Site of red cell cation leak induced by mercurial sulfhydryl reagents

It has been suggested that the human red cell anion transport protein, band 3, is the site not only of the cation leak induced in human red cells by treatment with the sulfhydryl reagent pCMBS ($\text{p-}\text{chloromercuribenzene sulfonate}$) but is also the site for the inhibition of water flux induced by the same reagent. Our experiments indicate that $\text{N-}\text{ethylmaleimide}$, a sulfhydryl reagent that does not inhibit water transport, also does not induce a cation leak. We have found that the profile of inhibition of water transport by mercurial sulfhydryl reagents is closely mirrored by the effect of these same reagents on the induction of the cation leak. In order to determine whether these effects are caused by band 3 we have reconstituted phosphatidylcholine vesicles containing only purified band 3. Control experiments indicate that these band 3 vesicles do not contain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ as measured by ATP dephosphorylation. pCMBS treatment caused a significant increase in the cation leak in this preparation, consistent with the view that the pCMBS-induced cation leak in whole red cells is mediated by band 3.     

Target analysis studies of red cell water and urea transport

Radiation inactivation was used to determine the nature and molecular weight of water and urea transporters in the human red cell. Red cells were frozen to -50 degrees C in a cryoprotectant solution, irradiated with 1.5 MeV electrons, thawed, washed and assayed for osmotic water and urea permeability by stopped-flow light scattering. The freezing and thawing process did not affect the rates of water or urea transport or the inhibitory potency of p-chloromercuribenzenesulfonate (pCMBS) on water transport and of phloretin on urea transport. Red cell urea transport inactivated with radiation (0-4 Mrad) with a single target size of 469 +/- 36 kDa. 40 microM phloretin inhibited urea flux by approx. 50% at each radiation dose, indicating that urea transporters surviving radiation were inhibitable. Water transport did not inactivate with radiation; however, the inhibitory potency of 2.5 mM pCMBS decreased from 86 +/- 1% to 4 +/- 9% over a 0-2 Mrad dose range. These studies suggest that red cell water transport either required one or more low-molecular-weight proteins, or is lipid-mediated, and that the pCMBS-binding site which regulates water flow inactivates with radiation. These results also suggest that red cell urea transport is mediated by a specific, high-molecular-weight protein. These results do not support the hypothesis that a band 3 dimer (190 kDa) mediates red cell osmotic water and urea transport.     

Chemical Modification of Membranes : 1. Effects of sulfhydryl and amino reactive reagents on anion and cation permeability of the human red blood cell

Four different amino-reactive reagents, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS),¹ 1-fluoro-2,4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and 2-methoxy-5-nitrotropone (MNT) decrease the anion permeability of the human red blood cell, as measured by sulfate fluxes, whereas the sulfhydryl agent, parachloromercuriphenyl sulfonic acid (PCMBS), does not. In contrast, PCMBS increases the cation permeability as measured by K⁺ leakage, whereas SITS does not. Of the other agents, FDNB increases the cation permeability to the same extent as PCMBS but MNT and TNBS produce smaller increases. PCMBS does not protect against FDNB as it does against other sulfhydryl agents (X-irradiation) and the FDNB effect on cations is attributed to amino groups. Studies of the binding of SITS indicate that it does not penetrate into the membrane and its failure to influence cation permeability is attributed to its inability to reach an internal population of amino groups. It is concluded that two ion permeability barriers, both involving proteins, are present in the red blood cell. The more superficial barrier contains amino groups and controls anion flow; the more internal barrier contains sulfhydryl and amino groups and controls cation flow. The amino groups contribute to the control of permeability by virtue of their positive charges, but the role of sulfhydryl groups is not clear. Only a small fraction of the membrane protein amino and sulfhydryl is involved in the barriers.     

Control of the mitochondrial inner membrane permeability by sulfhydryl groups

The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.     

Reflection coefficient and permeability of urea and ethylene glycol in the human red cell membrane

The reflection coefficient (sigma) and permeability (P) of urea and ethylene glycol were determined by fitting the equations of Kedem and Katchalsky (1958) to the change in light scattering produced by adding a permeable solute to a red cell suspension. The measurements incorporated three important modifications: (a) the injection artifact was eliminated by using echinocyte cells; (b) the use of an additional adjustable parameter (Km), the effective dissociation constant at the inner side of the membrane; (c) the light scattering is not directly proportional to cell volume (as is usually assumed) because refractive index and scattering properties of the cell depend on the intracellular permeable solute concentration. This necessitates calibrating for known changes in refractive index (by the addition of dextran) and cell volume (by varying the NaCl concentration). The best fit was for sigma = 0.95, Po = 8.3 X 10(-4) cm/s, and Km = 100 mM for urea and sigma = 1.0, Po = 3.9 X 10(-4) cm/s, and Km = 30 mM for ethylene glycol. The effects of the inhibitors copper, phloretin, p- chloromercuriphenylsulfonate, and 5,5'-dithiobis (2-nitro) benzoic acid on the urea, ethylene glycol, and water permeability were determined. The results suggest that there are three separate, independent transport systems: one for water, one for urea and related compounds, and one for ethylene glycol and glycerol.     

Water transport in human red cells: effects of 'non-inhibitory' sulfhydryl reagents

The water diffusional permeability of human red blood cells following exposure to various sulfhydryl group (SH) reagents have been studied using a nuclear magnetic resonance technique. Exposure of red blood cells up to 12 mM N-ethylmaleimide (NEM) or 10 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNE) alone does not affect water diffusion. In contrast, when DTNB treatment follows a preincubation of the cells with NEM, a small (18% at 37 degrees C) but significant inhibition of water permeability occurs. The NEM and DTNB treatment of the cells caused no change of the cell shape and volume or of the cell water volume. Consequently, the inhibition observed after NEM and DTNB treatment has a real significance.     

Comparative effects of two sulfhydryl reagents on the activities of a multifunctional red cell enzyme: bisphosphoglycerate mutase

The effects of two sulfhydryl reagents on the three activities of bisphosphoglycerate mutase have been compared. Under N-ethylmaleimide treatment all the activities were inhibited except for 60% of the non-stimulated phosphatase. With iodoacetamide the mutase and the stimulated-phosphatase activities were completely inhibited whereas the non-stimulated phosphatase and 60% of the synthase activities were unaffected. 2,3-bisphosphoglycerate protected all the activities of the enzyme against inactivation by the two sulfhydryl reagents whereas 3-phosphoglycerate protected them only against iodoacetamide. 2-phosphoglycolate had an identical effect to that of 3-phosphoglycerate except for its effect on the non-stimulated phosphatase activity, which was slightly enhanced under N-ethylmaleimide treatment.     

Interrelation of ethylene glycol,urea and water transport in the red cell

The reflection coefficient, σ_j, which measures the coupling between the jth solute and water transport across a semipermeable membrane, varies between 0 and 1.0. Values of σ_j significantly less than 1.0 provide irreversible thermodynamic proof that there is coupling between the transport of solute and solvent and thus that they share a common pathway. We have developed an improved method for measuring σ and have used it to determine that σ_{ethylene glycol} = 0.71 ± 0.03 and σ_urea = 0.65 ± 0.03, in agreement with many, but not all, previous determinations. Since both of these values are significantly lower than 1.0, they show that there is a common ethylene glycol/water pathway and a common urea/water pathway. Addition of first one and then two methyl groups to urea increases σ to 0.89 ± 0.04 for methylurea and 0.98 ± 0.4 for 1,3-dimethylurea, consistent with passage through an aqueous pore with a sharp cutoff in the 6–7 Å region.     

Osmotic water permeability of the human red cell. Dependence on direction of water flow and cell volume

The osmotic permeability coefficient (Pf) was measured with a stopped- flow light-scattering technique. There is an artifactual light- scattering signal produced by the initial mixing that decays with a half-time of approximately 0.2 s. This seriously interferes with the measurement of the osmotically induced change in cell volume, which has a similar half-time. This "injection artifact" is associated with the biconcave shape of the cells. It is negligible for cells that have been made nearly spherical by swelling them in 160 mosmol. The dependence of this artifact on the cell volume may explain the previously observed dependence of Pf on the cell volume. When cells are made echinocytic (and therefore spherically symmetric), this injection artifact becomes negligible at all cell volumes and Pf can be accurately measured. The Pf of echinocytic cells was nearly constant, varying by less than 10% with the direction of flow and the medium osmolarity (160-360 mosmol). The average value of Pf was 2.0 X 10(-2) cm/s (T = 23 degrees C).     

Effects of sulfhydryl and other reagents on the diffusional permeability of dog erythrocytes to small solutes

The effects of p-chloromercuriphenylsulfonic acid (PCMBS), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), phloretin and thiourea on the diffusional permeability of dog erythrocytes to tritiated water and to small 14C-labeled lipophilic and hydrophilic solutes were measured at 37 degrees C by means of the linear diffusion technique. Permeability to 3HHO was significantly decreased by PCMBS but was not affected by the other reagents. The permeability to the small hydrophilic solutes acetamide and urea was decreased by phloretin and thiourea but only the permeability to acetamide was reduced to a statistically significant extent by PCMBS. The permeability to the lipophilic solutes methanol, ethanol and antipyrine was not affected by any of these agents. We interpret these results as an indication that the small lipophilic solutes probably move through lipid areas, that the small hydrophilic solutes probably move through protein associated areas in the erythrocyte membrane and that pathways for the small hydrophilic solutes are distinct from those for water. While the pathways for water may be associated with membrane protein they do not appear to be associated specifically with band 3 protein as has been suggested for human erythrocytes. Diffusional water movement through the dog erythrocyte occurs by two distinct pathways.     

The release of membrane-bound calcium by radiation and sulfhydryl reagents

Effects of ionizing radiation and of sulfhydryl reagents on the ⁴⁵Ca binding of red cell membranes were studied. Corresponding effects of these agents on potassium leak from intact red cells were also determined. Essentially all the ⁴⁵Ca associated with the ghosts appeared to be bound. Calcium binding could be described by assuming two independent groups of binding sites with dissociation constants of about 6 × 10^?4 m and 2 × 10^?4 m. The total binding capacity was about 2.5 × 10^?4 moles/g ghost protein. Membrane calcium was decreased by radiation and by the two sulfhydryl reagents, p-chloromercuribenzoate (PCMB) and N-ethyl maleimide (NEM). The tightly bound calcium fraction appeared to be most affected by these agents. Changes in potassium leak evoked by varying doses of agents appeared to parallel effects on membrane calcium. These investigations suggest that the increased cation permeability observed after exposure or red cells to radiation or sulfhydryl reagents may be related to alterations in the calcium-binding properties of the cell membrane.     

Inactivation of human lactate dehydrogenase isozymes by sulfhydryl reagents

Human lactate dehydrogenase isozymes, LDH-1 and LDH-5, were inactivated at 25 degrees C and pH 7.5 by N-alkylmaleimides of varying chain length, and by fluorescein mercuric acetate. Second-order rate constants for the inactivation of LDH-5 by N-alkylmaleimides increased with increasing chain length of the maleimide derivative while essentially no chain-length effect was observed in the inactivation of LDH-1. Both isozymes were effectively inactivated by low concentrations of fluorescein mercuric acetate, and in both cases saturation kinetics were observed. Dissociation constants obtained from double-reciprocal plotting methods indicated a twofold better binding of fluorescein mercuric acetate to LDH-1. Protection from fluorescein mercuric acetate by NAD was observed with both enzymes.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Osmotic water permeability of human red cells

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.     

Inhibition of lectin-induced lymphocyte activation by diamide and other sulfhydryl reagents

Inhibition of lectin-induced lymphocyte activation by five reagents capable of combining with or oxidizing free sulfhydryl groups was examined. Each of the reagents tested was capable of inhibiting [methyl-³H]thymidine or [¹⁴C]uridine incorporation into trichloroacetic acid-insoluble material. Four of these reagents, iodoacetamide and N-ethylmaleimide (alkylating agents) and 5,5′-dithiobis (2-nitrobenzoic acid) and p-hydroxymercuriphenylsulfonic acid (sulfhydryl binding agents), inhibited activation when added to lymphocyte cultures together with lectin or at any time thereafter through 48 hr. In contrast, the sulfhydryl oxidizing agent diazine dicarboxylic acid bis[N,N-dimethylamide] (diamide) was effective only when added within 30–60 min of lectin or when added after 24 hr. This inhibition of lymphocyte activation was not due to decreased intracellular levels of reduced glutathione or to inhibition of binding of lectin to the lymphocyte. These results suggest that maintenance of free sulfhydryl groups is important during the early induction of lymphocyte activation and suggest that an obligatory step or steps in the activation sequence may involve sulfhydryl interactions.     

Inhibition of glycolipid biosynthesis in chloroplasts by ozone and sulfhydryl reagents

The metabolism of uridine 5′-pyrophosphate-galactose by spinach (Spinacia oleracea) chloroplast preparations was inhibited by ozone. The formation of digalactosyl diglyceride and trigalactosyl diglyceride was inhibited much more than the formation of monogalactosyl diglyceride, steryl glycoside, and acylated steryl glycoside. Essentially identical results were obtained when glycolipid synthesis was inhibited by N-ethyl maleimide, p-hydroxymercuribenzoate, and CdCl₂. Iodoacetate and iodoacetamide affected neither the total incorporation of sugar from uridine 5′-pyrophosphate-galactose nor distribution of the incorporated sugar in the various glycolipids.