Sialic acid, electrophoretic mobility and transmembrane potentials of the Amphiuma red cell.

Red cells from the giant salamander Amphiuma means are shown to contain sialic acid. The amount removed by the action of neuraminidase is equal to that released by acid hydrolysis, indicating that all of the sialic acid is present on the outer surface of the plasma membrane. These cells have a negative electrophoretic mobility and 100% enzymatic removal of sialic acid results in a 40% reduction in the mobility, suggesting that either a fraction of the sialic acid carboxyl groups are unavailable to the action of external electric fields, or other negatively charged groups contribute to the surface charge. A further reduction in mobility of normal and sialic acid-free cells is caused by an increased extracellular calcium concentration. The negative groups affected by calcium are most likely to be phosphate groups, since the isoelectric point of the cells is found to lie between the pK values for H2PO-4 groups and the carboxyl groups of sialic acid. Membrane potentials of single cells, from which 80% or more of the total sialic acid had been removed, were identical to those measured in normal cells, confirming that sialic acid plays little, if any, direct role in the maintenance of membrane potentials and ionic permeabilities.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

Cell surface charge alterations occurring during dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells

Dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells caused a decrease in net negative cell surface charge which began two days after exposure to the polar solvent and continued throughout the maturation process. Neuraminidase treatment caused a marked reduction in mobility of both untreated and dimethylsulfoxide-treated cells suggesting that sialic acid residues are the major anionogenic moieties of the surface membrane of Friend cells. A decrease in the content of total glycosidically bound sialic acid in dimethylsulfoxide-treated cells also occurred. The findings provide evidence to support an association between erythrodifferentiation of Friend cells and net negative surface charge dependent upon sialic acid residues.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Inhibition of ACTH-stimulated steroidogenesis in isolated rat adrenal cells treated with neuraminidase

The possible role of membrane sialic acid in the action of ACTH was investigated in rat adrenal cells. After treatment with neuraminidase, the cells showed a diminished steroidogenic response to ACTH while the response to cyclic AMP and dibutyryl cyclic AMP was unaffected. 11β-hydroxylation of deoxycorticosterone (DOC) was also not impaired. Dose response curves for three ACTH peptides (ACTH_1–39′, ACTH_1–24 and ACTH_1–10) with neuraminidase treated cells suggest that sialic acid residues on the glycoproteins of the plasma membrane may either impart affinity to the plasma membrane for ACTH molecule or facilitate transmission of the signal arising from ACTH-receptor interaction to the catalytic site of adenyl cyclase.     

Robonucelic acid within the cellular peripheral zone and the binding of calcium to ionogenic sites

Ribonuclease was shown to reduce the electrophoretic mobility of a line of cultured mammalian cells (RPMI no. 41), and Ehrlich ascites tumour cells. No reduction was detected in the case of human, mouse or embryonic chick erythrocytes. These data, taken with the various controls, support the hypothesis that RNA is a structural component of the peripheries of two types of cells, but not of erythrocytes from three species. Calcium-binding was studied in RPMI no. 41 cells, Ehrlich ascites tumour cells, and human and mouse eryhrocytes, by measurement of reduction in cellular electrophoretic mobility in suspending solutions containing various concentrations of calcium chloride. The effect of treating cells with neuraminidase and/or ribonuclease on calcium-binding was also studied. The results suggest that less calcium binds to the carboxyl groups of peripheral sialic acids than to the phosphates of peripheral, structural RNA. However, calcium apparently binds most avidly to as yet unidentified anionic sites.     

An improved method for the measurement of total lipid-bound sialic acids after cleavage of α2,8 sialic acid linkage withVibrio cholerae sialidase in the presence of cholic acid,SDS and Ca2+

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD₃ and GD₂) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca²⁺ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994).     

The ionic dependence of voltage-activated inward currents in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of Caenorhabditis elegans consists of a syncytium of radially orientated muscle cells that contract synchronously and rhythmically to ingest and crush bacteria and pump them into the intestine of the animal. The action potentials that support this activity are superficially similar to vertebrate cardiac action potentials in appearance with a long, calcium-dependent plateau phase. Although the pharyngeal muscle can generate action potentials in the absence of external calcium ions, action potentials are absent when sodium is removed from the extracellullar solution (Franks et al. 2002). Here we have used whole cell patch clamp recordings from the pharynx and show low voltage-activated inward currents that are present in zero external calcium and reduced in zero external sodium ions. Whilst the lack of effect of zero calcium when sodium ions are present is not surprising in view of the known permeability of voltage-gated calcium channels to sodium ions, the reduction in current in zero sodium when calcium ions are present is harder to explain in terms of a conventional voltage-gated calcium channel. Inward currents were also recorded from egl-19 (n582) which has a loss of function mutation in the pharyngeal L-type calcium channel and these were also markedly reduced in zero external sodium. Despite this apparent dependence on external sodium ions, the current was partially blocked by the divalent cations, cadmium, barium and nickel. Using single-channel recordings we identified a cation channel for which the open-time duration was increased by depolarisation. In inside-out patches, the single-channel conductance was highest in symmetrical sodium solution. Further studies are required to determine the contribution of these channels to the pharyngeal action potential.     

Study of carboxyl groups on the human blood platelet membrane

The authors have studied the whole carboxyl groups of the platelet membrane using liquid phase electrophoresis and an initial step of activation of the acid group by a water soluble carbodiimide (ethyl-l-diméthyl-aminopropyl-3-carbodiimide), followed by the action of a nucleophilic molecule. The calculation based on the theory of Gouy-Chapman have shown the existence of 24, 7.10(5) groups per cell. That is a number about 2.5 fold superior to the carboxyl groups of sialic acid as determined by the action of neuraminidase.     

Surface Anionic Groups in Symbiote-Bearing and Symbiote-Free Strains of Crithidia deanei1

The surface anionic groups of symbiote-bearing and symbiote-free strains of Crithidia deanei were compared by determining cellular electrophoretic mobility, by ultrastructural cytochemistry, and by identification of sialic acids by thin-layer and gasliquid chromatography. Symbiote-free Crithidia deanei has a highly negative surface charge (-0.9984 μm?s^-1? V^-1? cm), which is slightly reduced (-0.8527 μm?s^-1? V^-1? cm) by the presence of the endosymbiote. Treatment of both strains of C. deanei with neuraminidase decreased significantly the electrophoretic mobility of cells toward the cathodic pole, indicating the existence of exposed sialic acid residues responsible for the negative charge on the protozoan cell surface. Thin-layer and gas-liquid chromatography showed that N-glycolyl- and N-acetylneuraminic acids were present in both strains of C. deanei.     

A 1H STD NMR spectroscopic investigation of sialylnucleoside mimetics as probes of CMP-Kdn synthetase

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and CTP to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP _i . Saturation Transfer Difference (STD) NMR spectroscopy has been employed to investigate the sub-structural requirements of the enzyme’s binding domain. Sialylnucleoside mimetics, where the sialic acid moiety has been replaced by a carboxyl group and a hydrophobic moiety, have been used in NMR experiments, to probe the tolerance of the CMP-Kdn synthetase to such replacements. From our data it would appear that unlike another sialylnucleotide-recognising protein, the CMP-Neu5Ac transport protein, either a phosphate group or other functional groups on the sialic acid framework may play important roles in recognition by the synthetase. Dedicated to the memory of Professor Dr Yasuo Inoue     

Plasmodium lophurae: Membrane Proteins of Erythrocyte-free Plasmodia and Malaria-Infected Red Cells*

SYNOPSIS. Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitrogen decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm³, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm³ and 1.158 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230,000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Importance of Cationic Channels for Functioning of the Nuclear Envelope of Neurons as a Calcium Store

The membrane of the endoplasmic reticulum is, in fact, an extension of the nuclear envelope of eukaryotic cells; both these compartments can fulfill the function of intracellular calcium stores. Using a patch-clamp technique, we studied the biophysical properties of the channels expressed in the inner nuclear membrane of pyramidal neurons of the rat hippocampal CA1 area, in particular of large-conductance cationic channels and calcium channels of inositol trisphosphate receptors (the main channels in membranes of this type). As the results of the measurements showed, the activity of channels of both types demonstrates clearly pronounced voltage dependences. The probability of their open state (P _o) depends on the voltage inside the nuclear envelope lumen. At positive potentials, the activity of these channels is significantly more intense than at negative potentials. Moreover, channels of both types are reversibly blocked at considerable negative potentials. We believe that this property of ion channels in the nuclear envelope is an important factor responsible for the control of calcium signals in the cell nucleus. We propose a hypothesis on the mechanism underlying termination of Ca²⁺ release from such intracellular stores, which is based on the specificity of the voltage dependence of ion channels of the above-mentioned types.     

Effects of Photodynamic Action on the Cell Membrane of Euglena

The effects of photodynamic action on the cell membrane of Euglena gracilis were investigated by means of studies on dye binding and electrophoretic mobility. Molecular species of alkaline or acid dyes can penetrate the membrane to about the same extent. Once the cell has been injured by pbotodynamic action, ils ahility to exclude large ions is partially lost; it becomes greatly more permeable to dye anions. Binding of rose bengal induces an increased negative charge on Euglena cells which is reversed by subsequent photosensitized damage to the cells.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Effects of membrane potential on sodium-dependent calcium uptake by sarcolemma-enriched preparations from canine ventricle

Summary The effect of membrane potential on sodium-dependent calcium uptake by vesicles in an isolated cardiac sarcolemma preparation was examined. Initial time course studies showed that the reaction deviated from initial velocity conditions within minutes. This appeared to be due, in part, to loss of the sodium gradient. Assays carried out to 10 sec revealed a linear component of uptake (2 to 10 sec) and a faster component (complete by 2 sec). The latter was eliminated by loading the preparation with ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid (EGTA). This maneuver did not affect the slow component, and subsequent studies used preparations containing EGTA. Potassium Nernst potentials (E _K), established by potassium gradients in the presence of valinomycin, were varied from –100 to +30 mV by changing [K⁺] _o from 1.18 to 153.7mM ([K⁺] _i =50mM). The initial velocity of sodium-dependent calcium uptake was stimulated twofold by changingE _K from –100 to 0 mV and another twofold by raisingE _K from 0 to +30 mV. For the total range ofE _K and [K⁺] _o , 32 to 36% of the increase appeared to reflect stimulation by extravesicular potassium. The remainder appeared to be due to membrane potential. The profile of sodium-dependent calcium uptake versusE _K suggested that calcium influx through electrogenic sodium/calcium exchange may be much more affected by the positive region of the cardiac action potential than by the negative region.     

Cytochemistry of colloidal iron binding to the surface of Hela cells and human erythrocytes.

It seems from the literature that colloidal iron (C.I.) binding sites on cell surfaces cannot be completely removed by treatment with Vibrio Colerae alpha-neuraminidase. We wondered if C.I. particles bind to negative groups other than the carboxyl groups of sialic acids. Using HeLa cells from suspension cultures and fresh human erythrocytes, we examined, with the transmission electronmicroscope, the influence of the following enzymatic and histochemical treatments on C.I. staining: alpha-neuraminidase; hyaluronidase; ribonuclease; alpha-amylase; mild methylation (MM); MM + saponification (Sap.); MM + Sap +MM; MM + Sap + alpha-neuraminidase; active methylation (AM); AM + Sap; AM + Sap + AM; AM + Sap + alpha-neuraminiadase; CH3OH (80%); Sap. It seemed from these experiments that the carboxyl groups of alpha-neuraminidase sensitive sialic acids constitute the majority of binding sites for C.I. to these particular cells. The most interesting candidates for the residual binding of C.I. are carboxyl groups of alpha-neuraminidase resistant molecules, sulfon, sulfin, and sulfate groups.     

Two selective filters in the calcium channel of the molluscan neuron somatic membrane

Modification of the calcium channel of the somatic membrane of molluscan neurons under the influence of EDTA and other Ca-binding agents was investigated. The results showed that there are two selective filters in the calcium channel of this membrane. The first is located near the outer pore of the calcium channel and it binds bivalent cations in the order:pK _Ca:pK _Sr:pK _Ba:pK _Mg=6.6:5.5:4.8:4.2. This external filter regulates selectivity of the channel relative to the charge of the cation and it conjecturally contains several carboxyl groups. The second selective filter lies inside the channel and regulates permeability for ions with a single charge. It is suggested that the structure of the inner filter closely resembles that postulated by Hille for the selective filter of the sodium channel, and that it contains one carboxyl group. The results of investigation of the effect of Ca⁺⁺, Cd⁺⁺, and H⁺ on the fast sodium current of the somatic membrane showed that it is not blocked by these ions, but the decrease observed in its amplitude is connected with a change in the membrane surface potential and a corresponding change in the juxtamembranous concentration of carrier ions. On the basis of the experimental results it is postulated that the selective filter of the fast sodium channel of the molluscan neuron somatic membrane does not contain a carboxyl group.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 15, No. 4, pp. 420–427, July–August, 1983.     

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Studies on Electrogenesis under High K+ Concentrations in Internodal Cells of Chara corallina

+ concentration ([K⁺]_o) on the membrane potential (E_m) of Chara corallina was studied. E_m more negative than -100 mV was maintained even at 100 mM [K⁺]_o. Addition of Ca²⁺ to the external medium further increased this tendency. However, E_m responded sensitively to the increase in [K⁺]_o, when the electrogenic proton pump of the plasma membrane was inhibited by treating cells with dicyclohexylcarbodiimide, an inhibitor of proton pump. Analysis using equivalent circuit model of the plasma membrane suggested that the electrogenic proton pump was activated by the increase in [K⁺]_o. In the presence of 100 mM K⁺, action potentials were generated by electric stimuli. The ionic mechanism of generation of action potentials in the presence of K⁺ at high concentration was discussed. Received 3 October 2000/ Accepted in revised form 6 January 2001