The influence of water/air cooling on collateral tissue damage using a diode laser with an innovative pulse design (micropulsed mode)-an in vitro study.

Since the diode laser is a good compromise for the daily use in dental offices, finding usage in numerous dental indications (e.g., surgery, periodontics, and endodontics), the minimization of the collateral damage in laser surgery is important to improve the therapeutical outcome. The aim of this study was to investigate the effect of water/air cooling on the collateral thermal soft tissue damage of 980-nm diode laser incisions. A total of 36 mechanically executed laser cuts in pork liver were made with a 980-nm diode laser in micropulsed mode with three different settings of water/air cooling and examined by histological assessment to determine the area and size of carbonization, necrosis, and reversible tissue damage as well as incision depth and width. In our study, clearly the incision depth increased significantly under water/air cooling (270.9 versus 502.3 µm-test group 3) without significant changes of incision width. In test group 2, the total area of damage was significantly smaller than in the control group (in this group, the incision depth increases by 65 %). In test group 3, the total area of damage was significantly higher (incision depth increased by 85 %), but the bigger part of it represented a reversible tissue alteration leaving the amount of irreversible damage almost the same as in the control group. This first pilot study clearly shows that water/air cooling in vitro has an effect on collateral tissue damage. Further studies will have to verify, if the reduced collateral damage we have proved in this study can lead to accelerated wound healing. Reduction of collateral thermal damage after diode laser incisions is clinically relevant for promoted wound healing.

Reduction of collateral thermal impact of diode laser irradiation on soft tissue due to modified application parameters.

The aim of this study was to investigate the effect of different working modes (pulsed and micropulsed) and power settings of a standardized 980-nm diode laser on collateral thermal soft-tissue damage. A total of 108 bovine liver samples were cut with a diode laser at various settings in pulsed and micropulsed mode and histologically assessed to determine the area and depth of carbonization, necrosis and reversible tissue damage, as well as incision depth and width. Incision depth and width and the area and depth of carbonization, necrosis and reversible damage were correlated strongly with cutting speed. The area and depth of reversible damage were correlated with average power. The micropulsed mode produced a smaller zone of carbonization and necrosis and a smaller incision width. Setting the laser parameters in accordance with the absorption characteristics of the tissue reduced collateral thermal tissue damage while maintaining an acceptable cutting ability. Reducing collateral thermal damage from diode laser incisions is clinically relevant for promoting wound healing.

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.

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.

Effects of incorporated trypsin on anion exchange and membrane proteins in human red blood cell ghosts.

Varying concentrations of trypsin were sealed into human red cell ghosts and the effects on membrane proteins and sulfate equilibrium exchange were studied. After incubation for 45 min at 37 degrees C, pH 7.2, the following observations were made: above 10 ng/ml the ghosts undergo fragmentation without lysis. Dodecyl sulfate gel electrophoresis shows that the digestion of spectrin and of the protein in band 2.1 (nomenclature of Steck (1974) J. Cell. Biol. 62, 1-19) is nearly complete at 50 ng/ml, that of the protein in band 3 at 25 mug/ml. After digestion at 25 mug/ml, about 60% of the total protein of the membrane is released and the original bands of conventional dodecyl sulfate gel electropherograms of the remaining protein are nearly completely abolished. In their place three new bands appear representing peptides with molecular weights of 58 000, 48 000 and 34 000, respectively. Sometimes a fourth peptide with a molecular weight of approx. 13 000 is also observed. Using a radioactive labeling technique it is shown that the two peptides with the highest molecular weights are derived from the protein in band 3. Labeling with diazo[35S]sulfanilic acid indicates that in addition to the peptides in the described four Coomassie blue-stainable bands, other peptides with molecular weights up to 100 000 are still present in the exhaustively trypsinized ghosts. External trypsin has no effect on the sulfate equilibrium exchange in ghosts while internal trypsin causes inhibition. Inhibition becomes apparent at trypsin concentration exceeding those required to produce a complete digestion of spectrin. It remains incomplete, even at the highest intracellular concentrations which cause maximal effects on all membrane proteins, including the protein in band 3. Under these conditions strong further inhibition can be produced by agents which are known to inhibit anion transport in untreated red cells and ghosts. These agents include the penetrating 1-fluoro-2,4-dinitrobenzene and the nonpenetrating phlorizin, 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulfonic acid, 4,4'-diacetamido stilbene-2,2'-disulfonic acid, and 2-(4'-aminophenyl)-6-methylbenzenethiazol-3',7-disulfonic acid (APMB). Unlike the other nonpenetrating inhibitors APMB is not only capable of inhibiting at the outer but also at the inner membrane surface. Treatment with internal trypsin does not significantly reduce the inhibition by incorporated APMB. The described observations suggest that after exhaustive tryptic digestion of the major membrane proteins, the receptor sites for typical inhibitors of anion transport continue to exert their function.

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.

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.

Anion transport function of mouse erythroid band 3 protein (AE1) does not require acylation of cysteine residue 861.

Cys-861 of mouse band 3 is equivalent to Cys-843 of human band 3, the only acylated cysteine residue in the anion exchanger AE1 of the red blood cell (Hamasaki et al. (1992) Progress Cell Res. 2, 65-71). Mutation of Cys-861 to serine or methionine caused no significant changes of band 3-mediated anion exchange as measured after expression of the appropriate cRNAs in Xenopus oocytes. Susceptibility to inhibition of transport by 4,4'-dinitrostilbene-2,2'-disulfonate and PCMBS was not affected. We conclude that palmitoylation is not an absolute requirement for the successful execution the anion transport function by the hydrophobic domain of band 3 in the plasma membrane.

Mediation of inorganic anion transport by the hydrophobic domain of mouse erythroid band 3 protein expressed in oocytes of Xenopus laevis.

A cDNA clone of the mouse erythroid band 3 protein encoding the 556 amino acid residues of the hydrophobic domain from Thr-374 to the C-terminal Val-929 is shown by immunoprecipitation to be expressed in Xenopus oocytes. Measurements of 36Cl- efflux indicate that the translation product mediates Cl- transport, which is inhibitable reversibly by DNDS or H2DIDS, specific inhibitors of band 3-mediated transport. The apparent KI values are 3.6 microM and 0.094 microM, respectively, and hence similar to those found in the wild type band 3-mediated anion transport. The rapid reversible inhibition by H2DIDS slowly changes to irreversible inhibition. The rate of change increases with increasing pH, again similar as to the wild-type band 3. It is concluded that the hydrophobic domain of band 3 is capable of executing anion transport essentially similar to the full-length band 3, although minor differences with respect to transport and inhibition kinetics cannot be ruled out.

Identification by site-directed mutagenesis of Lys-558 as the covalent attachment site of H2DIDS in the mouse erythroid band 3 protein.

After functional expression of mouse erythroid band 3 by cRNA microinjection into Xenopus oocytes, 36Cl- efflux is irreversibly inhibited by H2DIDS. When a cRNA is injected that is derived from a cDNA in which the nucleotides encoding for lysine-558 were replaced by nucleotides encoding for asparagine, transport and inhibition of transport by H2DIDS still occur. However, when measured under conditions where no intramolecular crosslinking takes place the inhibition by H2DIDS is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 takes place at Lys-558.

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.

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.

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.

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-.

The effects of dansylation on the pH dependence of SO4(2-) and Cl- equilibrium exchange and on the H+/SO4(2-) cotransport across the red blood cell membrane.

Treatment of the erythrocyte membrane with dansyl chloride leads to the following effects: (i) SO4(2-) transport is enhanced, Cl- transport is reduced. At maximal acceleration of sulfate exchange, Cl- exchange is only partially inhibited. The two effects are lineary related suggesting that the Cl- and SO4(2-) transporting forms of band 3 are derived from the same pool. (ii) The maximum of the pH dependence of SO4(2-) equilibrium exchange as measured at low sulfate concentrations is replaced by a plateau. It now resembles the pH dependence of Cl- exchange in untreated red cells. The pH dependence of SO4(2-) equilibrium exchange as measured at high sulfate concentrations is virtually unchanged after dansylation. The pH dependence of the partially inhibited Cl- equilibrium exchange across the dansylated membrane as measured at high chloride concentrations remains similar as in the untreated red cells but is somewhat less pronounced. (iii) SO4(2-)/H+ cotransport remains essentially unaltered after modification by dansyl chloride. The effects of dansylation are discussed in terms of a model similar to the titratable carrier model originally proposed by Gunn (Gunn, R.B. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Rorth, M. and Astrup, P., eds.), pp. 823-827, Munksgaard, Copenhagen).

pH-dependence of inhibition by H2DIDS of mouse erythroid band 3-mediated Cl- transport in Xenopus oocytes. The effect of oligonucleotide-directed replacement of Lys-558 by an Asn residue.

The rapid reversible inhibition of band 3-mediated inorganic anion transport by 4,4'-diisothiocyanodihydrostilbene-2,2'-disfulfonate (H2DIDS) turns slowly into irreversible inhibition. This is due to covalent bond formation of the two isothiocyanate groups of the inhibitor with two lysine residues on band 3, called Lys a and Lys b. In the red cell membrane, the pK value of Lys a is about 2.5 pK units lower than the pK value of Lys b. Hence the susceptibility of Lys a to irreversible modification by H2DIDS far exceeds the susceptibility to Lys b. In the present paper, we have expressed in Xenopus oocytes cRNA's derived from cDNA clones encoding wild-type mouse band 3 and mouse band 3 in which Lys a (Lys-558) had been replaced by an Asn residue by oligonucleotide-directed mutagenesis. In accord with previous findings, in the oocytes both wild-type and mutated band 3 mediate Cl- exchange. After determining the uninhibited exchange rate the oocytes were exposed for a fixed length of time to H2DIDS at a concentration (20 microM) which saturates all H2DIDS binding sites with reversibly bound H2DIDS (KI = 0.3 microM and 1.1 microM, respectively, for wild-type and mutant). Exposure was terminated by washing with a medium in which H2DIDS was replaced by bovine serum albumin to remove free and reversibly bound H2DIDS from the extracellular phase. Subsequent measurements of Cl- efflux yielded a measure for the irreversible inhibition that persisted. Since the transition from reversible to irreversible H2DIDS binding was found to follow first-order kinetics it was possible to calculate rate constants. From the pH dependence of the rate constants, pK values were calculated. These calculations could be made since in the wild-type, in which Lys a and Lys b are present, the exposure to H2DIDS could be confined to a pH range in which little if any covalent binding to Lys b takes place. The data could be represented by a single pK value of 8.3. In the mutant, Lys a is missing. Hence, covalent reaction can only take place with Lys b. Measurements over the appropriate pH range could be described by a single pK of 10.8. These values are 0.8-0.9 pK units higher than those previously obtained in experiments with band 3 in the red cell membrane (Kampmann et al. (1982) J. Membr. Biol. 70, 199-216).(ABSTRACT TRUNCATED AT 250 WORDS)

Three different actions of phenylglyoxal on band 3 protein-mediated anion transport across the red blood cell membrane.

Phenylglyoxalation of the red blood cell membrane leads to three superimposed effects on band 3 protein-mediated anion equilibrium exchange as measured by means of radiosulfate: (1) a shift of the curve relating transport activity to pH towards lower pH values, possibly in combination with an increase of the maximal transport activity. This is accompanied by effect (2), the abolishment of a chloride-stimulated component of anion transport seen at low pH values. Effect (3) consists of inhibition of anion equilibrium exchange. Effect (1) prevails when phenylglyoxalation is performed at low concentrations of PG and low pH, while effect (3) predominates when exposure to PG is executed at high pH and high concentration of PG. Effect (1) is associated with a decrease of the Ki values for inhibition and binding of the reversibly acting stilbene disulfonates DNDS and DBDS. The inhibition observed as a consequence of effect (3) is linearly related to a decrease of the capacity of band 3 to combine with the stilbene disulfonate DBDS. The results are interpreted on the assumption that PG is capable of reacting with two or possibly three distinct binding sites in band 3. Reaction with one of them leads to effect (1) and, perhaps, to effect (2); reaction with the other to effect (3). The latter is possibly due to modification of Arg 730, which is homologous to Arg 748 in mouse band 3. Site-directed mutagenesis of this arginine residue showed that it is required for band 3-mediated anion transport.

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.

Effect of site-directed mutagenesis of the arginine residues 509 and 748 on mouse band 3 protein-mediated anion transport.

Using site-directed mutagenesis, the arginine residues 509 and 748 in mouse band 3 protein were substituted by Lys, Thr, and Cys, or by Lys and Gln, respectively. After expression in Xenopus oocytes of the cRNAs encoding wild type band 3 or any one of the band 3 mutants, chloride equilibrium exchange was measured. When the flux measurements were performed two to three days after microinjection of the cRNAs, in contrast to the wild type, neither one of the mutants was able to accomplish transport, with the possible exception of the mutants R509K and R748K both of which showed some transport activity of doubtful significance. Immunoprecipitates revealed that the Arg 748 mutants were expressed similar to the wild type band 3 while no expression of the Arg 509 mutants could be detected. When the flux measurements were performed only 3 h after microinjection of the cRNAs, transport activity was observed in the oocytes that had received cRNAs encoding wild type band 3. In some oocytes of a population, a very slight transport activity was brought about by cRNA encoding Arg 509 mutants. No transport activity could be detected after injection of the Arg 748 mutant. Immunoprecipitation demonstrated the successful biosynthesis of wild type band 3 and of both the Arg 509 and the Arg 748 mutants. The experiments suggest that mutation of Arg 748 leads to biosynthesis of an inactive form of the band 3 protein, while that of Arg 509 results in expression of an abnormally folded, possibly functionally more or less intact form, which is proteolytically degraded within less than one day.

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.