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M L Jennings

Publications and source records attributed to M L Jennings.

51 records · Page 3Linked to original sources
PubMed1985

Functions of extracellular lysine residues in the human erythrocyte anion transport protein.

The extracellular lysine residues in the human erythrocyte anion transport protein (band 3) have been investigated using chemical modification with the impermeant homobifunctional active ester bis(sulfosuccinimidyl)-suberate (BSSS). This agent forms covalent intra- and intermolecular cross-links in human band 3 in intact cells (Staros and Kakkad. 1983. J. Membr. Biol. 74:247). We have found that the intermolecular cross-link has no detectable effect on the anion transport function of band 3. The intramolecular cross-link, however, causes major changes in the characteristics of the anion transport. These functional alterations are caused by the modification of lysine residues at the stilbene disulfonate binding site. BSSS pretreatment at pH 7.4 irreversibly inhibits Cl-Br exchange by at least 90% when the transport is assayed at extracellular pH above 8. In the same BSSS-pretreated cells, however, the Cl-Br exchange rate is activated by lowering the pH of the flux medium (intracellular pH fixed at 7). The flux is maximal at pH 5-6; a further lowering of the extracellular pH inhibits the anion exchange. This acid-activated Cl-Br exchange in the BSSS-treated cells is mediated by band 3, as indicated by phenylglyoxal and phloretin inhibition of the flux. Thus, the BSSS pretreatment has little effect on the maximal Cl-Br exchange flux catalyzed by band 3, but it shifts the alkaline branch of its extracellular pH dependence by approximately 5 pH units. BSSS also eliminates the self-inhibition of Cl-halide exchange by high extracellular Br or I concentrations. These results indicate that the BSSS-modified lysines do not participate directly in anion translocation, but that one of the lysines normally provides a positive charge that is necessary for substrate anion binding. This positive charge is removed by the BSSS treatment but can be replaced by lowering the extracellular pH. The results also provide insight regarding the halide selectivity of the maximal rate of chloride-halide exchange: the native selectivity (Br much greater than I) is nearly abolished by BSSS treatment, which suggests that the selectivity results from the very strong binding of iodide to an outward-facing modifier site.

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

Absence of significant sodium-hydrogen exchange by rabbit erythrocyte sodium-lithium countertransporter.

Red blood cells of several mammalian species (e.g., human, rabbit, bovine) possess a countertransport system for Li and Na. We have used rabbit red blood cells to determine the extent of sodium-proton exchange by this countertransporter. Ouabain-insensitive 22Na efflux into phosphate-buffered K-acetate media of varying Na concentrations was determined at extracellular pH 7.5 and 6.9. To maintain the intracellular pH at approximately 7.2, most of the acetate in the pH 6.9 medium was replaced by gluconate, a nonpenetrating anion. Extracellular Na strongly (greater than 10-fold) stimulated the 22Na efflux in both high- and low-pH media, but the K1/2 for this stimulation was higher at pH 6.9. This is consistent with a competitive binding of H to the outward-facing transport site. However, the lower pH itself, in a Na-free medium, stimulated the 22Na efflux only very slightly. The 22Na efflux stimulated by lowering the extracellular pH to 6.9 was less than 1% of the stimulation produced by 100 mM extracellular Na. It is concluded that, although H appears to bind to the substrate site of the Na-Li exchanger, there is no significant H transport in the physiological pH range.

Animals↗
PubMed1984

Erythrocyte band 3 protein: evidence for multiple membrane-crossing segments in the 17 000-dalton chymotryptic fragment.

We have investigated the topology of the band 3 protein of the human erythrocyte membrane by a combination of chemical labeling and proteolytic cleavage. The N-terminal third of the membrane-bound domain of band 3 is a 17 000-dalton chymotryptic fragment that is known to traverse the membrane an odd number of times. At least three lysine residues on this fragment can be labeled by reductive methylation of intact cells, under conditions that cause labeling of exofacial, but not intracellular, lysine residues. One of the labeled lysines is the one that reacts with anionic aryl isothiocyanates, and another is very close to the C terminus of the fragment. Both these are on the C-terminal 11-kilodalton CNBr peptide. The third labeled lysine is on the 6-kilodalton N-terminal CNBr peptide, which had not been previously known to have an extracellular site. Control experiments using a stilbenedisulfonate derivative demonstrate that the labeled 6-kilodalton CNBr peptide is not a degradation product of the 11-kilodalton CNBr fragment. Also, the exofacial lysine on the 6-kilodalton peptide can be labeled by reductive methylation even when the stilbenedisulfonate site is occupied by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate, which blocks band 3 mediated transport of BH4 into the cells. This is further indication that the labeled lysine is accessible from the extracellular water. These data are the first direct evidence that the 17-kilodalton chymotryptic fragment spans the membrane more than once.

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

Peptides of human erythrocyte band 3 protein produced by extracellular papain cleavage.

The structure of the human red blood cell membrane band 3 protein has been investigated by proteolysis of intact cells with papain. Papain cleaves the 35,000-dalton chymotryptic peptide (CH35) of band 3 into two integral fragments of Mr 7,500 (P7) and 28,000 (P28). The papain cleavage of CH35 causes inhibition of band 3-catalyzed Cl transport. The peptide P28 carries the band 3 carbohydrate and also contains the 2 cysteine residues in CH35. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) reacts covalently with a lysine residue on P28, rather than P7, in native band 3. However, if the cells are first digested with papain and then reacted with H2DIDS, there is significant covalent reaction with P7, producing a covalent cross-link between P7 and the 60,000-dalton chymotryptic peptide (CH60). Graded papain digestion experiments and end group determinations indicate that the alignment of the band 3 peptides is N-CH60-P7-P28-COOH. The NH2-terminal sequence of P7 is identical with a segment of a peptic fragment of band 3 recently sequenced (Brock, C.J., Tanner, M.J.A., and Kempf, C. (1983) Biochem. J. 213, 577-586). This published sequence, plus our sequence data on CH35 and P7, show that papain cleaves the outer surface of CH35 at two sites, which are 6 residues and 71 residues from the chymotrypsin cleavage site. The 65-residue peptide (P7) between these sites is the best characterized segment of band 3 thus far described: its sequence and location in the band 3 primary structure are now known, and both ends of the peptide are unambiguously exofacial.

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

A rabbit erythrocyte membrane protein associated with L-lactate transport.

Chemical Labeling has been employed to attempt to identify the lactate transport protein of rabbit erythrocytes, which have a very high capacity for stereoselective lactate transport. The lactate transport protein catalyzes lactate/proton cotransport (or lactate/hydroxyl exchange) at physiological pH, as demonstrated by uphill proton fluxes induced by lactate gradients. However, lactate/lactate and lactate/pyruvate exchange are considerably more rapid than lactate/proton cotransport. Although the lactate transporter is less sensitive to inhibition by the stilbenedisulfonate derivative H2DIDS (4,4'diisothiocyano-2,2'-dihydrostilbenedisulfonate) than is the inorganic anion exchanger (band 3), H2DIDS is nonetheless a reasonably potent inhibitor of the lactate transport. A 1-h treatment with 10(-4) M H2DIDS irreversibly inhibits lactate/lactate exchange by greater than 80%. This inhibition appears to be related to the labeling (by [3H]H2DIDS) of an integral membrane polypeptide of Mr = 43,000. This [3H]H2DIDS-labeled polypeptide is absent from human erythrocytes, which have a 100-fold lower Vmax for lactate transport than do rabbit erythrocytes. These experiments suggest that the polypeptide of Mr = 43,000 is a component of the lactate transport system.

Animals↗
PubMed1982

Reductive methylation of the two 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate-binding lysine residues of band 3, the human erythrocyte anion transport protein.

The bifunctional anion transport inhibitor 4,4'p-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) can react covalently with both chymotryptic peptides of band 3, the human erythrocyte anion transport protein. The functional roles of the two sites of covalent H2DIDS attachment have been investigated using reductive methylation with formaldehyde and borohydride, which is a substrate for band 3. Reductive methylation of outward facing lysing residues of intact cells can block the covalent H2DIDS reaction with both the 60,000- and the 38,000-dalton chymotryptic peptides (CH60 and CH38, respectively). The methylation does not interfere with reversible H2DIDS binding. Methylation of all copies of and 3 at both sites of covalent H2DIDS attachment inhibits Cl-Cl exchange by 75%. The same treatment does not detectably alter the apparent affinity for extracellular substrate (Br). The extent of transport inhibition by reductive methylation correlates very well with the extent of blockage of the Amino acid analysis indicates that the H2DIDS-binding lysine on CH38 rather than some coincidentally modified lysine is responsible for the inhibition. This lysine is therefore likely to be closely associated with the anion transport pathway.

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

Stoichiometry of a half-turnover of band 3, the chloride transport protein of human erythrocytes.

The kinetics of human red blood cell Cl transport have been studied under nonequilibrium conditions to determine whether or not an outward Cl gradient can recruit the transport protein from an inward-facing to an outward-facing configuration. Three kinds of evidence are consistent with this outward recruitment. First, the initial net Cl efflux into a Cl-free phosphate medium is independent of the intracellular Cl concentration in the range 20-170 mM. Second, an outward Cl gradient strongly enhances the inhibitory potency of DNDS (4,4'-dinitro-2,2'-stilbene disulfonate), which suggests that DNDS binds primarily to outward-facing states. Finally, we have estimated the number of Cl ions transported during the putative outward recruitment. Resealed red cell ghosts containing only 70 muM 36Cl were resuspended at 0 degrees C in a Cl-free, HCO3-free Na2SO4 medium. In the first 10 s, or approximately 10(6) Cl ions per ghost, followed by a much slower further loss of Cl. The rapid loss of 10(6) Cl ions per ghost, which is abolished by pretreatment with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), appears to represent the Cl that is transported during the first half-turnover of the transport cycle. These data are strong evidence that the influx and efflux events in the catalytic cycle for anion transport do not take place simultaneously, and that the stoichiometry of the transport cycle is close to one pair of anions exchanged per band 3 monomer.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1981

Modification by papain of the structure and function of band 3, the erythrocyte anion transport protein.

Extracellular papain is known to inhibit the anion transport function of the band 3 protein of the human red blood cell membrane. Previous work [Jennings, M. L., & Passow, H. (1979) Biochim. Biophys. Acta 554, 498-519] had suggested that this inhibition may result from the removal by papain of 5 000-10 000 daltons from the 35 000-dalton chymotryptic peptide of band 3. The present work shows, however, that papain also removes a small peptide from the C terminus of the 60 000-dalton chymotryptic peptide. The C-terminal amino acid sequence of this peptide is -Lys-Thr-Tyr. Whether or not this newly discovered action of papain is responsible for inhibiting anion transport is unknown. The effects of extracellular papain on the band 3 function have been characterized in detail. Papain inhibits Cl-Cl exchange in a high Cl medium by almost 90%. This inhibition appears to result from inhibition of the efflux step in the catalytic cycle for the transport, because papain does not inhibit the anion transport when it is assayed under influx-limited conditions. Moreover, since papain has no detectable effect on the dissociation constant for extracellular substrate (SO4) binding, the material removed by papain cannot be involved closely in the outward-facing substrate site. In contrast, removal of this material strongly (12-fold) reduces the affinity of the inhibitor 4,4'-dinitro-2,2'-stilbenedisulfonate for outward-facing sites. Therefore, stilbenedisulfonate binding involves portions of the band 3 molecule which are not intimately related to substrate binding.

Adult↗
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↗
PubMed1976

Proton fluxes associated with erythrocyte membrane anion exchange.

Transient extracellular pH changes accompany the exchange of chloride for sulfate across the erythrocyte membrane. The direction of the extracellular pH change during chloride efflux and sulfate influx depends on experimental conditions. When bicarbonate is present, the extracellular pH drops sharply at the outset of the anion exchange and tends to follow the partial ionic equilibrium described by Wilbrandt (W. Wilbrandt, 1942. Pfluegers Arch. 246:291). When bicarbonate is absent, however, the anion exchange causes the pH to rise, indicating that protons are cotransported with sulfate during chloride-sulfate exchange. The pH rise can be reversed by the addition of HCO(-3) (4 muM) or 2,4-dinitrophenol (90 muM). This demonstrates that the proton-sulfate cotransport can drive proton transport uphill. The stoichiometry of the transport is that one chloride exchanges for one sulfate plus one proton. These results support the titratable carrier model proposed by Gunn (Gunn, R.B. 1972, In: Oxygen Affinity of Hemoglobin and Red Cell Acid-Base Status. M. Rorth and P. Astrup, editors. p. 823. Munksgaard, Copenhagen) for erythrocyte membrane anion exchange.

Anions↗
PubMed1976

Interaction between phloretin and the red blood cell membrane.

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.

Binding Sites↗