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.     

Localization of Erythrocyte Membrane Sulfhydryl Groups Essential for Glucose Transport

The reactions of three organic mercurial compounds, chlormerodrin, parachloromercuribenzoate (PCMB), and parachloromercuribenzenesulfonate (PCMBS) with intact red blood cells, hemolyzed red cells, hemoglobin solutions, and hemoglobin-free ghosts have been characterized. Both PCMB and PCMBS react with only 2 to 3 sulfhydryl groups per mole of hemoglobin in solution, whereas chlormerodrin reacts with 6 to 7. In hemoglobin-free ghosts, however, all three reagents react with a similar number of sulfhydryl groups, approximately 4 x 10^-17 moles per cell, or about 25 per cent of the total stromal sulfhydryl groups, which react with inorganic mercuric chloride. In the intact cell the membrane imposes a diffusion barrier; chlormerodrin and PCMB penetrate slowly, whereas PCMBS does not. Kinetic studies of chlormerodrin binding to intact cells reveal that the majority of stromal sulfhydryl groups is located inside the diffusion barrier, with only 1 to 1.5 per cent (or 1 to 1,400,000 sites per cell) located outside of this barrier. Reaction of PCMBS with intact cells is limited to this small fraction on the outer membrane surface. All three reagents are capable of inhibiting glucose transport in the red cell. With chlormerodrin and PCMBS it was demonstrated that the inhibition results from interactions with the sulfhydryl groups located on the outer surface of the membrane.     

Attachment of immunoglobulin to liposomal membrane via protein carbohydrate

A general method has been developed for the covalent attachment of immunoglobulin molecules to the outer layer of liposomal membranes. Aldehyde groups are generated by the mild oxidation with periodate or galactose oxidase of the carbohydrate groups on the constant region of the heavy chain. The oxidized protein is then reacted with a hydrazide group linked to a membrane component. Detailed studies were carried out with monomers of a monoclonal human IgM and two monoclonal murine IgM antibodies specific for the 1-dimethylaminonaphthalene-5-sulfonyl (Dns) group. Two hydrazide-containing hydrophobic reagents were used: N^α-lauroyl, N-Dns-lysine hydrazide and lauric acid hydrazide. The number of protein aldehyde groups formed was assayed by reaction with N-(2,4-dinitrophenyl)-β-alanylglycylglycine hydrazide. Measurement of the intrinsic affinity for Dns-lysine of the processed anti-Dns IgMs demonstrated no substantial impairment of the specific reactivity of the antibody either from the oxidation step or the subsequent attachment to small unilamellar vesicles. The extent of attachment of antibody to small unilamellar vesicles was evaluated with respect to the mol% of hydrazide in the membrane, the duration of the incubation period for the aldehyde-hydrazide reaction and the ratio of protein to hydrazide. The yield of attached protein was significantly dependent on each of these experimental parameters over the ranges tested. Under the most favorable conditions the extent of covalent attachment of IgMs to small unilamellar vesicles was 535 μg of protein per μ mol of phospholipid, corresponding to 0.3 mol% of protein. Under these conditions, 61% of the total protein was associated with the small unilamellar vesicle fraction after fractionation by gel filtration. The attachment of the antibody to small unilamellar vesicles did not destroy the integrity of the vesicles, as demonstrated by the retention of carboxyfluorescein following initial encapsulation during the formation of small unilamellar vesicles.     

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled byN-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites are preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.     

Uptake of proteins by red blood cells

During hypotonic hemolysis red cells can take up ¹²⁵I-myoglobin and ¹²⁵I-immunoglobulin G. Cells which contain these proteins have distinctive cell morphology and are called gray ghosts. The association of protein with gray ghosts is fairly stable: these cells retain half of the proteins after 3 days. Passive diffusion of protein into the internal cell volume is the most plausible mechanism for uptake, and several lines of evidence indicate that the loaded proteins are freely diffusable within the red cells. Bacteriophage T4 is not taken up during hemolysis so uptake through large gaps in the red cell membrane with subsequent resealing seems unlikely. If an efficient procedure for fusing loaded gray ghosts to culture cells can be devised, it will be possible to introduce selected macromolecules into the cytoplasm of culture cells quite easily.     

N-chloroacetyl-[125I]iodotyramine: an alkylating agent with high specific activity

The preparation of iodinated N-chloroacetyltyramine and its evaluation as a specific sulfhydryl reagent are described. N-Chloroacetyltyramine was synthesized by a carbodiimide-mediated condensation of chloro- or iodoacetic acid and tyramine·HCL, and the crystalline product was iodinated in a reaction with chloramine T to yield either a 3,5-[¹²⁵I]diiodotyramine derivative, or a trace-iodinated product when carrier-free ¹²⁵I was employed. These iodinated derivatives react specifically with sulfhydryl groups, as judged by their ability to label reduced but not unreduced ribonuclease A and immunoglobulin E. Specific activities of 1 Ci/mmole in ¹²⁵I or ¹³¹I can be readily achieved with both the diiodinated and trace-iodinated (carrier-free) derivatives, and the specific activity of the former can be used directly to quantitate sulfhydryl groups in subnanomolar quantities of protein. N-Chloroacetyl ¹²⁵I-labeled tyramine prepared by trace iodination with carrier-free ¹²⁵I is more useful when very high specific activities (100–1000 mCi/μmol) are required. The utility of these reagents is discussed.     

Enzymatic inactivation of human alpha-1-proteinase inhibitor by neutrophil myeloperoxidase.

Peanut agglutinin was acylated with a new heterobifunctional, cleavable photosensitive crosslinking reagent, N-[4-(p-azidophenylazo)benzoyl]-3-aminopropyl-N′-oxysuccinimide ester. The lectin derivative binds specifically and reversibly to neuraminidase-treated human erythrocyte ghosts and upon irradiation covalent attachment of over 35% of the bound lectin occurs. The affinity-crosslinked ghosts were solublized in deoxycholate, immunoprecipitated with anti-peanut agglutinin antiserum, and analyzed by sodium dodecylsulfate polyacrylamine gel electrophoresis. Bands containing both peanut agglutinin and membrane glycoproteins were detected with apparent molecular weights of 58 000, 85 000, 110 000 and 135 000. Upon subsequent cleavage with sodium dithionite, asialoglycophorin A (apparent M.W. 41 000 and 85 000) and a second glycoprotein (apparent M.W. 58 000 – 61 000) were tentatively identified as the receptors for peanut agglutinin in the intact membrane.     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

Anion transport in red blood cells and arginine-specific reagents: The location of [14C]Phenylglyoxal binding sites in the anion transport protein in the membrane of human red cells

The reaction of phenylglyoxal, a reagent specific for arginine residues, with erythrocyte membrane at pH 7.4 results in complete inhibition of sulfate equilibrium exchange across human red cells. The inactivation was found to be concentration and time depenent. The binding sites of this reagent in the anion transport protein (band 3) under these conditions were determined by using [¹⁴C]phenylglyoxal. The rate of incorporation of the radioactivity into band 3 gave a good correlation with the rate of inactivation. Under conditions where the transport is completely inhibited about 6 mol [¹⁴C]phenylglyoxal are incorporated into 1 mol band 3. Treating the [¹⁴C]phenylglyoxalated ghosts at different degrees of inactivation with extracellular chymotrypsin showed that about two-thirds of these binding sites are located on the 60 kDa fragment.     

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyte ghosts

Incubation of erythrocyte ghosts with carbonylcyanide m-chlorophenylhydrazone (CCCP) plus Ca²⁺ resulted in inactivation of the Ca²⁺-stimulated ATPase activity. Omission of Ca²⁺ or lowering of the temperature below 25 °C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of β-mercaptoethanol did not influence the Ca²⁺-dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca²⁺-containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(β-aminoethyl acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca²⁺-stimulated, Mg²⁺-ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg²⁺-ATPase slightly. CCCP was much more specific for the Ca²⁺-stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca²⁺ for inactivation was also quite specific.     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn²⁺ and is used in vivo.The mean lifetime of water inside human erythrocytes was found to be 17 ms at 24°C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

A sensitive gel filtration method for determination of protein sulfhydryl groups with 14C-chloromercuribenzoate

A gel filtration method employing ¹⁴C-chloromercuribenzoic acid is described for the quantitative determination of sulfhydryl groups in microgram quantities of protein. The method has been applied to several native proteins, hemoglobin, monoamine oxidase, and yeast cytochrome c. In all cases values in close agreement with known literature values were obtained. Horse heart cytochrome c and lysozyme, which have no sulfhydryl groups, did not bind the mercurial reagent. Modifications of the method are described for determining the sulfhydryl content of denatured proteins in the presence of sodium lauryl sulfate. The precision of the method was found to be compatible with known methods for determining the sulfhydryl composition of proteins.     

Distribution of negatively charged liposomes on rat liver hepatocytes and non-hepatocytes in cell suspension

The in vitro interactions between negatively charged multilamellar liposomes and purified rat liver parenchymal and non-parenchymal cells were studied. The liposomes were labelled with [¹⁴C]cholesterol and contained [³H]methotrexate. For both cell types the time course of liposomal attachment to the cells slowed down gradually after a rapid initial phase lasting ca 90 min. The rate of attachment at 4 °C was 3–7 times lower than that at 37 °C, and the metabolic inhibitors dinitrophenol and iodoacetic acid caused reduction of 20–30%. Up to 45% of the cell-associated liposomal radioactivity could be detached within 1 h incubation with unlabelled liposomes. Whereas liver parenchymal cell suspension seemed to exhibit similar characteristics in vitro as in vivo, the non-parenchymal cells in vitro showed a 20–50-fold reduction in the rate of liposomal attachment compared to in vivo.     

Interactions Between Sendai Virus and Human Erythrocytes

Concentrated Sendai virus, when adsorbed to erythrocytes at 4 C, caused invaginations in the plasma membrane. Following elevation of the temperature to 37 C, the plasma membrane became fused with the viral envelope before dissolution of the virions and rupture of the cells. Cell lysis was accompanied by rapid and total loss of hemoglobin to the extracellular space. Following aqueous pyridine extraction, the hemoglobin-free ghosts remaining were found to be devoid of N-acetylneuraminic acid and to have solubility properties different from those of normal erythrocyte ghosts. By the action of viral neuraminidase, bound N-acetylneuraminic acid was also liberated from purified virus receptor substance whose electrophoretic mobility was thereby substantially reduced. Cu⁺⁺ selectively inhibited hemolysis and neuraminidase without interfering with hemagglutination and attachment. Neuraminidase appeared to be essential for Sendai virus hemolysis; viral particle size may also be a critical factor in this process.     

On the mechanism for the red-cell accumulation of mefloquine,an antimalarial drug

The accumulation of the antimalarial drug mefloquine by human red blood cells has been studied by ¹⁹F-NMR spectroscopy. The uptake process was nonlinearly dependent on the external drug concentration. Concentrations inside cells as high as 60-times greater than those in the extracellular phosphate-buffered-saline were observed. Red-cell ghosts were also found to accumulate mefloquine with high-affinity binding sites for the drug. Hemoglobin was found to bind mefloquine with low affinity, but due to the high concentration of this protein it is a significant drug compartment in the red cell. Analysis of the ¹⁹F-NMR chemical shifts and linewidths of mefloquine in the presence of red cells, red-cells ghosts and hemoglobin indicates restricted mobility of the drug in the membrane-bound state and slow exchange with the extracellular medium. This is a significant characteristic of the reaction in connection with the prophylactic activity of the drug. Exchange of the drug between hemoglobin and the red-cell membrane, however, is fast and may play an important role in the bioavailability of the drug to the parasite.     

Membrane effects of imidoesters in hereditary stomatocytosis

The marked increase in cation (Na⁺, K⁺) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). ⁴⁵Ca influx in intact RBC, ⁴⁵Ca efflux in red ghosts, and ⁴⁵Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT³²P–only 20–40% of normal control values in both short-term (5 min) and more extended (60 min) incubations–is not improved by DMA. After reaction of ¹⁴C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes is corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3–9 Å, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.     

Inhibition of K+-sensitive p-nitrophenylphosphatase activity on rhesus-positive red cells after incubation with IgG-anti-rhesus-D

The incubation of ghosts derived from human Rhesus-positive red cells with IgG-anti-Rhesus-D inhibited the K⁺-sensitive p-nitrophenylphosphatase activity. This enzyme has a partial function in the (Na⁺ + K⁺)-ATPase system related to the phosphorylation step, which is important for active potassium transport through the red cell membrane. The specificity of the impairment by the antigen-antibody reaction in the Rhesus-D system was proved by the following controls. Ghosts obtained from Rhesus-negative red cells incubated by IgG-anti-Rhesus-D and those of Rhesus-positive red cells treated with non-immune serum did not show any reduction of the K⁺-p-nitrophenylphosphatase activity. The ghost preparation with lanthanum carried out after hypotonic hemolysis of the washed red cells in 2 mM LaCl₃ at pH 6 was the most suitable procedure to explore this topic in comparison to other techniques for preparing ghosts of red cells.     

Clustering of erythrocytes by fibrinogen is inhibited by carnitine: evidence that sulfhydryl groups on red blood cell membranes are involved in carnitine actions.

Carnitine is bound by intact red blood cells, by red blood cell ghosts, and by glutaraldehyde-fixed human erythrocytes in a non-saturable, temperature-dependent manner. Binding of carnitine by these preparations is blocked by sulfhydryl reagents. Incubation or preincubation of red blood cell preparations with carnitine inhibits the aggregation of erythrocytes otherwise elicited by fibrinogen. Identical effects are obtained with red blood cell ghosts. In contrast, choline, even at high concentrations, is inactive in preventing the aggregation of erythrocytes. We discuss possible mechanisms by which carnitine favors the dispersion of red blood cells, and we present data indicating that sulfhydryl groups on erythrocyte membranes are required to permit these carnitine actions to be manifested.     

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.