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1.
Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle.Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca 2+/μl of water space. After KCl washing both contained less than 4 nmol Ca 2+/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca 2+ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca 2+ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca 2+-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca 2+-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’. 相似文献
2.
The effect of taurine on ATP-dependent calcium transport was examined in guinea-pig cardiac ventricle homogenates and in microsomal preparations enriched in sarcoplasmic reticulum. Taurine (5?50 mM) did not affect ATP-dependent calcium binding or uptake in either of these preparations or alter the rate of decay of calcium uptake activity. Taurine (20 mM) also did not affect the oxalate-dependent calcium uptake stimulation noted in the presence of cyclic AMP-dependent protein kinase and cyclic AMP. The mechanism by which taurine alters cardiac function remains to be elucidated. 相似文献
3.
Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose gradients (27-32-34-38-45%, wt/wt). All the fractions were characterized in terms of calcium content, Ca 2+/Mg 2+-ATPase activity, and radioligand binding of [ 3H]-PN 200-110 and [ 3H]ryanodine, specific to dihydropyridine-sensitive calcium channels and ryanodine receptors, respectively. Gradient fractions of muscles stimulated for 24 h underwent acute changes in the pattern of protein bands. First, light fractions from longitudinal sarcoplasmic reticulum, enriched in Ca 2+-ATPase activity, R 1 and R 2, were greatly reduced (67% and 51%, respectively); this reduction was reflected in protein yield of crude microsomal fractions prior to gradient loading (25%). Second, heavy fractions from the sarcoplasmic reticulum were modified, and part (52%) of the R 3 fraction was shifted to the R 4 fraction, which appeared as a thick, clotted band. Quantification of [ 3H]-PN 200-110 and [ 3H]-ryanodine binding revealed co-migration of terminal cisternae and t-tubules from R 3 to R 4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may be part of a physiological response to fatigue. 相似文献
4.
Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G 1 form. 相似文献
5.
Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose gradients (27-32-34-38-45%, wt/wt). All the fractions were characterized in terms of calcium content, Ca2+/Mg2+-ATPase activity, and radioligand binding of [3H]-PN 200-110 and [3H]ryanodine, specific to dihydropyridine-sensitive calcium channels and ryanodine receptors, respectively. Gradient fractions of muscles stimulated for 24 h underwent acute changes in the pattern of protein bands. First, light fractions from longitudinal sarcoplasmic reticulum, enriched in Ca2+-ATPase activity, R1 and R2, were greatly reduced (67% and 51%, respectively); this reduction was reflected in protein yield of crude microsomal fractions prior to gradient loading (25%). Second, heavy fractions from the sarcoplasmic reticulum were modified, and part (52%) of the R3 fraction was shifted to the R4 fraction, which appeared as a thick, clotted band. Quantification of [3H]-PN 200-110 and [3H]-ryanodine binding revealed co-migration of terminal cisternae and t-tubules from R3 to R4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may be part of a physiological response to fatigue. 相似文献
6.
On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca 2+-ATPase is selectively extracted while approximately half of the initial Mg 2+-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca 2+-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg 2+-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca 2+-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg 2+-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca 2+-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures. 相似文献
7.
The relationship between sarcoplasmic reticulum phospholipid and Ca(2+) binding by sarcoplasmic reticulum membranes was explored. Ca(2+) bound in the absence of ATP was defined as "ATP-independent Ca(2+) binding," and the additional amount of Ca(2+) bound in the presence of ATP was defined as "ATP-dependent Ca(2+) binding." The latter was found to be very sensitive to the loss of sarcoplasmic reticulum phospholipid; the amount of Ca(2+) bound was reduced when as little as 3% of the phospholipid was destroyed by phospholipase C. Further destruction of membrane phospholipid up to a 40% loss caused little or no further reduction of this Ca(2+) binding. However, when the destruction of phospholipid exceeded 40%, further loss of this Ca(2+) binding occurred, and there was an almost complete loss of this function when more than 60% of the sarcoplasmic reticulum phospholipid was destroyed. 相似文献
8.
To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma. 相似文献
9.
Cardiac microsomes represent a heterogeneous fraction which contains mitochondrial, plasma membrane and lysosomal enzymes in addition to markers believed to originate in the sarcoplasmic reticulum. The exact composition of this fraction depends on the method of preparation in that prolonged homogenization of ventricular myocardium increases both the yield of microsomal protein and the proportion of the mitochondrial contaminant. Ultracentrifugation of cardiac microsomes on density gradients made with sucrose alone is of limited value in isolating fragmented sarcoplasmic reticulum. Because of aggregation of the microsomes, zonal ultracentrifugation in sucrose permits isolation of material with only slight enhancement in the activity of markers for the sarcoplasmic reticulum. In the presence of LiBr, used under conditions which inhibit the damaging effects of this salt on the activities studied, aggregation of the microsomal fraction is reduced and density gradient fractionation is more effective. The fragmented sarcoplasmic reticulum prepared by zonal centrifugation in 0.5 M LiBr contains less than 1/5 the level of mitochondrial enzymes found in the original microsomes while the rate of Ca2+ uptake is enhanced 2-fold and the extent of Ca2+ uptake is enhanced 4-fold over that in the crude microsomal fraction. The sarcoplasmic reticulum markers were concentrated in a region of the gradient containing approx. 5% of the original protein that did not correspond to an obvious protein peak. 相似文献
10.
Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na + + K +)-ATPase, a plasma membrane marker enzyme, and not with Ca 2+-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane. 相似文献
11.
The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development. 相似文献
12.
Protein activator of cyclic nucleotide phosphodiesterase from bovine brain (9 μg/0.5 ml) was found to stimulate ATP-dependent Ca 2+ - transport in dog cardiac microsomal preparations enriched in sarcoplasmic reticulum. Both oxalate-dependent calcium uptake and ATP-dependent calcium binding were increased. Cyclic AMP-dependent protein kinase (Sigma, type 1, 25 μg/0.5 ml) and cyclic AMP (1 μM) also stimulated calcium uptake and the presence of a maximal stimulatory concentration of the phosphodiesterase activator produced an additive elevation of calcium uptake indicating separate mechanisms of action and potentially different modulatory roles for these two systems in the control of calcium transport. 相似文献
13.
Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca 2+ binding and accumulating activities as well as (Mg 2+ + Ca 2+)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca 2+ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca 2+ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca 2+ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca 2+ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca 2+ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca 2+ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca 2+ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca 2+ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca 2+ accumulation ( V (nmol Ca 2+/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca 2+ required for half-maximal velocities did not differ significantly with age ( K0.5 for Ca 2+ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca 2+ binding also indicated that the velocity of Ca 2+ binding but not the concentration of Ca 2+ required for half-maximal binding was altered due to aging. At identical Ca 2+ concentrations, the combined Ca 2+ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca 2+ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca 2+ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg 2+ + Ca 2+)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca 2+ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca 2+ transport. Further, the age-related increment in the Ca 2+ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca 2+ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca 2+ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart. 相似文献
14.
Microsomal membrane vesicles isolated from the petals of young carnation ( Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca 2+ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca 2+ stimulation of ATP hydrolysis was not discernible above the high background of Ca 2+-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca 2+ concentration, suggesting that Ca 2+ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca 2+ at half maximal velocity of uptake (S 0.5) was 12.5 micromolar and the Hill coefficient ( nH) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca 2+, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca 2+ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca 2+ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca 2+ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S 0.5 or nH, but Vmax for ATP-dependent Ca 2+ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca 2+ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene. 相似文献
15.
A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca 2+-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca 2+-binding protein and a M 55 protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca 2+-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of 32P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca 2+ uptake. The Ca 2+-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca 2+-pump and Ca 2+-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca 2+-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles 32P-labelled phosphoenzyme per mg protein. The Ca 2+-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca 2+ binding by sarcoplasmic reticulum vesicles and by the Ca 2+-pump and Ca 2+-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca 2+-pump protein contained nonspecific high-affinity Ca 2+ binding sites with a capacity of 90–100 and 55–70 nmoles Ca 2+ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca 2+ per mg protein. The binding constants for nonspecific and specific Ca 2+ binding by both preparations were approx. 1 μM ?1. The Ca 2+-binding protein nonspecifically bound 900–1000 nmoles Ca 2+ per mg protein with a binding constant of about 0.25 μM ?1. 相似文献
16.
Endoplasmic reticulum membranes were isolated from roots of garden cress ( Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca 2+ uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca 2+ uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca 2+ transport (ruthenium red, LaCl 3 and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanide m-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5′-triphosphate, respectively. The results indicate a Ca 2+ transport function located on the endoplasmic reciculum of garden cress roots. 相似文献
17.
Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment. 相似文献
18.
Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate. 相似文献
19.
The binding of calcium by isolated sarcoplasmic reticulum from cow uterus was studied. Sarcoplasmic reticulum was prepared by differential centrifugation. Three fractions were obtained: I, sedimented between 2,500–15,000 x g; II at 40,000 x g; and III, at 150,000 x g. Fraction II was further purified on a sucrose density gradient. All three fractions contained considerable amounts of intrinsic calcium, mostly in fraction I. Calcium binding in the presence of ATP 1 and Mg also was greatest in fraction I, followed by fraction II, with less in fraction III. Without ATP no calcium was taken up. 5 and 10 m M sodium azide partially inhibited calcium binding in fraction I, but not in fraction II, suggesting the presence of some mitochondria or mitochondrial fragments in fraction I. Calcium binding in fraction II was completely inhibited by 3 m M salyrgan; this fraction thus appears to be sarcoplasmic reticulum. ATPase activity was found in all three fractions, highest in fraction II. It is computed that calcium binding in fractions I and II, on the basis of a 50% yield of protein, is sufficient to elicit contraction by supplying calcium to the contractile proteins of the smooth muscle cell and to regulate relaxation and contraction. 相似文献
20.
The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na + + K +)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low Km phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent Km for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg 2+, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg 2+ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle. 相似文献
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