共查询到20条相似文献,搜索用时 15 毫秒
1.
Varda Shoshan-Barmatz 《The Journal of membrane biology》1988,103(1):67-77
Summary The relationship between Ca2+ release from sarcoplasmic reticulum, induced by elevated pH, tetraphenylboron (TPB–) or chemical modification, and the change in the surface charge of the membranes as measured by the fluorescence intensity of anilinonaphthalene sulfonate (ANS) is examined. The stimulated Ca2+ release is inhibited by dicyclohexylcarbodiimide and external Ca2+. TPB–, but not tetraphenylarsonium (TPA+), causes a decrease in ANS– fluorescence, with 50% decrease occurring at about 5 m TPB–. The decrease in ANS– fluorescence as well as the inhibition of Ca2+ accumulation induced by TPB– are prevented by TPA+. A linear relationship between the decrease in membrane surface potential and the extent of the Ca2+ released by TPB– is obtained. Similar levels of [3H]TPB– bound to sarcoplasmic reticulum membranes were obtained regardless of whether or not the vesicles have taken up Ca2+. The inhibition of Ca2+ accumulation and the [3H]TPB– incorporation into the membranes were correlated. Ca2+ release from sarcoplasmic reticulum, by pH elevation, chemical modification or by addition of NaSCN (0.2 to 0.5m) or the Ca2+ ionophore ionomycin, is also accompanied by a decrease in ANS– fluorescence intensity. However, chemical modification and elevated pH affects the surface potential much less than SCN– or TPB– do. These results suggest that the enhancement of Ca2+ release by these treatments is not due to a general effect on the membrane surface potential, but rather through the modification of a specific protein. They also suggest that membrane surface charges might play an important role in the control mechanism of Ca2+ release. 相似文献
2.
Summary Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca2+. Vescle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca2+-concentrations and is half maximal around 10–6
m. Other divalent cations (Ba2+, Sr2+, and Mg2+) were ineffective. Mg2+ inhibits Ca2+-induced fusion. Therefore, the fusion of secretory vesiclesin vitro is Ca2+ specific and exhibits properties similar to the exocytotic process of various secretory cells.Various substances affecting secretionin vivo (microtubular inhibitors, local anethetics, ionophores) were tested for their effect on membrane fusion in our system.The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca2+, and in its Ca2+-specificity. Chemical and enzymatic modifications of the vesicle membrane indicate that glycoproteins may account for these differences. 相似文献
3.
A mitochondria-free membrane fraction prepared from rat myometrium accumulated 45Ca2+ in the presence of oxalic acid and ATP. The rate of transport of Ca2+ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca2+-transport. ATPase activity was stimulated by Mg2+; slight additional stimulation was obtained in the presence of Na+ and K+ but not in the presence of Ca2+. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca2+-transport suggest it has little to do with Ca2+ accumulation by the membranes.Cyclic AMP-induced increase in Ca2+-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10−4 M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca2+-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility. 相似文献
4.
Jing Gao Hiroshi Takeuchi Hisanori Umebayashi Zhao Zhang Miho Matsuda Masato Hirata 《Advances in enzyme regulation》2010,50(1):237-246
Exocytosis plays an essential role in fundamental cellular events by secreting neurotransmitters, hormones, and cytokines. Although the minimal molecular components termed SNARE that govern membrane fusion have been identified, the precise mechanisms behind the finely-tuned regulation of exocytosis executed by many molecules in addition to the actions of SNARE remain to be fully identified. Here, we evaluated a model system for assaying catecholamine secretion from permeabilized rat pheochromocytoma PC12 cells, in which the structural integrity required was preserved adequately. Among several chemical reagents used for the cell permeabilization and freezing-thawing procedures, the treatment of cells with digitonin at concentrations of 7.5–15 μM was most suitable for the secretion assay, as it was considered to cause mild disruption of the plasma membrane, enabling free access to small molecules such as Ca2+ and ATP to the minimal membrane fusion machinery. No additional cytosolic proteins were required to reconstitute the secretion. In this assay model, ATP was necessary to maintain the priming state before Ca2+-triggered exocytosis but was not required for the Ca2+-triggered membrane fusion process itself. The present study provides a useful cell model for exploring novel molecules that may be implicated in exocytosis such as those playing regulatory roles in addition to the “minimal membrane fusion machinery for exocytosis”, which does not require any additional special apparatus. 相似文献
5.
Summary The roles that Ca2+, calmodulin, and ATP play in the redistribution of conconavalin A (Con A) binding sites on the surface of mouse T-lymphoma cells were examined. Membranes of cells labeled with fluorescein-conjugated Con A (Fl-Con A) were made permeable (skinned) to ions and proteins by incubation in a solution containing no added Ca2+, 7mm EGTA, and ATP. The intracellular ionic and protein concentrations could then be varied, and the degree of Con A receptor capping monitored simultaneously. A graded increase (9.0 to 30%) was found in the number of capped cells with increasing Ca2+ concentration from 10–6–10–4.9
m. Increasing concentrations of trifluoperazine, chlorpromazine, and promethazine (1.5×10–6 to 1.0×10–4
m) in cell suspensions containing 10–4
m Ca2+ produced graded inhibition of capping in the same order that the drugs bind to calmodulin. Removal of extracellular Ca2+ dissociated (reversed) some of the caps into patches, thus reducing their number (12%). ATP was required for either capping or cap dissociation to occur. Addition of calmodulin (3.9×10–8–6.3×10–7
m) to the cell suspension increased the Ca2+ sensitivity. These results provide direct evidence that capping of Con A receptors is a reversible process (i) regulated by intracellular Ca2+ concentration, (ii) requiring ATP as an energy source, and (iii) susceptible to the influence of calmodulin. These findings are consistent with the hypothesis that the collection of surface receptor patches into cap structures is controlled by the interaction of actomyosin filaments, which in turn is regulated by a Ca2+-calmodulin-activated control system. 相似文献
6.
Jens R. Coorssen Paul S. Blank Masahiro Tahara Joshua Zimmerberg 《The Journal of cell biology》1998,143(7):1845-1857
Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion. 相似文献
7.
Malcolm Weller 《Molecular and cellular biochemistry》1978,20(2):95-102
Summary Preparations of human erythrocyte membranes have been made which are in the form of sealed vesicles and which behave as osmometers on suspension in solutions of simple inorganic salts. Using these preparations the permeability of the membranes to Na+, K+, Mg2+ and Ca2+ was measured. Cyclic AMP (but not cyclic GMP) increased the permeability of the membranes to Ca2+ with a half maximal effect at a concentration of 25µm but did not affect the permeability to the other ions tested. Phosphorylation of proteins in the erthrocyte membrane lowered the permeability to Ca2+ without affecting the permeability to the other ions tested and there was a good correlation between the time course of protein phosphorylation and decrease in Ca2+ permeability.It is postulated that the system through which cyclic AMP causes an initial rapid rise in Ca2+ permeability followed by increased phosphorylation of membrane proteins and reduced Ca2+ permeability may have a widespread occurrence in biological systems and serve to control the concentration of Ca2+ in the cytoplasm. 相似文献
8.
A chicken pectoralis muscle membrane fraction enriched in a Mg2+- or Ca2+-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg2+ or Ca2+ but not by Sr2+, Ba2+, Co2+, Ni2+ or Pb2+. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% (w/w) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca2+ ATPase. Also unlike sarcoplasmic reticulum Ca2+ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca2+ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb2+ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na+ + K+) ATPase and sarcoplasmic reticulum Ca2+ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle. 相似文献
9.
《Neurochemistry international》2009,54(6-8):401-407
The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca2+ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca2+-independent step, termed docking and followed by fusion step that is triggered by Ca2+. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed. 相似文献
10.
The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca2+ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca2+-independent step, termed docking and followed by fusion step that is triggered by Ca2+. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed. 相似文献
11.
A model of the active transport of ions through internal membranes of mitochondria is proposed. If concentrations of ions
in a cell are known, this model allows calculating concentrations of all main ions (H+, Ca+2, K+, Mg2+, Na+, Cl−) in the mitochondrion matrix and the resting potential across the membrane. The theoretical values satisfactorily agree with
available experimental data on the concentrations and the potentials, including different operating regimes of the adenosine
triphosphate (ATP) synthetase (the main regime, short circuiting or ATP synthetase blocking). The active transport of Mg2+ ions in exchange for protons was assumed. In accordance with the model, the ATP synthetase operation is possible only if
the stoichiometric coefficient of protons is 3. 相似文献
12.
R. H. Ashley 《The Journal of membrane biology》1989,111(2):179-189
Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k
d
3nm.B
max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl– and Ca2+, respectively. The reconstituted Ca2+ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca2+ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca2+/TRIS+ permeability ratio of 11.0. They also conducted other divalent cations (Ba2+>Ca2+>Sr2+>Mg2+) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca2+ channels, sharing some of the characteristics of Ca2+ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca2+ release from internal membrane stores. 相似文献
13.
Gerhard Meissner 《生物化学与生物物理学报:生物膜》1973,298(4)
The binding of ATP and Ca2+ by the Ca2+ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca2+ pump protein has been found to contain one specific ATP and two specific Ca2+ binding sites per phosphorylation site. ATP binding is dependent on Mg2+ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca2+. In the presence of Mg2+ and the absence of Ca2+, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca2+ binding. In the absence of ATP, Ca2+ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg2+, Sr2+ and La3+, in that order, decrease Ca2+ binding by the Ca2+ pump protein. The affinity of the Ca2+ pump protein for both ATP and Ca2+ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca2+ pump protein-MgATP2− and Ca2+ pump protein-calcium complexes are approx. 0.25 and 0.5 μM−1, respectively. The unphosphorylated Ca2+ pump protein does not contain a Mg2+ binding site with an affinity comparable to those of the ATP and Ca2+ binding sites.The affinity of the Ca2+ pump protein for Ca2+ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca2+ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca2+. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca2+.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca22+-MgATP2− complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca2+ and probably Mg2+. 相似文献
14.
The Ca2+-pumping ATPase of erythrocyte plasma membranes of hypertensive humans (HTN) show, in the absence ofcalmodulin, a low Vmax comparable to that of the enzyme of the erythrocyte membranes of normotensive humans (NTN). Although the addition of calmodulin (1.5 gper ml) increased the maximum activity of the calcium pump of membranes of HTN and NTN individuals by at least 2-fold and 4-fold, respectively, the activator protein partially purifed from the erythrocytes of HTN individuals enhanced the activity of the enzyme in a fashion similar to that of the protein obtained from the haemolysate of NTN individuals. A determination of the dependence of the activity of the pump on concentration of ATP revealed that the Km (ATP) of the enzyme of membranes of HTN individuals is 52% higher than that of the enzyme of membranes of NTN individuals, while the Vmax (1.75±0.28 mol ATP mg protein–1 h–1) of the pump is 46% lower in the membranes of HTN humans than that of the enzyme of membranes of normal individuals (3.25 ±0.42 mol ATP mg protein–1 h–1) . It seems likely from these results that elevated erythrocyte Ca2+ concentration associated with essential hypertension may be due to a defective interaction between the Ca2+-pumping ATPase and the calmodulin Ca2+ complex, 相似文献
15.
Ca2+-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific
preparation to study the mechanisms by which Ca2+ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins
have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide
(IA). IA was found to enhance the kinetics and Ca2+ sensitivity of both CV-plasma membrane and CV–CV fusion. If Sr2+, a weak Ca2+ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca2+, suggesting that IA acts at the Ca2+-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol
sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with
fusion competency. 相似文献
16.
Scott R. Whittemore Robert H. Lenox Edith D. Hendley Dr. Yigal H. Ehrlich 《Neurochemical research》1981,6(7):775-785
The effect of (–)-isoproterenol on adenylate cyclase activity were studied in rat cerebral cortical membranes prepared and assayed in the presence of calcium ions. In assays carried out in the presence of high Mg2+ concentrations (5–10 mM) and of Ca2+ in the micromolar range, addition of 1–100 M (–)-isoproterenol caused over 50% inhibition of adenylate cyclase activity. Since these conditions are optimal for supporting endogenous phosphorylative activity in synaptic membranes, we tested whether the observed effects are mediated by changes in the phosphorylation of specific proteins in these membranes. This was done by preincubation of lysed synaptosomes under phosphorylating conditions in the presence and absence of isoproterenol followed by extensive washes and analysis of cyclic AMP formation in resuspended membranes. Addition of (–)-isoproterenol to the preincubation resulted in a 30% decrease of adenylate cyclase activity in the reincubation. Inclusion of [-32P]ATP in the preincubation and examination of the phosphorylation state of specific proteins in membranes entering the reincubation revealed that (–)-isoproterenol inhibited the phosphorylation of a specific protein band with apparent molecular weight of 47,000 (designated band F). These results support the hypothesis that alterations in membrane protein phosphorylation induced by neurotransmitters play a role in the regulation of adenylate cyclase activity. 相似文献
17.
Njanoor Narayanan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,678(3):442-459
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 Ca2+ binding and accumulating activities as well as (Mg2+ + Ca2+)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca2+ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca2+ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca2+ 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 Ca2+ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca2+ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca2+ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca2+ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca2+ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca2+ accumulation (V (nmol Ca2+/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca2+ required for half-maximal velocities did not differ significantly with age (K0.5 for Ca2+ (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 Ca2+ binding also indicated that the velocity of Ca2+ binding but not the concentration of Ca2+ required for half-maximal binding was altered due to aging. At identical Ca2+ concentrations, the combined Ca2+ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca2+ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca2+ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg2+ + Ca2+)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca2+ 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 Ca2+ transport. Further, the age-related increment in the Ca2+ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca2+ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca2+ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart. 相似文献
18.
Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration. 相似文献
19.
Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves 总被引:3,自引:0,他引:3
Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(K
m
(Ca2+)=0.4 m) and ATP(K
m
(ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl– or NO
3
–
. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl– was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO
4
3–
which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl–-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl–-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl–-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves. 相似文献
20.
Summary ATP-dependent45Ca2+ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl2 and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na+,K+)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca2+ uptake was maximal with 10 and 2 mol/liter free Ca2+, and half-maximal with 0.9 and 0.5 mol/liter free Ca2+. It was maximal at 3 and 0.2 mmol/liter free Mg2+ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg2+ was replaced by Mn2+ or Zn2+ ATP-dependent Ca2+ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn2+ could replace Mg2+ for Ca2+ uptake by 20%. Other divalent cations such as Ba2+ and Sr2+ could not replace Mg2+ in Ca2+ uptake. Ca2+ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca2+ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca2+ uptake. The sequence was K+>Rb+>Na+>Li+>choline+ in plasma membranes and Rb+K+Na+>Li+>choline+ for rough endoplasmic reticulum. The anion sequence was Cl–Br–I–>SCN–>NO
3
–
>isethionate– >cyclamate–>gluconate–>SO
4
2–
glutarate– and Cl–>Br>gluconate>SO
4
2–
>NO
3
–
>I–>cyclamate–SCN–, respectively. Ca2+ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K+ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca2+ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca2+ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca2+ from the cell that might be involved in the regulation of the cytosolic Ca2+ level. 相似文献