Aser Rothstein

The nature of the membrane sites controlling anion permeability of human red blood cells as determined by studies with disulfonic stilbene derivatives.

SummaryThe disulfonic acid stilbene derivative SITS reported to be covalently bonded to the membrane of the red blood cell, was found to be largely reversibly bound. Reversal of its specific inhibitory effect on anion permeability was attained by washing the cells with buffer containing albumin. The small fraction of covalently bonded SITS could be increased by prolonging the time of exposure of the cells or by multiple exposures. A series of other disulfonic stilbene derivatives was synthesized. All of them specifically inhibited anion permeability whether or not they are capable of forming covalent bonds. Their inhibitory effectiveness, however, varied over a 5,000-fold range, allowing certain conclusions to be made concerning the chemical architecture of the binding site. Certain of the compounds were almost entirely covalently bonded. One of them was labeled with125I and used to determine to which membrane proteins the compound is bound. Over 90% was found in a protein band on acrylamide gels of 95,000 mol wt. The most effective compound against sulfate permeability was equally effective against chloride permeability, producing a maximum inhibition of over 95%. The residual anion fluxes respond differently to pH and temperature than do the fluxes of unmodified cells.

Sulfhydryl Groups in Membrane Structure and Function

Publisher Summary The sulfhydryl group is one of the most reactive and ubiquitous ligands in biological systems. It is found in most proteins and also in a few low molecular-weight substances such as glutathione, CoA, lipoate, thioglycolate, and free cysteine. It is the most studied of ligands, particularly in relation to its role in enzymic activity and properties of proteins. It is also involved in many membrane functions, because chemical agents with a degree of specificity for sulfhydryl can disturb many functions attributed to the cell membrane. The sulfhydryl group not only constitutes a unique marker for delineating the general role of proteins in membrane functions, it can also serve as a marker for specific functional proteins through the use of radioactive reagents that form stable bonds with sulfhydryls. The various factors that are assumed to play a role in determining reactivity of particular sulfhydryl groups in proteins are discussed in the chapter. The mechanism of action of sulfhydryl agents and the functional role of membrane sulfhydryl groups are also discussed.

The transport of Zn2+, Co2+ and Ni2+ into yeast cells

Abstract Ni 2+ , Co 2+ , Zn 2+ can be taken up into a non-exchangeable pool by yeast cells, by a system that also transports Mg 2+ and Mn 2+ . The affinity series is Mg 2+ , Co 2+ , Zn 2+ > Mn 2+ > Ni 2+ > Ca 2+ > Sr 2+ . The uptake is small in starved cells, but is enhanced in the presence of glucose. It is remarkably stimulated (5–20 fold) if cells are pretreated with phosphate and glucose. The uptakes are the same under aerobic or anaerobic conditions suggesting that fermentative reactions can supply the energy for transport. Uptake is reduced at low pH (below 5.0), but a H + exchange system is not involved. Instead, 2 K + (or 2 Na + in Na + -loaded cells) are secreted for each divalent cation absorbed.

Synthesis of tritiated 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells.

SummaryThe potent and specific inhibitor of anion permeability, 4,4′-diisothicyanostilbene-2,2′-disulfonic acid (DIDS) was synthesized in tritiated form ([3H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([3H]H2DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [3H]DIDS was substantially faster than that of [3H]H2DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [3H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×106 molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.

Changes in the properties of human erythrocyte membrane protein after solubilization by butanol extraction

1. 1.|Human erythrocyte membrane protein is readily solubilized and freed of most of the membrane lipid after a single extraction of an aqueous suspension of hemoglobin-free ghosts with n-butanol. The technique yields a mean recovery of 83.1% ± 3.8 (S.D., n = 10) of the membrane protein in the butanol-saturated water phase. 2. 2.|The mean amount of lipid present in the water phase after a single extraction is 4.8% ± 3.9 (S.D., n = 6) of the weight of protein. Loss of lipid is symmetrical except that a relatively high proportion of phosphatidyl serine remains. Analysis of the chemical composition of the protein indicates that it is a glycoprotein, which contains hexose, hexosamine, fucose and sialic acid. Carbohydrates account for about 9% of the protein by weight. 3. 3.|Approximately 98% of the protein has a single electrophoretic mobility and appears as a single peak in the void volume after gel column chromatography. Ultracentrifugal analysis, however, demonstrates heterogeneity of the protein. The protein is soluble at neutral pH in salt-containing aqueous media and has an isoelectric point between pH 4.0 and 5.0. 4. 4.|Solubilization of membrane protein by treatment with n-butanol results in a minimal (5%) increase in p-chloromercuriphenylsulfonate titratable SH− groups. 5. 5.|The cation-independent nucleosidetriphosphatase and alkaline monoester phosphydrolase activities of intact stroma were recovered to the extent of 73% to 107% in the solubilized protein. Recoveries of acid phosphohydrolase were 40% at pH 7.0 and 30% at pH 6.0. 6. 6.|The prominent cation activation of both nucleosidetriphosphatase and acid phosphohydrolase activities in stroma was virtually lost on solubilization of the protein. In the case of nucleosidetriphosphatase the concentration of butanol necessary for loss of activation by Mg2+ or by Mg2+, Na+ and K+ was sufficiently high (above 500 mM) to cause disintegration of structure.

The cation permeability of erythrocytes in low ionic strength media of various tonicities

SummaryThe steady state passive efflux of salt from human red blood cells was measured in various low ionic strength media in which the osmotic pressure ranged from 200 to 600 milliosmolar. Sucrose was used as the nonpenetrating nonelectrolyte. If the flux is plotted against the log of the salt concentration, the data for each tonicity can be fitted by three straight-line segments separated by two sharp inflections, one at low external salt concentrations (0.1 to 0.3mM), confirming observations of LaCelle and Rothstein, and a second at higher salt concentrations (20 to 50 mM). As the osmolarity of the medium is increased, the inflection in every case seems to be uniquely determined by the membrane potential calculated from the Nernst equation with use of the chloride ratio. One inflection occurs at about 45 mV and the second at 170 mV in experiments at five different tonicities. Calculations from the Goldman equation suggest that the inflections represent potential-dependent changes to new permeability states. The osmotic pressure of the medium also influences the permeability. The coefficient is systematically reduced as the osmotic pressure is increased.

Water-soluble proteins of the human red cell membrane

SummaryProcedures were developed for preparation of red cell membranes almost free of hemoglobin but with minimal loss of membrane proteins. Two water-soluble protein fractions are described, each constituting about 25% of the ghost protein. The first is ionically bonded and can be solubilized in water rapidly at pH 7.0 and more slowly at higher ionic strength solutions, with a minimal rate at 20mm. This fraction contains four major components with molecular weights ranging from 30,000 to 48,000. The second fraction can only be solubilized at an appreciable rate if Ca++ is absent and at higher pH (9.0). It is predominantly a single molecular weight component (150,000). It tends to aggregate at higher ionic strength and in the presence of Ca++. Evidence is presented suggesting that the water-soluble proteins are present at the inner face of the membrane. The lipids remain in a water-insoluble residue that contains four major protein components ranging in molecular weight from 30,000 to 100,000. The latter is the predominant component. Only the residue contains the Na+−K+-activated ATPase, the cholinesterase, antigenic activity and most of the sialic acid and carbohydrate. The first water-soluble fraction contains a Mg++-activated ATPase. The extraction of the water-soluble proteins is accompanied by anatomical changes resulting finally in the formation of small membranous vesicles.

The relationship of the cell surface to metabolism. XIII. The cation-binding properties of the yeast cell surface

Abstract The binding of exogenous bivalent and univalent cations by the yeast cell is rapid and reversible, obeying a simple mass-law equation. Bivalent cations, especially UO 2 ++, are bound more firmly than are univalent cations. There are at least two species of binding sites, tentatively identified as phosphoryl and carboxyl groups. These are located at the periphery of the cell, isolated from endogenous cations by a permeability barrier.

Volume regulation of Chinese hamster ovary cells in anisoosmotic media.

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.

Effects of quinine on Ca++-induced K+ efflux from human red blood cells

SummaryThe Ca++-mediated increase in K+-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to known concentrations of Ca++ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K+ concentration. In K+-free solutions the concentration of quinine to achieve 50% inhibition (K50) was 5 μm, but at 5mm K+ the required concentration was increased 20-fold to 100 μm. An increase in internal Na+ had the opposite effect, allowing a high potency of quinine despite the presence of external K+. Alterations in the internal K+ level, on the other hand, were without effect on theK50, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl− by NO3−, a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K+ from an external binding site, the reported K+-activation site for the Ca++-mediated K+-permeability.