertussis toxin. These results suggest that a GTP-binding protein is involved in coupling the action potential of transverse tubules to Ca2+ release from the terminal cisternae. 相似文献
18.
Long-term stabilization and crystallization of (Ca2+ + Mg2+)-ATPase of detergent-solubilized erythrocyte plasma membrane. 总被引:1,自引:0,他引:1
Conditions which were optimal for the stabilization of Ca2(+)-transporting ATPase in solubilized sarcoplasmic reticulum membranes (Piku?la, S., Mullner, N., Dux, L. and Martonosi, A. (1988) J. Biol. Chem. 263, 5277-5286) were also found conducive for preservation of (Ca2+ + Mg2+)-ATPase activity in detergent-solubilized erythrocyte plasma membrane for up to 60 days. Of particular importance for the stabilization of calmodulin-stimulated Ca2(+)-dependent activity of (Ca2+ + Mg2+)-ATPase of solubilized erythrocyte plasma membrane was the presence of Ca2+ (10-20 mM), glycerol, anti-oxidants, proteinase inhibitors and appropriate detergents. Among eight detergents tested octaethylene glycol dodecyl ether, polyoxyethylene glycol(10) lauryl alcohol and polydocanol were found to be promotive in long-term preservation of the enzyme activity. Under these conditions (Ca2+ + Mg2+)-ATPase of erythrocyte ghosts became highly stable and developed microcrystalline arrays after storage for 35 days. Electron micrographs of the negatively stained and thin sectioned material indicated that crystals of purified, detergent-solubilized, lipid-stabilized erythrocyte (Ca2+ + Mg2+)-ATPase differ from those of Ca2(+)-ATPase of detergent-solubilized sarcoplasmic reticulum microsomes. 相似文献
19.
Ca2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers 总被引:5,自引:4,他引:1
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Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19 degrees C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/H+ exchanger, or gramicidin D, an H+ greater than K+ greater than Na+ channel-former, evoked rapid force development and stimulated release of approximately 30% of the accumulated 45Ca within 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+-specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Ca efflux. 5 min pretreatment of the skinned fibers with 50 microM digitoxin, a permeant glycoside that specifically inhibits the Na+,K+ pump, inhibited monensin and gramicidin D stimulation of 45Ca efflux; similar pretreatment with 100 microM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: monensin and gramicidin D stimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+,K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; the transverse tubule-mediated stimulation of 45Ca efflux appears to be entirely Ca2+ dependent. 相似文献
20.
The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum. 相似文献