Showing papers on "Membrane published in 1982"
TL;DR: Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm.
Abstract: A new, fluorescent, highly selective Ca2+ indicator , "quin2", has been trapped inside intact mouse and pig lymphocytes, to measure and manipulate cytoplasmic free Ca2+ concentrations, [Ca2+]i. Quin2 is a tetracarboxylic acid which binds Ca2+ with 1:1 stoichiometry and an effective dissociation constant of 115 nM in a cationic background mimicking cytoplasm. Its fluorescence signal (excitation 339 nm, emission 492 nm) increases about fivefold going from Ca-free to CA-saturated forms. Cells are loaded with quin2 by incubation with its acetoxymethyl ester, which readily permeates the membrane and is hydrolyzed in the cytoplasm, thus trapping the impermeant quin2 there. The intracellular quin2 appears to be free in cytoplasm, not bound to membranes and not sequestered inside organelles. The fluorescence signal from resting cells indicates a [Ca2+]i of near 120 nM. The millimolar loadings of quin2 needed for accurately calibrated signals do not seem to perturb steady-state [Ca2+]i, but do somewhat slow or blunt [Ca2+]i transients. Loadings of up to 2mM are without serious toxic effects, though above this level some lowering of cellular ATP is observed. [Ca2+]i was well stabilized in the face of large changes in external Ca2+. Alterations of Na+ gradients, membrane potential, or intracellular pH had little effect. Mitochondrial poisons produced a small increase in [Ca2+]i, probably due mostly to the effects of severe ATP depletion on the plasma membrane. Thus intracellulary trapped chelators like quin2 offer a method to measure or buffer [Ca2+]i in hitherto intractable cell types.
2,146 citations
TL;DR: The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum as well as applying it to various subfractions of a rat liver microsomal fraction.
Abstract: A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na2CO3 followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form. Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al. (1974, J. Cell Biol. 61:213-231). The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum. In the accompanying paper (1982, J. Cell Biol. 93:103-110) the procedure is applied to peroxisomes and mitochondria.
1,713 citations
TL;DR: Mechanisms for the transfer of newly synthesized polypeptides to their sites of function in different subcellular membranes and organelles are considered, and models in which specific features of the polypePTides serve as signals to direct them along selected sub cellular pathways to their final destination are discussed.
Abstract: Because of the high degree of organizational complexity of eucaryotic cells, it is clear that the implementation of their genetic programs must, in many cases, involve a complex sequence of cotranslational and posttranslational events that are necessary to transfer polypeptides from their sites of synthesis to their sites of function. It is difficult to envisage the existence of a single general mechanism that would ensure that all polypeptides released from ribosomes attain their correct subcellular destination . This is because there are, in a eucaryotic cell, at least as many possible destinations for a newly synthesized polypeptide as there are different compartments and membrane systems. Moreover, it is not likely that completed and fully folded polypeptides, with their charged and polar residues exposed to the aqueous environment, could freely traverse hydrophobic barriers constituted by phospholipid bilayers, which are the universal feature of all cell membranes (208). Instead, it may be expected that special mechanisms have evolved that direct polypeptides to specific membranes and, when necessary, assist them in their passage across the hydrophobic barriers . These mechanisms must involve specific receptors for structural features ofthe polypeptides and may entail conformational changes or even extensive structural modifications of the polypeptide, as well as the expenditure of energy . In this paper we consider mechanisms for the transfer of newly synthesized polypeptides to their sites of function in different subcellular membranes and organelles, and discuss models in which specific features ofthe polypeptides serve as signals to direct them along selected subcellular pathways to their final destination . These signals may act during translation or after synthesis of the polypeptide is completed and may or may not be removed from the initial product of translation . Transient or permanent signals within polypeptides destined to membranes would also account for the final characteristic orientation of membrane proteins with respect to the phospholipid bilayer . Several other reviews discussing various aspects of this subject have recently appeared (15, 44, 53, 117a, 127, 243a) .
798 citations
TL;DR: It is concluded that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylatecyclase system.
Abstract: GTP and isoproterenol activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in washed membranes prepared from C6 gliomas cells was enhanced by incubation with islet-activating protein, one of the pertussis toxins, if the incubation mixture was supplemented with NAD and ATP. The action of the protein was observed immediately after its addition and increased progressively in magnitude as the protein concentration or the incubation time increased. There was simultaneous incorporation of radioactivity from the ADP-ribose moiety of variously labeled NAD into the membrane protein with a molecular weight of 41,000. We conclude that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylate cyclase system.
770 citations
Patent•
05 Apr 1982
TL;DR: In this article, the authors describe colloidal sized particles composed of magnetic iron oxide (Fe 3 O 4 ) coated with a polysaccharide, preferably dextran, or a derivative thereof having pendant functional groups.
Abstract: The invention relates to colloidal sized particles composed of magnetic iron oxide (Fe 3 O 4 ) coated with a polysaccharide, preferably dextran, or a derivative thereof having pendant functional groups. The particles have a magnetic moment, are electron dense, and are stable and non-aggregating under physiological conditions. They can be covalently bonded to antibodies, enzymes and other biological molecules and used to label and separate cells, cellular membranes and other biological particles and molecules by means of a magnetic field.
513 citations
TL;DR: This work has studied the response of membrane patches from neuroblastoma cells to intracellular Ca2+ ions, and found a class of nonselective cation channels activated by micromolar concentrations of Ca2+, which suggests that this hitherto scarcely recognized channel species may be more widely distributed than previously supposed.
Abstract: Recent work suggests an important role for intracellular agents in controlling ion channels in the membranes of nerve cells and other excitable tissues. Calcium ions1,2, cyclic nucleotides3 and protein kinases4,5 can all act on the inner surface of the membrane to influence ion channel activity. Earlier studies of these effects on intact cells could not, however, effectively control or measure conditions on the inner membrane surface. The technique of recording from detached membrane patches6,7permits free access to the intracellular face of ion channels and makes it possible to study them in isolation from the many components of the cytoplasm. With this technique, I have studied the response of membrane patches from neuroblastoma cells to intracellular Ca2+ ions, and have found a class of nonselective cation channels activated by micromolar concentrations of Ca2+ on the intracellular face of the membrane. These channels are almost equally permeable to Na+, K+, Li+ and Cs+ ions, but are practically impermeable to Ca2+ ions. Similar channels were first found recently in cultured heart cells8, where they probably account for the previously reported ‘transient inward’ current9. Their discovery in neuronal cells as well as heart cells suggests that this hitherto scarcely recognized channel species may be more widely distributed than previously supposed.
442 citations
TL;DR: Despite the apparent heterogeneity, amino acid sequence analysis and comparison of proteolytic fragments suggest that all forms of the glycoprotein are derived from the same polypeptide chain.
431 citations
TL;DR: Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper and found to be greatly dissimilar.
Abstract: Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper (1982, J. Cell Biol. 93:97-102). The polypeptide compositions of these membranes were determined by SDS PAGE and found to be greatly dissimilar. The peroxisomal membrane contains 12% of the peroxisomal protein and consists of three major polypeptides (21,700, 67,700 and 69,700 daltons) as well as some minor polypeptides. The major peroxisomal membrane proteins as well as most of the minor ones are absent from the endoplasmic reticulum (ER). Conversely, most ER proteins are absent from peroxisomes. By electron microscopy, purified peroxisomal membranes are approximately 6.8 nm thick and have a typical trilaminar appearance. The phospholipid/protein ratio of peroxisomal membranes is approximately 200 nmol/mg; the principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine as in ER and mitochondrial membranes. In contrast to the mitochondria, peroxisomal membranes contain no cardiolipin. All the membranes investigated contain a polypeptide band with a molecular mass of approximately 15,000 daltons. Whether this represents an exceptional common membrane protein or a coincidence is unknown. The implications of these results for the biogenesis of peroxisomes are discussed.
356 citations
TL;DR: An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes, and has been visualized by rotary shadowing as an extended, flexible rod.
Abstract: An immunoreactive analogue of erythrocyte spectrin has been purified from brain membranes. This protein co-sediments with and cross-links actin filaments, associates with spectrin-binding sites on erythrocyte membranes, and has been visualized by rotary shadowing as an extended, flexible rod. The brain spectrin comprises 3% of the total membrane protein, and may have a major role in mediating linkage of actin to membranes.
328 citations
TL;DR: Differential scanning calorimetry (DSC), and to a lesser extent differential thermal analysis (DTA), are powerful yet relatively rapid and inexpensive thermodynamic techniques for studying the thermotropic phase behavior of lipids in model and biological membranes, without the introduction of exogenous probe molecules.
TL;DR: Evidence is provided for a large increase in H+ ion permeability in depolarized cells and it is suggested that this increase in proton conductance may be the basis for the ‘nonspecific’ currents previously described in perfused molluscan neurones.
Abstract: Until now the only reported effect of depolarization on the intracellular pH (pHi) of excitable cells is an acidification of the cell cytoplasm1,2. It seems unlikely that this could be a direct effect of membrane potential because pHi is known to be regulated by an electroneutral mechanism3,4 and in most cells H+ ions are not in equilibrium with the membrane potential (Em). In any case the membrane conductance to H+ ions would be expected to be small because they are at such low concentrations on either side of the cell membrane. But it is possible that the H+ ion permeability of the membrane increases on depolarization just like that of other ions in the bathing medium (Na+, K+ and Ca2+ for example). To test this idea we have made pHi measurements on molluscan neurones under voltage-clamp. Our findings, presented here, provide evidence for a large increase in H+ ion permeability in depolarized cells. We suggest that this increase in proton conductance may be the basis for the ‘nonspecific’ currents previously described in perfused molluscan neurones5,6 and we assess the physiological significance of this newly discovered pathway.
TL;DR: The permeability of P. aeruginosa outer membrane was determined by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm, showing that this membrane has a low permeability to most hydrophilic compounds.
Abstract: Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.
TL;DR: Lipid analog diffusion is slightly enhanced in blebs as expected of evanescent lipid protein interaction, and strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.
Abstract: Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported. These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton. On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) approximately 3 x 10(-9) cm2/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane. In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D approximately less than 10(-10) cm2/s with substantial fractions virtually nondiffusible (D less than 10(-12) cm2/s). Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction. This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.
TL;DR: The result suggests that [3H]AMPA might represent binding to an excitatory GLU receptor, which is saturable and reversible at physiological pH and the highest blevel in striatal membranes.
Abstract: Binding of [3H]AMPA to rat brain membranes was investigated. The binding was saturable and reversible at physiological pH. Computer-aided Scatchard analysis of the binding data, as determined by using L-glutamic acid (L-GLU) to define nonspecific binding, suggested the presence of two independent binding sites, with KDS of 9 and 2440 nM, respectively. Additional freezing, thawing and washing sequences gave membranes with only one binding site, with a KD of 278 nM. [3H]AMPA binding exhibited the highest level in striatal membranes. A series of analogues of GLU and aspartic acid (ASP) were tested as inhibitors of [3H]AMPA binding. L-ASP and compounds which interact predominantly with N-methyl-D-aspartic acid (NMDA) receptor sites were inactive as inhibitors of [3H]AMPA binding, whereas L-GLU and compounds which interact predominantly with glutamic acid diethyl ester receptor sites were inhibitors with the same order of potency as that shown by the excitatory action in vivo. The result suggests that [3H]AMPA might represent binding to an excitatory GLU receptor.
TL;DR: Golgi fractions isolated from rat liver homogenates have been resolved into membrane and content subfractions by treatment with 100 mM Na2CO3 pH 11.3, and the phospholipids of both membrane andcontent were found to be uniformly labeled with [33P]phosphate administered in vivo.
Abstract: Golgi fractions isolated from rat liver homogenates have been resolved into membrane and content subfractions by treatment with 100 mM Na2CO3 pH 11.3. This procedure permitted extensive extraction of content proteins and lipoproteins, presumably because it caused an alteration of Golgi membranes that minimized the reformation of closed vesicles. The type and degree of contamination of the fractions was assessed by electron microscopy and biochemical assays. The membrane subfraction retained 15% of content proteins and lipids, and these could not be removed by various washing procedures. The content subfraction was contaminated by both membrane fragments and vesicles and accounted for 5 to 10% of the membrane enzyme activities of the original Golgi fraction. The lipid compositions of the subfractions was determined, and the phospholipids of both membrane and content were found to be uniformly labeled with [33P]phosphate administered in vivo.
TL;DR: The membrane surfaces within the rod outer segment of the toad, Bufo marinus, were exposed by rapid-freezing followed by freeze-fracture and deep-etching, showing a fine granularity and suggesting that the particles represent protrusions of the integral membrane protein, rhodopsin, into the intradisk space.
Abstract: The membrane surfaces within the rod outer segment of the toad, Bufo marinus, were exposed by rapid-freezing followed by freeze-fracture and deep-etching. Platinum-carbon replicas of disk membranes prepared in this way demonstrate a distinct sidedness. The membrane surface that faces the lumen of the disk shows a fine granularity; particles of approximately 6 nm are packed at a density of approximately 30,000/micron 2. These dimensions suggest that the particles represent protrusions of the integral membrane protein, rhodopsin, into the intradisk space. In addition, when rhodopsin packing is intentionally perturbed by exhaustive digestion with phospholipase C, a concomitant change is observed in the appearance of the luminal surface granularity. The cytoplasmic surface of the disk rarely displays this rough texture; instead it exhibits a collection of much larger particles (8-12 nm) present at approximately 10% of the concentration of rhodopsin. This is about the size and concentration expected for certain light-regulated enzymes, cGMP phosphodiesterase and GTP-binding protein, which are currently thought to localize on or near the cytoplasmic surface of the disk. The molecular identity of the 8-12-nm particles will be identified in the following companion paper. A further differentiation of the cytoplasmic surface can be seen around the very edge, or rim, of each disk. This rim has relatively few 8-12-nm particles and instead displays short filamentlike structures connecting it to other membranes. These filaments extend between adjacent disks, across disk incisures, and from disk rims to the nearby plasma membrane.
TL;DR: It was concluded that the MIP26 preparation was homogeneous and bound unequivocally to lens communicating junctions, indicating that MIP 26 is a component of these structures.
Abstract: Plasma membranes of vertebrate lens fiber cells contain a major intrinsic polypeptide with an apparent molecular weight of 26,000 (MIP26). These plasma membranes are extremely rich in communicating junctions, and it has been suggested that MIP26 is a component of them. MIP26 was purified from cow lenses using preparative SDS gel electrophoresis followed by hydroxylapatite column chromatography. From gel electrophoresis patterns and aggregational properties it was concluded that the MIP26 preparation was homogeneous. The purified MIP26 was used to produce monospecific antibodies in rabbits as assessed by double immunodiffusion and crossed immunoelectrophoresis of purified MIP26 and solubilized lens plasma membranes against the antiserum. Indirect immunocytochemical studies were performed on open and closed lens plasma membrane vesicles by incubation in anti-MIP antiserum followed by ferritin-conjugated goat antirabbit IgG. The conjugate bound unequivocally to lens communicating junctions, indicating that MIP26 is a component of these structures.
TL;DR: The proteins of the outer membrane from rat liver mitochondria have been subfractionated by means of density gradient centrifugation and it was observed that a polypeptide of Mr 32 000 renders asolectin vesicles permeable to ADP and forms pores in bilayer membrane.
TL;DR: Pathways independent of the anion exchange system and insensitive to its specific inhibitors may account for slow movements of C1[63] and SCN[31].
Abstract: Transport of anions across the red blood cell membrane has been studied intensively in recent years under the impact of the successful identification and molecular characterization of the transport protein mediating chloride/bicarbonate exchange. The features of this electrically silent exchange process which is mediated by a 95-K Dalton intrinsic membrane protein, termed band 3 according to its position on SDS-polyacrylamide gels, have recently been treated comprehensively [67]. The transport system is characterized by a broad acceptance of substrates encompassing "regular" monoand divalent inorganic anions (halides, oxyanions) [67], but also more exotic passengers [24, 35, 74]. Numerous organic anions most likely also permeate the erythrocyte membrane in this way, e.g., dicarboxylates [25, 39, 76], aliphatic and aromatic sulfonates [17, 18, 33, 45, 64, 67, 76]; (B. Deuticke, unpublished results) as well as organic phosphates [9, 48] and even certain amino acids [108]. The question of parallel, alternative pathways of anion transport in the erythrocyte membrane has hitherto met less attention. Pathways independent of the anion exchange system and insensitive to its specific inhibitors may account for slow movements Of C1[63] and SCN[31]. Alternative routes have mainly been discussed for monocarboxylates. Being the anions of weak and often rather lipophilic acids, they have long been postulated [41, 54] to permeate by nonionic diffusion
04 Feb 1982
TL;DR: In this communication some of the known structural and functional changes in membranes due to heating will be summarized.
Abstract: A number of major questions remain unanswered regarding the relationship between cellular membranes and the viability of cells exposed to hyperthermia. Foremost, are membranes involved at all in hyperthermic killing, radiosensitization, or thermotolerance of mammalian cells after heating to hyperthermic temperatures in the range of 41-45?C? If so, which membranes are implicated: plasma, nuclear, mitochondrial, lysosomal, endoplasmic reticular, etc? Damage might be restricted to only one area of a membrane due to membrane heterogeneity (1). In particular, what specific molecular alterations occur in membranes when they are heated to hyperthermic temperatures? In this communication some of the known structural and functional changes in membranes due to heating will be summarized.
TL;DR: In this paper, a model of diffusion controlled mass transfer in liquid surfactant membranes is developed for uniform emulsion globules having no internal circulation, where the solute is assumed to react instantaneously and irreversibly with the internal reagent at a reaction surface which advances into the globule as the reagent is consumed.
Abstract: A model of diffusion controlled mass transfer in liquid surfactant membranes is developed for uniform emulsion globules having no internal circulation. The solute is assumed to react instantaneously and irreversibly with the internal reagent at a reaction surface which advances into the globule as the reagent is consumed. A perturbation solution to the resulting non-linear equations is presented. In general, the zero-order, or pseudo-steady state solution alone often gives an adequate representation of the process. Experimental data on the batch extraction of phenol from waste water are in good agreement with the model predictions.
TL;DR: The concomitant hydrophilic–amphiphilic transition is the result of a conformational reorganization of C9 and allows insertion of poly C9 into membranes in the form of a transmembrane protein channel.
Abstract: The ninth component of complement C9 can undergo circular polymerization in the fluid phase and on lipid membranes. The concomitant hydrophilic-amphiphilic transition is the result of a conformational reorganization of C9 and allows insertion of poly C9 into membranes in the form of a transmembrane protein channel. The ultrastructure of poly C9 resembles that of membrane lesions caused by complement.
TL;DR: Light-membrane fractions obtained by hypoosmotic lysis of neurospora crassa mitochondria exhibit buoyant densities and marker-enzyme activities characteristic of outer mitochondrial membranes.
Abstract: Light-membrane fractions obtained by hypoosmotic lysis of neurospora crassa mitochondria exhibit buoyant densities and marker-enzyme activities characteristic of outer mitochondrial membranes. SDS PAGE of these membrane fractions indicates that a polypeptide of M(r) 31,000 is the main protein component. Under negative-stain electron microscope examination many of the membranes in these fractions appear as large (0.5-1- mum diameter), collapsed vesicles. The surfaces of flattened, open (i.e., ripped) vesicles often exhibit extended two-dimensional arrays of subunits are arranged into hexagons within each parallelogram unit cell, 12.6x11.1 nm (lattice angle = 109 degrees).
TL;DR: This chapter reviews the evidence for lipid domains in membranes and points out the functional effects of their putative selective perturbation, and presents evidence that the phospholipid of plasma membranes is not necessarily homogeneously fluid, but may well be organized into co-existing domains relatively gel and fluid in nature.
Abstract: According to the \"fluid mosaic\" model of membrane organization,' amphipathic globular or intrinsic proteins are embedded in a homogeneous sea of fluid bilayer phospholipid, in which they are free to diffuse laterally. This model has been useful in stimulating thinking regarding membrane structure, but evidence from recent studies has led to some refinements of the model. In particular, it has been suggested that certain intrinsic proteins may interact with submembranous peripheral and cytoskeletal proteins. This may in some cases restrict2 or, in other cases, promote3,'5 lateral movement of the intrinsic membrane proteins. In addition, we and others have presented evidence that the phospholipid of plasma membranes is not necessarily homogeneously fluid, but may well be organized into co-existing domains relatively gel [ordered acyl chains] and fluid (disordered acyl chains) in nature.4 The physical state of the lipids surrounding various proteins may control protein conformation, regulate interactions between proteins, and regulate protein function. It is the purpose of this chapter to review our evidence for lipid domains in membranes, and to point out the functional effects of their putative selective perturbation. The biological studies provide indirect supporting evidence for the concept.