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H Passow

Publications and source records attributed to H Passow.

At least 37 records · Page 2Linked to original sources
PubMed1989

Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis.

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Inhibition of inorganic anion transport across the human red blood cell membrane by chloride-dependent association of dipyridamole with a stilbene disulfonate binding site on the band 3 protein.

The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Endogenous D-glucose transport in oocytes of Xenopus laevis.

Endogenous glucose uptake by the oocytes of Xenopus laevis consists of two distinct components: one that is independent of extracellular Na+, and the other one that represents Na+-glucose cotransport. The latter shows similar characteristics as 2 Na+-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na+, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulate transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na+-independent and the Na+-dependent transport systems. The effect on the Na+-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.

Animals↗
PubMed1988

Temperature dependence of anion transport in the human red blood cell.

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1988

Comparison of murine band 3 protein-mediated Cl- transport as measured in mouse red blood cells and in oocytes of Xenopus laevis.

Murine band 3 protein was expressed in oocytes of Xenopus laevis after microinjection of the mRNA from the spleens of anemic mice. The 36Cl- efflux from the oocytes was compared with the chloride fluxes measured in murine red cells. In both oocytes and red cells, the band 3-mediated chloride transport showed the following features: the selective inhibitor of band 3-mediated anion transport, 4,4'-dinitrostilbene-2,2'-disulfonate exerts its effects only when applied to the outside and not when applied to the inside of the membrane. The K1/2 for inhibition by external 4,4'-dinitrostilbene-2,2'-disulfonate was of the order of 1.5 to 2.0 mumol/l. Flufenamate and persantine also produce similar inhibitory effects. Decreasing the pH from 7.4 to 6.0 leads to some inhibition. It is concluded that essential features of the mode of action of murine erythroid band 3 protein in the plasma membrane of the oocyte are similar to the mode of action in the bilayer of the red blood cell of the mouse.

Animals↗
PubMed1987

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane.

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.

Calcium↗
PubMed1987

Potential dependence of the "electrically silent" anion exchange across the plasma membrane of Xenopus oocytes mediated by the band-3 protein of mouse red blood cells.

Mouse erythroid band-3 protein was incorporated into the plasma membrane of Xenopus oocytes by microinjection of poly(A)+-mRNA from spleens of anemic mice. Subsequently, the efflux of microinjected 36Cl was continuously followed in single oocytes in a perfusion chamber the bottom of which was formed by the window of a Geiger-Müller tube. During the flux measurements, the membrane potential was clamped to different holding potentials. The efflux increased over the voltage range of -10 to -100 mV by a factor of about 1.5. Since the membrane potential cannot act as a driving force of anion exchange, it is suggested that the observed slight potential dependence is related to a recruitment of the anion-loaded transport protein by the electrical field, thereby changing the steady-state distribution between inwardly and outwardly facing anion binding sites of the transport molecules. The experimental data are discussed in terms of ping-pong kinetics, assuming that the potential dependence is primarily due to an effect of the electrical field in the membrane on the rate-limiting interconversion of inwardly and outwardly oriented anion binding sites. The results are compatible with the assumption that in the oocyte membrane the substrate-loaded band-3 molecules are preferentially inwardly oriented, and that the transition from the inwardly to the outwardly oriented conformation is associated with a reorientation of an effective charge of 0.1 elementary charge. During progesterone-induced maturation of the oocytes, several endogenous transport systems change their activity drastically. The mouse band-3 protein in the oocyte membrane also undergoes activity changes; however, these changes do not seem to involve direct regulation by specific metabolic processes. They can be explained as a consequence of the depolarization of the membrane potential associated with the maturation process.

Animals↗
PubMed1987

Sodium-dependent and sodium-independent phosphate uptake by full-grown, prophase-arrested oocytes of Xenopus laevis before and after progesterone-induced maturation.

The uptake of inorganic phosphate by full-grown, prophase-arrested oocytes occurs by two essentially independent transport systems, which accomplish the delivery of extracellular phosphate into two different cellular compartments. One transport system is sodium-dependent, the other is sodium-independent. Both systems can be inhibited by sulfhydryl reagents and the sodium-dependent system becomes inhibited during progesterone-induced oocyte maturation.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Lead-induced activation and inhibition of potassium-selective channels in the human red blood cell.

The selective increase of net K+ permeability in human red cells brought about by either Ca2+ or lead was studied using a light scattering technique to measure net K+ fluxes in cell suspensions and the patch-clamp technique to study K+ transport in individual K+-selective channels of the red cell membrane. Using ultrapure solutions it was demonstrated that the effect of lead is neither the indirect consequence of a lead-induced increase of the accessibility of the receptor sites of the K+-selective channels to traces of Ca2+ that are present as contamination in analytical grade reagents nor to the release of Ca2+ from intracellular Ca2+ stores. It is further shown that in cell-free membrane patches low concentrations of lead (10 microM) in Suprapur solutions evoke the same single-channel events as added Ca2+ and that this activity can be inhibited by high concentrations of lead (100 microM), similar to the net KCl efflux measured by means of the light scattering technique. It is concluded, therefore, that both Ca2+ and lead independently activate the same K+-selective channels in the red cell membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

pH dependence of phosphate transport across the red blood cell membrane after modification by dansyl chloride.

Dansylation of the red blood cell membrane inhibits monovalent anion transport as measured by means of 36C1 and enhances divalent anion transport as measured by means of 35SO4 (Legrum, Fasold and Passow (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1573-1590 and Lepke and Passow (1982) J. Physiol. (London) 328, 27-48). In the present work the effect of dansylation on phosphate equilibrium exchange was studied over the pH range where the ratio between monovalent and divalent phosphate anions varies. At high pH, phosphate equilibrium exchange was enhanced; at low pH, exchange was inhibited. The pH maximum of phosphate equilibrium exchange, seen at pH 6.3 in untreated ghosts is now replaced by a plateau. The inverse effects of dansylation on the rates of exchange at high and low pH suggest that both monovalent and divalent phosphate anions are accepted as substrates by the anion transport protein. A tentative attempt to obtain a quantitative estimate of the ratio of monovalent and divalent phosphate transport indicates that in the untreated red cell membrane over the pH range 7.2-8.5 the transport of HPO42- is negligible compared to the transport of H2PO4-.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

Enhancement of divalent anion transport across the human red blood cell membrane by the water-soluble dansyl chloride derivative 2-(N-piperidine)ethylamine-1-naphthyl-5-sulfonylchloride (PENS-Cl).

Sulfate transport across the red cell membrane is enhanced by the newly synthesised, water-soluble and nonpenetrating dansyl chloride derivative 2-(N-piperidine)ethylamine-1-naphthyl-5-sulfonylchloride (PENS-Cl). The transport is only enhanced if the potentiating agent 2-(4-aminophenyl-3-sulfonic acid)-6-methylbenzothiazol-7-sulfonic acid (APMB) is present during incubation with PENS-Cl. The enhanced flux is reduced by the anion-transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS) to about the same low level as in untreated controls. In contrast to dansyl chloride, PENS-Cl does not increase cation leakage from the red cells. The effects of PENS-Cl on sulfate transport resemble those produced by dansyl chloride. However, it can be shown that PENS-Cl only reacts with one subset of sites that are modified by dansyl chloride and involved in bringing about the enhancement of sulfate transport. This subset does not include the sites accessible to dansyl chloride in the absence of APMB. It comprises only a fraction of the sites exposed to dansyl chloride in the presence of APMB. Very little labelling of proteins of the red cell membrane can be seen after exposure of ghosts to the PENS-Cl, while dansyl chloride labels all major proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Mediation of anion transport in oocytes of Xenopus laevis by biosynthetically inserted band 3 protein from mouse spleen erythroid cells.

mRNA from the spleens of anemic mice was purified by oligo(dT)-cellulose chromatography and fractionated by density gradient centrifugation. After injection into oocytes of Xenopus laevis, two of the four fractions obtained led, after 16 h of incubation at 20 degrees C, to the expression of mouse band 3 protein, as demonstrated by immunoprecipitation with polyclonal antibodies against mouse band 3. Flux measurements showed an approximately 2- to 4-fold increment of 36Cl- uptake, which could be abolished by two different stilbene disulfonates, specific inhibitors of band 3-mediated anion transport in red blood cells.

Animals↗
PubMed1984

Anion transport in single erythrocyte ghosts measured by fluorescence microphotolysis.

Fluorescence microphotolysis was used to measure in single resealed human erythrocyte ghosts the band 3-mediated transport of the fluorescent anion N-(7-nitrobenzofurazan-4-yl)-taurine (NBD-taurine). Transport was reduced to less than 5% of the control by the specific inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The accuracy of the determination of the rate constant for NBD-taurine influx was approximately +/- 15% as calculated from repetitive measurements in individual ghosts. The sample population distribution of the rate constant was slightly skewed towards values larger than the mean. The rate of NBD-taurine transport showed an optimum near pH 7. The Arrhenius plot was linear in the range from 28.5 degrees C to 51 degrees C with an apparent activation enthalpy of 21.4 kcal/mol.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation of Xenopus oocytes.

During progesterone-stimulated maturation of defolliculated full-grown Xenopus oocytes, the activities of the transport systems for L-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms. A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8-12 X 10(9) molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K+ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na-K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na-K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg++ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na+-dependent incorporation of 32P from gamma-[32P]-ATP that can be reversed by the addition of K+. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.

Animals↗
PubMed1984

Sodium-alanine cotransport in oocytes of Xenopus laevis: correlation of alanine and sodium fluxes with potential and current changes.

The sodium-dependent L-alanine transport across the plasma membrane of oocytes of Xenopus laevis was studied by means of [14C]-L-alanine, 22Na+ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 2:1 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 2:1. The sodium-L-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess of D-alanine over L-alanine caused about 60% inhibition.

Alanine↗