Plasma Membrane Of Animal Cells Research Articles

Determination of ATP Leakage from Cultured Cells in Toxicity Testing: A Two-Step Bioluminescent Assay

The intact plasma membrane of animal cells in culture is considered to be almost impermeable to ATP. Thus, leakage of intracellular ATP to the extracellular medium is indicative of membrane damage. We have developed a rapid and simple method for determination of ATP leakage, using L-1210 and ELD cells incubated with velleral or isovelleral (two terpenes). ATP was determined by a bioluminescent firefly luciferin-luciferase reaction in a two-step assay in the same sample. Extracellular ATP (= leakage) was measured directly in cell suspensions, and total ATP was determined in cell lysate suspensions after addition of a detergent. ATP leakage (% of total ATP) increased after incubation with both terpenes, in a dose-dependent manner.

Immunological approaches to gap junctions: Studies of immuno-localization, membrane protein topology, and function

It is now widely accepted that the plasma membranes of epithelial cells display a marked degree of polarity. This polarity is reflected in a number of parameters, e.g. the localization of certain proteins to basolateral membranes. Polarity is also illustrated by the presence of discrete membrane specializations, including desmosomes, tight junctions and gap junctions. These specializations provide strong support for the idea of membrane "domains" within the polarized membrane of the epithelial cell. This presentation relates to the proteins found in gap junction membranes. The issues include a clear identification of these proteins in different cells and an analysis of how a cell-to-cell channel is constructed by these proteins.The plasma membranes of most animal cells contain cell-to-cell channels, which provide for the direct, passive exchange of small molecules between cells. Collections of these channels are identified as "gap junctions." This form of intercellular communication is thought to be important in a wide variety of biological processes, including cellular differentiation, proliferation, and tissue homeostasis.

Partial characterization of the adsorbed protein layer on Dirofilaria immitis (Nematoda) cuticle.

Electron microscopy coupled with ferritin-conjugated indirect immunolabeling was used to locate sites of adsorbed host protein on cuticular surfaces of the adult canine heartworm, Dirofilaria immitis. The epicuticle appeared as a trilaminated structure. At high magnifications, the outermost layer of this structure was resolved into a trilaminar layer, which might correspond to the plasma membrane of animal cells. A ruthenium red-positive layer was external to the epicuticle. Ferritin-antibody conjugates showed evidence of adsorbed dog albumin, dog immunoglobulin class G (IgG), and dog complement fraction 3 (C3) on the surface. Ferritin adsorption to control surfaces was minimal. Possible causes and effects of interfacial host-protein adsorption are discussed in an attempt to bring insight to the hemocompatible nature of the parasitic cuticle.

Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit.

We carried out experiments designed to generate a novel cell-surface protein from a small glycosylated secretory protein. DNA encoding the entire precursor of human chorionic gonadotropin (hCG, alpha subunit) was fused precisely to DNA encoding the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. When expressed in animal cells this DNA encoded the 92-amino acid hCG-alpha subunit anchored in cellular membranes by an extension composed of the 49 carboxyl-terminal amino acids of vesicular stomatitis virus glycoprotein. This hybrid protein was transported efficiently to the plasma membrane of animal cells. The two asparagine-linked glycans on the anchored form of hCG-alpha were large and heterogeneous when compared to those on the secretory form. Experiments employing in vitro mutagenesis and the glycosylation inhibitor tunicamycin established that the presence of at least one of the two asparagine-linked glycans was required for expression of the anchored molecule on the cell surface. However, as reported previously, secretion of hCG-alpha occurred in the absence of glycosylation. Also, mutations eliminating the second glycosylation site (at amino acid 78) in both the anchored or secreted forms apparently led to partial denaturation or a conformational change interfering with transport of the protein.

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to min, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate- or linolenate-supplemented medium, 71.8% and 75.5% of the plasma membrane sterol is exofacial, respectively.

Interactive laser cytometry: Non-invasive microscopic approaches for the measurement of dynamic processes in animal and plant cells and tissue

All aspects of cellular metabolic, biosynthetic, and replicative activity depend on the movement of signaling messenger molecules, macromolecules, and organelles between cellular compartments. On another level, individual cellular activity is coordinated and synchronized in tissues by the movement of molecules through trans-membrane structures forming aqueous channels between contiguous cells. The types of movement found in the cell can be discussed in terms of: a) diffusion, b) flow, and c) energy driven transport. Diffusive processes can be 1-, 2-, or 3-dimensional. An example of each type of diffusion would be the movement of control proteins along DNA/RNA strands (1-D), the lateral transport of proteins in membranes (2-D), and the intercellular movement of molecules through gap junctions or piasmodesmata (3-D). Flow processes have been most frequently observed in the bulk movement of animal cell plasma membrane and cytoplasmic streaming in plants, while energy driven transport has been most vividly demonstrated for the intracellular transport of vesicles along microtubule tracks.

Spatial relationship and conformational changes between the cardiac glycoside site and beta-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase.

(Na,K)-ATPase, the enzyme responsible for active transport of Na and K across the plasma membranes of animal cells, consists of a catalytic subunit (alpha) and a glycoprotein subunit (beta) with unknown function. We have determined the distance between fluorescent probes directed to specific sites on the alpha- and beta-subunits and ligand-induced changes in the fluorescence of a probe specifically attached to the beta-subunit. The cardiac glycoside site on the alpha-subunit was labeled with anthroylouabain [Fortes, P. A. G. (1977) Biochemistry 16, 531-540]. The oligosaccharides on the beta-subunit were labeled with lucifer yellow carbohydrazide [Lee, J. A., & Fortes, P. A. G. (1985) Biochemistry 24, 322-330]. Resonance energy transfer from anthroylouabain to lucifer yellow was measured by steady-state and time-resolved fluorescence spectroscopy. The distance between these probes was determined from the efficiency of energy transfer. The average distance between anthroylouabain and lucifer yellow was 47 A and was independent of the number of acceptor molecules attached to the beta-subunit. The measured distance corresponds to the distance between the cardiac glycoside site and the center of the labeled oligosaccharides on the beta-subunit within one alpha beta dimer. The distance was the same (47 A) when anthroylouabain was bound with ATP or Pi as phosphorylating ligands but increased to 49 A in the presence of vanadate. The change in average distance provides quantitative evidence of a conformational difference between the complexes of cardiac glycosides with (Na,K)-ATPase induced by phosphorylating ligands or by vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA.

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.

Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells.

Recently published data indicate that the alpha-subunit of Na,K-ATPase, a transmembrane protein of animal cell plasma membranes, is synthesized as a soluble precursor. In the present experiments we demonstrate that an apparent "soluble" form can indeed be detected in crude cytosolic fractions prepared by centrifugation from MDCK cells disrupted by sonication. We find, however, that this form has no precursor-product relationship with membrane-associated alpha-subunit. The quantity of unsedimentable alpha-subunit can be greatly diminished by increasing the centrifugal field employed to remove membranous vesicles from the cytosol fraction. Sonication of membrane pellets generates alpha-subunit which, like the "soluble" form, resists pelleting. Finally, cytosol fractions prepared from cells disrupted by sonication contain membrane-bound vesicles which can be immunoadsorbed on Staphylococcus aureus cells coated with a monoclonal antibody directed against alpha-subunit. We find, therefore, that the previously observed soluble precursor of alpha-subunit is actually a component of small unpelleted membrane vesicles generated by harsh disruption conditions. When cells are disrupted by less violent techniques no newly synthesized alpha-subunit can be detected in the cytosol fraction. We calculate that to escape detection under our experimental conditions a bona fide soluble precursor of alpha-subunit must have a cytosolic t1/2 less than 20 s. We conclude that the alpha-subunit is most probably inserted into the bilayer cotranslationally.

Plasma Membrane Proton Pumps in Animal Cells

Proton pumping in animal cell plasma membranes is mediated by at least three distinct types of transport systems and has a variety of functions. The gastric proton pump develops chemical gradients exceeding 6 pH units, secreting acid from the gastric mucosa to help digest food. Another ATPase/H+ pump, which regulates whole-body acid and bicarbonate excretion in kidney distal tubules, can develop acid gradients of 3 pH units. And ATP-independent Na+/H+ exchange systems and multiple-ion exchangers operate both to control intracellular pH and to maintain whole-body proton-bicarbonate balance.

Plasma membrane‐microfilament interaction in animal cells

AbstractMicrofilament interactions with the plasma membranes of animal cells appear to vary with cell type and localization. In the erythrocyte, actin oligomers are associated with the membrane via spectrin and ankyrin. The ends of stress fibers in cultured cells, such as fibroblasts, are attached to the plasma membrane at focal adhesion sites and may involve the protein vinculin as a linking protein. In intestinal brush border microvilli a 110,000 dalton protein links the microfilament bundles to sites on the microvillus. A transmembrane complex containing actin stably associated with a cell surface glycoprotein can be isolated from ascites tumor cell microvilli and can be obtained still associated with microfilaments by gentle extraction and gradient centrifugation of the microvilli. These varied interaction mechanisms are believed to be needed to satisfy the different structural and dynamic requirements of the particular systems.

On the structural organization of biological pumps and channels

The plasma membranes of most animal cells contain transport proteins which function to provide passageways for the transported species across essentially impermeable lipid bilayers. The channel is a passive transport system which allows the movement of ions and low molecular weight molecules along their concentration gradients. The pump is an active transport system and can translocate cations against their natural concentration gradients. The actions and interplay of these two kinds of transport proteins control crucial cell functions such as active transport, excitability and cell communication. In this paper, we will describe and compare several features of the molecular organization of pumps and channels. As an example of an active transport system, we will discuss the structure of the sodium and potassium ion-activated triphosphatase [(Na+ +K+)-ATPase] and as an example of a passive transport system, the communicating channel of gap junctions and lens junctions.

Specific receptor for ceruloplasmin in membrane fragments from aortic and heart tissues.

Membrane fragments from aortic and heart tissues of immature chicks were observed to bind highly purified, 125I-labeled chick ceruloplasmin. The binding reaction exhibited a linear Scatchard plot for both tissues and showed for each an apparent dissociation constant (Kd) of about 10(-8) M. On the basis of Scatchard analyses, aorta contained 1.5 pmol of receptors/mg of membrane protein, whereas receptors in the membranes from heart tissue were at least 5 times more dense. The binding of chick ceruloplasmin to aorta membranes was trypsin sensitive and neuraminidase insensitive, and showed both saturation and reversibility. Various sialoglycoproteins in 500 molar excess had very little effect on the binding. The asialo derivatives of these proteins likewise did not inhibit the binding. Human ceruloplasmin was found to bind very weakly to the chick membranes. Asialo chick ceruloplasmin bound with the same efficacy as native chick ceruloplasmin. Heat-denatured chick ceruloplasmin, however, was very ineffectual in displacing native 125I-ceruloplasmin from the membranes. These studies provide the first evidence for a homologous membrane receptor for native ceruloplasmin in the plasma membranes of animal cells.

Plasma Membrane ATPase of Fungi and Plants as a Novel Type of Proton Pump

Publisher Summary This chapter presents the evidence obtained at different levels of biological organization that characterizes the plasma membrane ATPase of fungi and plants as a novel type of proton pump. It presents a parallel discussion of studies with fungal and plant cells to stress the similarities of their bioenergetic mechanisms that plant physiologists and microbial biochemists may have failed to appreciate. The membranes of bacteria, mitochondria, and chloroplasts contain a similar ATPase involved in proton transport. These enzymes are sensitive to azide and dicyclohexylcarbodiimide and, in the case of mitochondria and photosynthetic bacteria, to oligomycin. They do not form phosphorylated intermediates and they have alkaline pH optima and very complicated structures. These enzymes have a hydrophilic catalytic part with five different subunits protruding from the membrane and a hydrophobic part embedded in the membrane with three different subunits that form a proton channel blocked by dicyclohexyl-carbodiimide. A different type of ATPase is found in the microsomes and plasma membranes of animal cells. These enzymes catalyze the exchange of sodium for potassium and of protons for potassium and calcium transport. The ATPase activities are dependent on the presence of the transported cations and the pH optima are neutral.

Dibutyryl cAMP-inducible alkaline phosphatase in animal cell plasma membranes: fluorescence detection of mutant clones on polyester cloth.

We have developed a rapid screening assay that allows us to estimate the alkaline phosphatase content of mouse L-M cell colonies immobilized on polyester cloth. This permitted the identification and isolation of two mutant clones with increased constitutive alkaline phosphatase activity and six clones that fail to express this activity when treated with dibutyryl cyclic AMP. Both of the strains with increased constitutive activity have basal enzymatic activities that are 6- to 7-fold higher than the activity of the parental strain. The extents to which the cyclic nucleotide further induces alkaline phosphatase in these two strains are different, however, indicating that they represent two classes of mutants. Studies using amino acids and synthetic peptides as alkaline phosphatase inhibitors suggest that only one alkaline phosphatase isoenzyme predominates, in both the parental and the mutant cell lines, with or without induction by cyclic nucleotide. Comparison to mouse tissues indicates that our cell lines express an isozyme resembling that found in kidney and bone. The six clones that fail to express alkaline phosphatase activity when treated with dibutyryl cyclic AMP also have extremely low basal levels of the enzyme. All of these mutant strains continue to synthesize protein when treated with dibutyryl cyclic AMP and undergo growth cessation and morphological changes in the presence of this agent. Thus, the mutations all appear to affect factors specific to the expression of alkaline phosphatase activity rather than factors that affect general cellular responsiveness or permeability to dibutyryl cyclic AMP. The characterization of these strains may help elucidate mechanisms of eukaryotic membrane protein biogenesis, enzyme induction, and regulation of gene expression by cyclic nucleotides.

Radiation effects on cell membranes

The recent developments in the field of membrane biology of eukaryotic cells result in revival of relevant radiobiological studies. The spatial relations and chemical nature of membrane components provide rather sensitive targets. Experimental data are presented concerning the effects of relatively low doses of X-irradiation and low concentration of tritiated water (HTO) on variou receptor function - concanavalin A, cationized ferritin, poliovirus - of plasma membranes of animal and human cells which point to early and temporary disturbances of the composite structures and functions of membranes. References are given to the multifold roles of radiation-induced membrane phenomena on the development and regeneration of radiation injuries. Invited plenary lecture presented at 16th Annual Meeting of the European Society for Radiation Biology, Krakow, Poland, 7–10 September 1981

Glycosphingolipids in cellular interaction, differentiation, and oncogenesis.

The idea that glycosphingolipids (or, briefly, glycolipids) are ubiquitous components of plasma membrane and display cell type-specific patterns perhaps stemmed from the classical studies on glycolipids of erythrocyte membranes.(1,2) Subsequently, plasma membranes of various animal cells were successfully isolated and analyzed; all were characterized by their much higher content of glycolipid than was found in intracellular membranes.(3–8) It is generally assumed that glycolipids are present at the outer leaflet of the plasma membrane bilayer, although this assumption is based only on experiments with surface-labeling by galactose oxidase-NaB[3H]4 of intact and lysed erythrocyte membranes and inside-out vesicles.(9,10) Obviously, further extensive studies of the organization of glycolipid in other eukaryotic cell membranes are necessary. Interestingly, the majority of neutral glycolipids present in plasma membranes are cryptic (see Section 4.2.1).

Proteins of Animal Cell Plasma Membranes, Volume 2 (Annual Research Reviews)

Proteins of Animal Cell Plasma Membranes, Volume 2 (Annual Research Reviews)

Evolution of membrane bioenergetics.

One of the first problems encountered by primitive cells was that of volume regulation; the continuous entry of ions, (eg, NaCl) and water in response to the internal colloid osmotic pressure threatening to destroy the cell by lysis. We propose that to meet this environmental challenge cells evolved an ATP-driven proton extrusion system plus a membrane carrier that would exchange external protons with internal Na+. With the appearance of the ability to generate proton gradients, additional mechanisms to harness this source of energy emerged. These would include proton-nutrient cotransport, K+ accumulation, nucleic acid entry, and motility. A more efficient system for the uptake of certain carbohydrates by vectorial phosphorylation via the PEP-phosphotransferase system probably appeared rather early in the evolution of anaerobic bacteria. The reversal of the proton-ATPase reaction to give net ATP synthesis became possible with the development of other types of efficient proton transporting machinery. Either light-driven bacterial rhodopsin or a redox system coupled to proton translocation would have served this function. Oxidation of one substrate coupled to the reduction of another substrate by membrane-bound enzymes evolved in such a manner that protons were extruded from the cell during the reaction. The progressive elaboration of this type of redox proton pump permitted the use of exogenous electron acceptors, such as fumarate, sulfate, and nitrate. The stepwise growth of these electron transport chains required the accretion of several flavoproteins, iron-sulfur proteins, quinones, and cytochromes. With modifications of these four basic components a chlorophyll-dependent photosynthetic system was subsequently evolved. The oxygen that was generated by this photosynthetic system from water would eventually accumulate in the atmosphere of the earth. With molecular oxygen present, the emergence of cytochrome oxidase would complete the respiratory chain. The proton economy of membrane energetics has been retained by most present-day microorganisms, mitochondria, chloroplasts, and cells of higher plants. A secondary use of the energy stored as an electrochemical difference of Na+ for powering membrane events probably also evolved in microorganisms. The exclusive age of the Na+ economy is distinctive of the plasma membrane of animal cells; the Na+-K+ ATPase sets up an electrochemical Na+ gradient that provides the energy for osmoregulation, Na+-nutrient co-transport, and the action potential of excitable cells.

Annual Research Reviews. Proteins of Animal Cell Plasma Membranes Vol. I

Annual Research Reviews. Proteins of Animal Cell Plasma Membranes Vol. I