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H Passow

Publications and source records attributed to H Passow.

At least 55 records · Page 3Linked to original sources
PubMed1984

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes.

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS). Using appropriate values for the probability of a channel in the open state, we estimated that 25% of the cells in the population contain 11-55, and 75% of the cells 1-5 channels per cell that are activated in the time average (20 degrees C, pH 7.6).

Calcium↗
PubMed1984

Characterization of anion transport system in trout red blood cell.

Anion transport in the trout red blood cell is mediated by a membrane protein that selectively binds dihydro-4,4'-dithiocyanostilbene-2,2'-disulfonic acid (3H2DIDS) and that forms on sodium dodecyl sulfate (SDS)-polyacrylamide gel electropherograms a band with the same diffuse structure at the same location as the band 3 protein of the mammalian red blood cells. There exists a linear relationship between binding of H2DIDS to this protein and the inhibition of anion equilibrium exchange. At maximal inhibition about 8 X 10(6) molecules/cell are bound to the protein. The kinetics of anion transport in the trout red blood cell differ from those of mammalian red blood cells. In addition to a H2DIDS-sensitive component of sulfate transport there exists a considerable H2DIDS-insensitive component with a relative magnitude that decreases with increasing temperature. At 23 degrees C, it amounts to about 25%. The temperature dependence of the H2DIDS-sensitive component is about 15 kcal/mol instead of 32 as in human red blood cells. Cl- transport increases with increasing pH. Above pH 7.4, the rate of transport becomes too fast to be measurable with either inhibitor stop or filtration technique. SO2-4 transport is nearly pH independent over the pH range 6.5 to 7.8 and the net entry of SO2-4 in exchange against intracellular Cl-, as followed in the absence of CO2, is accompanied by little if any proton uptake. Net proton uptake becomes measurable only at temperatures above 40 degrees C. Possibly at lower and more physiological temperatures, the band 3 protein in the red blood cell of the trout accomplishes part of the SO2-4 movements without cotransporting protons.

Animals↗
PubMed1983

Inhibition of anion transport across the red cell membrane by dinitrophenylation of a specific lysine residue at the H2DIDS binding site of the band 3 protein.

The inhibition of anion transport by dinitrophenylation of the red cell membrane is brought about by the modification of a single lysine residue located on the 17-kDa segment of the band 3 protein. This residue is identical with Lys a, which is also capable of reacting with one of the two isothiocyanate groups of 4,4'-diisothiocyano dihydro-stilbene-2,2'-disulfonate (H2DIDS). The rate of reaction between Lys a and 1-fluoro-2,4-dinitrobenzene is reduced when a second lysine residue on the 35-kDa segment of the band 3 protein becomes dinitrophenylated. This latter residue is not identical with Lys b which is known to be present on the 35-kDa segment and involved in the cross-linking of this segment with the 17-kDa segment by H2DIDS.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Band-3 protein-mediated anion conductance of the red cell membrane. Slippage vs ionic diffusion.

The band 3 protein-mediated, valinomycin-induced KCl efflux continues to increase with increasing [KCl] when the Cl-/Cl- equilibrium exchange becomes saturated. This suggests the existence of a band 3-mediated component of Cl- flux that contributes to the electrical conductance without being related to slippage; i.e., equilibration of the unloaded transport protein between the two membrane surfaces.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

The kinetics of intramolecular cross-linking of the band 3 protein in the red blood cell membrane by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid (H2DIDS).

The two isothiocyanate groups of the anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2-2'-disulfonate (H2DIDS) may react covalently with two lysine residues called a and b that reside on the chymotryptic 60,000 Dalton and 35,000 Dalton segments, respectively, of the band 3 protein of the human erythrocyte membrane. Under suitable conditions, the reaction leads to the establishment of intramolecular cross-links between a and b (M.L. Jennings & H. Passow, 1979, Biochim. Biophys. Acta 554:498-519). In the present work, the time course of the reactions with a and b, and of the establishment of the cross-link were investigated experimentally and compared with simple mathematical models of the reaction sequence. The rates of reaction with a and b were found to increase with increasing pH. Regardless of pH, the rate of reaction with a exceeds that with b several-fold. Once the H2DIDS molecule has reacted with a, the rate of the subsequent reaction of the other isothiocyanate group with b is reduced by about 1/30. The reactions that follow the unilateral attachment to site b are not yet clear. A more detailed analysis of the time course of the cross-linking reaction suggests that a satisfactory description of the kinetics requires the assumption that the H2DIDS binding site may exist in two different states, and that the transition from one state to the other is associated with changes of the reactivities of either lys a alone or of both lys a and b. This led to the formulation of the two-states model of the H2DIDS binding site, which is supported by other pieces of independent evidence. The analysis of the pH dependence of the rate of thiocyanylation of b shows that the apparent pK value of that lysine residue is about 9.9 to 10.0 and hence slightly lower than the intrinsic pK of a lysine residue in an aqueous environment.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Inverse effects of dansylation of red blood cell membrane on band 3 protein-mediated transport of sulphate and chloride.

1. Dansylation of the red cell membrane produces inverse effects on SO(4) (2-) and Cl(-) equilibrium exchange. The former is enhanced by several orders of magnitude (Legrum, Fasold & Passow, 1980), the latter is inhibited. Both effects are potentiated after dansylation in the presence of 2-(4-amino-3-sulphophenyl)-6-methyl-7-benzothiazol sulphonic acid (APMB), a disulphonic acid that combines non-covalently with the 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulphonic acid (H(2)DIDS) binding site of the anion transport protein.2. After dansylation the maximum of the pH dependence of SO(4) (2-) exchange near pH 6.3 is replaced by a plateau. When dansylation is performed in the presence of APMB, the plateau is reached at a much higher level at around pH 7.0 and resembles that observed by Funder & Wieth (1976) for Cl(-).3. The mutual interactions between the transfer site, the H(2)DIDS binding site, and the as yet unidentified danysl chloride binding sites were studied in detail. Occupation of the H(2)DIDS binding site by the non-covalently binding agents 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS), 4,4'-bis(acetamido) stilbene-2,2'-disulphonate (DAS) or APMB inhibit the enhanced SO(4) (2-) exchange across the previously dansylated membrane. The apparent K(I) value remains the same as in untreated membranes for DNDS, is reduced to 1/3 for DAS, and to 1/60 for APMB. Conversely, when dansylation is carried out while the H(2)DIDS binding site is occupied by DNDS, APMB or DAS, the enhancement of SO(4) (2-) exchange (as measured after removal of excess dansyl chloride and the additional agent) is prevented by DNDS, augmented by APMB and not affected by DAS. This suggests that the agents stabilize different conformations of the H(2)DIDS binding site that are associated with different accessibilities of the dansyl chloride binding sites.4. The SO(4) (2-) equilibrium exchange as measured at a fixed Cl(-) concentration is enhanced when the Cl(-) concentration at which dansylation is carried out is increased, indicating allosteric interactions between anion binding and the exposure of the dansyl chloride binding sites.5. The enhanced K(+) efflux from dansylated red cells is independent of the described modifications of the dansylation reaction by APMB, DAS or DNDS, demonstrating that there exists no simple correlation between the changes of anion and cation movements that are induced by dansylation.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1981

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of 35S thiocyanate.

Up to a SCN- concentration of about 110 mM, the concentration dependence of SCN- equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits a trans acceleration by Cl- and can be inhibited by H2DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced in trans acceleration experiments with Cl- and insensitive to H2DIDS but susceptible to inhibition by phloretin. At SCN- concentrations exceeding 110 mM, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered. The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0 mM at pH 7.4 to 13.3 mM at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19 x 10(-12) to 22 x 10(-12) moles x cm-2 x sec-1. The nonsaturable flux component also increases slightly. In accordance with previous observations of Wieth (J. Physiol. (London) 207:563-580, 1970), we find that SCN- increases K+ and Na+ permeability. The induced cation-permeability is considerably smaller than the SCN- exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1980

Role of external potassium in the calcium-induced potassium efflux from human red blood cell ghosts.

The exposure of red cell ghosts to external Ca++ and K+ leads to a rapid net K+ efflux. Preincubation of the ghosts for various lengths of time in the absence of K+ in the external medium prior to a challenge with maximally effective concentrations of Ca++ and K+ renders the ghosts unresponsive to that challenge with a half-time of about 7-10 min. Preincubation at a range of K+ concentrations for a fixed length of time (60 min) prior to the challenge revealed that K+ concentrations of about 500 microM or more suffice to maintain the K+ channel in a maximally responsive state for at least 60 min. These K+ concentrations are considerably lower than the K+ concentrations required to make the responsive channel respond with a maximal rat of K+ efflux. Thus external K+ is not only necessary to induce the permeability change but also to maintain the transport system in a functional state. The presence of Mg++ or ethylenediamine-tetraacetic acid (EDTA) in the K+-free preincubation media preserves the responsiveness to a challenge with Ca++ plus K+. In contrast to external K+, the presence of external Ca++ does not reduce but rather enhances the loss of responsiveness. An excess of EDTA prevents the effects of Ca++ while washes with EDTA after exposure to Ca++ do not reverse them. In red cell ghosts that contain Ca++ buffers, the transition from a responsive to a nonresponsive state incubation in the absence of external K+ is enhanced. The effects of incubation in the presence of Ca++ in K+-free media are reversed; external Ca++ now reduces the rate at which the responsiveness is lost. The loss of responsiveness after incubation in K+-free media prior to a challenge with external K+ and internal Ca++ does also take place when K+-efflux from red cell ghosts is measured by means of 42K+ into media that have the same K+ concentrations as the ghost interior. This confirms that the effects of K+-free incubation are due to the modification of the K+-selective channel rather than to an inhibition of diffusive Cl--efflux.

Calcium↗
PubMed1980

Creatine and creatinine transport in old and young human red blood cells.

The time course of creatine influx or efflux as measured in populations of red cells or red cell ghosts with normal age distribution does not follow simple two-compartment kinetics. This suggests that the contributions of individual cells to transport as measured in the populations as a whole are not uniform. In agreement with this inference, fractionation of red cell populations with respect to cell age shows that transport in young cells is considerably faster than in old cells. The dependence of creatine transport on creatine concentration in the medium follows an equation that can be interpreted to represent a super-imposition of a saturable component (apparent Km = 0.02 mM) and another component that cannot be saturated up to a creatine concentration of 5.0 mM. In contrast to the non-saturable component, the saturable component depends on the energy metabolism of the cell and can be inhibited by beta-guanidinopropionic acid and the proteolytic enzyme pronase. This latter finding suggests that the saturable component represents active transport that is mediated by a transport protein. The non-saturable component is little, if at all, dependent on cell age while the saturable component is higher in young cells than in old cells. Phloretin inhibits both components of creatine flux, but the maximal inhibition that can be achieved at high concentration is only 70--80%. Under the experimental conditions used for the study of creatine transport, creatinine equilibration between cells and medium follows the kinetics expected for a steady-state two-compartment system. Creatinine flux is proportional to creatine concentration over the concentration range studied (up to 5 mM). It cannot be inhibited by beta-guanidinopropionic acid or pronase.

Biological Transport↗
PubMed1980

Anion transport across the red blood cell membrane and the conformation of the protein in Band 3.

Measuring the rate of dinitrophenylation of a specific lysine residue (called a) that is allosterically linked to the transfer site, it could be demonstrated that the anion transport protein may exist in two different conformational states, designated cis and trans. In the cis conformation a is easily accessible for reaction with dinitrofluorobenzene; in the trans conformation, a is less accessible. In the presence of the substrate anion Cl, the equilibrium between the cis and trans conformation is towards the cis conformation. Reversibly acting inhibitors of anion transport arrest the transport system, either predominantly in the cis or in the trans conformation. Phlorizin and certain positively charged derivatives of furosemide produce arrest in cis conformation, internal 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonate (APMB) and Ca++ in trans conformation. Within this frame of reference, the different susceptibilities of the transfer site to internal and external 4,4' diacetamido-2,2'-stilbene disulfonate (DAS) are interpreted on the assumption that the conformation of the transfer site changes during the transition of the transport protein from the cis to the trans conformation, so that in the trans conformation a reaction with DAS is no longer possible.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1980

Enhancement of anion equilibrium exchange by dansylation of the red blood cell membrane.

Dansylation of resealed red cell ghosts enhances the band 3 protein-mediated equilibrium exchange of sulfate ions. After dansylation, the specific anion transport inhibitor 4,4'-diisothiocyanato-dihydrostilbene 2,2'-disulfonate (H2DIDS) is still capable of combining with its original binding site on the band 3 protein and of producing the same high degree of inhibition of sulfate exchange as in the untreated red cell ghost. Nevertheless, dansylation causes allosteric effects at the H2DIDS-binding site that exhibit themselves by an increased susceptibility to dinitrophenylation of one of the amino acid residues that is involved in the covalent bond formation with H2DIDS and a decrease of the apparent KI values for two reversibly acting inhibitors that are known to produce their effects at the H2DIDS-binding site of the band 3 protein. The degree of enhancement of divalent anion exchange depends on both the pH that existed during dansylation and the pH at which the anion equilibrium exchange across the dansylated membrane is measured. The effect of dansylation reaches a broad maximum around ph 6.6. In untreated ghosts, divalent anion equilibrium exchange passes through a maximum around pH 6.3. After dansylation under optimal conditions at pH 6.6, anion equilibrium exchange as measured below the maximum of pH 6.3 is much less enhanced than above the maximum. Under suitable experimental conditions, the maximum may be replaced by a plateau that extends up to at least pH 8.5. At this pH, the enhancement is about 100-fold. Thus, the pH dependence of divalent anion exchange becomes more similar to that of monovalent anion exchange. The apparent activation enthalpies for sulfate-equilibrium exchange across the modified membrane, as measured at pH 6.3 and 7.9, are indistinguishable, independent of temperature between 0 and 37 degrees C and amount to 146 kj/mol. This is similar to the activation enthalpies measured in the untreated membrane. The mode of action of dansyl chloride is discussed on the basis of currently considered mechanisms of divalent anion transport, for which the pertinent equations are presented.

Anions↗
PubMed1979

Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate.

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents. Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a cross-linking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport, Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the noninhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control anion transport.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1979

A comparison of the inhibitory potency of reversibly acting inhibitors of anion transport on chloride and sulfate movements across the human red cell membrane.

The effects of a variety of chemically diverse, reversibly acting inhibitors have been measured on both Cl- and SO2-4 equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8 degrees C) and in the same medium for both the Cl- and SO2-4 tracer fluxes. Under these conditions the rate constant for Cl--Cl- exchange is about 20,000 times larger than that for SO2-4--SO2-4 exchange. Despite this large difference in the rates of transport of the two anions, eight different reversibly acting inhibitors have virtually the same effect on the Cl- and SO2-4 transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl- and SO2-4 self-exchange. In addition, the slowly penetrating disulfonate 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid (APMB) is 5-fold more effective from the outer than from the inner membrane surface in inhibiting both Cl- and SO2-4 self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl- and the slowly penetrating divalent anion SO2-4 are transported by the same system.

Biological Transport, Active↗
PubMed1977

Anion transport across the red blood cell membrane and the protein in band 3.

The paper reviews existing evidence for the participation of the protein in band 3 (nomenclature of Steck, [1]) in anion transport across the red cell membrane and discusses the possible role of common binding sites on band 3 for 1-fluoro-2,4-dinitrobenzene, 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid and dihydro 4,4'-diisothiocyanato stilbene-2,2'-disulfonic acid in the transport process.

Affinity Labels↗