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 SO₄^2? equilibrium exchange across the human red cell membrane. The measurements were carried out under the same conditions (pH 6.3, 8°C) and in the same medium for both the Cl^? and SO₂⁴ tracer fluxes. Under these conditions the rate constant for Cl^?-Cl^? exchange is about 20 000 times larger than that for SO₄^2?-SO₄^2? 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 SO₄^2? transport. The proteolytic enzyme papain also has the same inhibitory effect on both the Cl^? and SO₄^2? 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 SO₄^2? self-exchange. We interpret these results as evidence that the rapidly penetrating monovalent anion Cl^? and the slowly penetrating divalent anion SO₄^2? are transported by the same system.     

Further observations on metabolic responses in hamster cells: changes in UDPG dehydrogenase activity.

The effects of phloretin, H₂DIDS (4,4′-diisothiocyano-1,2-diphenylethane-2,2′-disulfonate) and SO₄^?2 on anion transport in Ehrlich ascites tumor cells was studied in an effort to determine whether Cl^? and SO₄^?2 share a common transport mechanism. Sulfate, in the presence of constant extracellular Cl^? (100 mM), reduces Cl^? self-exchange by 43% (40 mM SO₄^?2) and Cl^??SO₄^?2 exchange by 36% (25 mM Cl^?/O SO₄^?2) compared to 25 mM Cl^?/50 mM SO₄^?2. Phloretin blocks without delay and to the same extent the self-exchange of both Cl^? and SO₄^?2. For example, at 10^?4 M phloretin, anion transport is inhibited 28% which increases to 78% at 5 × 10^?4 M. Reversibly bound H₂DIDS also inhibits the self-exchange of both Cl^? and SO₄^?2. However, at all H₂DIDS concentrations tested (0.5 ? 10 × 10^?5 M) SO₄^?2 transport was far more susceptible to inhibition than that of Cl^?. H₂DIDS when irreversibly bound to the cell inhibits SO₄^?2 but not Cl^? transport The results of these experiments are consistent with the postulation that both Cl^? and SO₄^?2 are transported by a common mechanism possessing two reactive sites.     

Characteristics of anion transport in cat and dog red blood cells

Summary Self-exchange of chloride and sulfate in dog and cat red cells has been measured under equilibrium conditions. The rates of efflux for these anions are approximately twofold higher in dog compared to cat red blood cells. Although the rates differ, the anion exchange systems of these two red cell types exhibit many common properties. The dependence of³⁵SO₄ efflux on the intracellular SO₄ concentration, the pH dependence and the inhibition of³⁵SO₄ efflux by Cl and SITS are almost identical in dog and cat red cells. Nystatin treatment was used to study the dependence of³⁶Cl efflux on internal Cl. Chloride efflux exhibits saturation in both cell types with dog red cells possessing a higherV _max andK _1/2 than cat red cells. The number of anion transport sites was estimated by extrapolation to the number of molecules of dihydro DIDS (H₂DIDS, where DIDS is 4,4-diisothiocyano-2,2 stilbene-disulfonic acid) which were bound at 100% inhibition of transport. The results indicate that either the turnover numbers for anion transport differ in dog, cat, and human red cells or that there is heterogeneity in the function of the membrane components which bind H₂DIDS.     

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: I. Kinetic properties of NBD-taurine transfer in symmetric conditions

Summary The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H⁺-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H⁺ on both sides of the red blood cell membrane.     

Kinetic characteristics of bicarbonate-chloride exchange across the neonatal human red cell membrane

The kinetics of HCO₃^?/Cl^? exchange across red cell membrane of newborn infants was studied using a stopped-flow rapid reaction apparatus with a glass pH electrode attached. The measured apparent permeability P is (1.35±0.08 (S.E.)) · 10^?4 cm/s (n=30) for newborns, compared with (3.1 ± 0.4) · 10^?4 cm/s (n=15) for adults. These correspond to half-times of 0.2 s for newborns and 0.1 s for adults indicating that neonatal red cells exchange Cl^? for HCO₃^? only half as fast as do adult cells. The temperature dependence of the exchange rate was studied from 2 to 42°C. From the Arrhenius plot the activation energy of the exchange process in neonatal red cells changes from 22.9 kcal/mol (low temperature) to 4.8 kcal/mol (physiological temperature) at a transition temperature of 17°C. These values are lower than the corresponding values for adult red cells, 34.7 and 10.2 kcal/mol. HCO₃^?/Cl^? exchanges in both adult and neonatal red cells are inhibited by phlorizin. Inhibition constants K_i are 0.8 mM and 2.5 mM for adults and newborns, respectively. The differences in the values of the HCO₃^?/Cl^? exchange rate constant and the activation energy of the exchange process between neonatal and adult red cells indicate that there is a modification of HCO₃^?/Cl^? transport system in the neonatal red cell membranes.     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

Sulfate flux in high sodium cat red cells

The transport of radioactive sulfate in cat red cells has been studied. The rate constant for ³⁵SO₄ inward movement under steady-state conditions is 0.24 ± 0.02/hr. This movement was found to be sensitive to osmotic changes in cell volume and to the nature of anions in the incubation medium; it increases with increasing cell volume and decreases with decreasing cell volume. The anions SCN, NO₃, and I were found to inhibit the uptake of ³⁵SO₄. Furthermore, 1-fluoro-2,4-dinitrobenzene at a concentration of 1 mM inhibits (>90%) this uptake. The inward movement of erythritol-¹⁴C shows qualitatively the same dependence on cell volume as ³⁵SO₄, but it is insensitive to the nature of the anion present in the bathing medium. It was also found that the usually observed inhibition of radioactive Na uptake by SCN in cat red cells can be reversed when cell volume is increased.     

Anion transport in dog, cat, and human red cells. Effects of varying cell volume and Donnan ratio

Membrane potential and the rate constants for anion self-exchange in dog, cat, and human red blood cells have been shown to vary with cell volume. For dog and cat red cells, the outward rate constants for SO4 and Cl increase while the inward rate constant for SO4 decreases as cells swell or shrink. These changes coincide with the membrane potential becoming more negative as a result of changes in cell volume. Human red cells exhibit a similar change in the rate constants for SO4 and Cl efflux in response to cell swelling, but shrunken cells exhibit a decreased rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent increase in PNa. If this increase in PNa is prevented by ATP depletion or if the outward Na gradient is removed, the response to shrinking is identical to human red cells. These results suggest that the volume dependence of anion permeability may be secondary to changes in the anion equilibrium ratio which in red cells is reflected by the membrane potential. When the membrane potential and cell volume of human red cells were varied independently by a method involving pretreatment with nystatin, it was found that the rate of anion transport (for SO4 and Cl) does not vary with cell volume but rather with membrane potential (anion equilibrium ratio); that is, the rate constant for anion efflux is decreased and that for influx is increased as the membrane potential becomes more positive (internal anion concentration increases) while the opposite is true with membrane hyperpolarization (a fall in internal anion concentration).     

Sulfate transport in rabbit ileum: Characterization of the serosal border anion exchange process

Summary The preceding paper [30] shows that transepithelial ileal SO₄ transport involves Na-dependent uptake across the ileal brush border, and Cl-dependent efflux across the serosal border. The present study examines more closely the serosal efflux process. Transepithelial mucosa (m)-to-serosa (s) ands-to-m fluxes (J _ms,J _sm) across rabbit ileal mucosa were determined under short-circuit conditions. SO₄ was present at 0.22mm. In standard Cl, HCO₃ Ringer's,J _ms ^SO4 was 81.3±5.3 (1se) andJ _ms ^SO4 was 2.5±0.2 nmol cm^–2 hr^–1 (n=20). Serosal addition of 4-acetamido-4-isothiocyanostilbene-22-disulfonate (SITS), 44-diisothiocyanostilbene-22-disulfonate (DIDS) or 1-anilino-8-naphthalene-sulfonate (ANS) inhibited SO₄ transport, SITS being the most potent. Several other inhibitors of anion exchange in erythrocytes and other cells had no effect on ileal SO₄ fluxes. In contrast to its effect on SO₄ transport, SITS (500 m) did not detectably alter Cl transport.Replacement of all Cl, HCO₃ and PO₄ with gluconate reducedJ _ms ^SO4 by 70% and increasedJ _ms ^SO4 by 400%. A small but significantJ _net ^SO4 remained.J _ms ^SO4 could be increased by addition to the serosal side of Cl, Br, I, NO₃ or SO₄. The stimulatory effect of all these anions was saturable and SITS-inhibitable. The maximalJ _ms ^SO4 in the presence of Cl was considerably higher than in the presence of SO₄ (73.1 and 42.2 nmol. cm^–2 hr^–1, respectively;p<0.001). TheK _1/2 value for Cl was 7.4mm, 10-fold higher than that for SO₄ (0.7mm). Omitting HCO₃ and PO₄ had no measurable effects on SO₄ fluxes.This study shows that (i) SO₄ crosses the serosal border of rabbit ileal mucosa by anion exchange; (ii) the exchange process is inhibited by SITS, DIDS and ANS, but not by several other inhibitors of anion exchange in other systems; (iii) SO₄ may exchange for Cl, Br, I, NO₃ and SO₄ itself, but probably not for HCO₃ or PO₄; (iv) kinetics of the exchange system suggest there is a greater affinity for SO₄ than for Cl, although the maximal rate of exchange is higher in the presence of Cl; and, finally (v) SITS has little or no effect on net Cl transport.     

Temperature-dependent changes of chloride transport kinetics in human red cells

Chloride self-exchange in human red cells was studied between 0 degrees C and 38 degrees C. At higher temperatures the flow-tube method was used. Although the general features of chloride transport at 0 degrees C and 38 degrees C are similar, the following differences were found: (a) the maximum pH of chloride self-exchange flux was lowered 0.6 pH unit from 7.8 to 7.2 when temperature was increased from 0 degrees C to 38 degrees C; (b)the apparent half-saturation constant increased from 28 mM at 0 degrees C to 65 mM at 38 degrees C; (c) chloride transport at body temperature is slower than predicted by other investigators by extrapolation from low-temperature results. Chloride transport increased only 200 times when temperature was raised from 0 degrees C to 38 degrees C, because the apparent activation energy decreased from 30 kcal mol(-1) to 20 kcal mol(-1) above a temperature of 15 degrees C; (d) a study of temperature dependence of the slower bromide self-exchange showed that a similar change of activation energy occurred around 25 degrees C. Both in the case of Cl(-) (15 degrees C) and in the case of Br(-) (25 degrees C), critical temperature was reached when the anion self-exchange had a turnover number of about 4x10(9) ions cell (-1)s(-1); (e) inhibition of chloride transport by DIDS (4,4’- diisothiocyano-stilbene-2,2’-disulfonate)revealed that the deflection persisted at 15 degrees C at partial inhibition (66 percent) presumably because DIDS inactivated 66 percent of the transport sites. It is suggested that a less temperature- dependent step of anion exchange becomes rate limiting at the temperature where a critical turnover number is reached.     

Optimization of parameters for improvement of phenol degradation by Rhodococcus UKMP-5M using response surface methodology

Phenol is a toxic compound and is one of the major pollutants contained in the waste water from petroleum and its downstream industries. Response surface methodology (RSM) was used to optimize medium composition and culture condition for enhancement of growth of Rhodococcus UKMP-5M and phenol degradation rate in shake flask cultures. Phenol and (NH₄)₂SO₄ concentrations as well as temperature were the most significant factors that influenced growth and phenol degradation. Central composite design (CCD) was used for optimization of these parameters with growth, and degradation rates were used as the responses. Cultivation with 0.5 g/L phenol and 0.3 g/L (NH₄)₂SO₄ and incubation at 36 °C greatly enhanced growth of Rhodococcus UKMP-5M, where the final cell concentration increased from 0.117 g/L to 0.376 g/L. On the other hand, the degradation rate was greatly increased in cultivation with 0.7 g/L phenol and 0.4 g/L (NH₄)₂SO₄ and incubation at 37 °C. In this cultivation, the time taken to degrade 1 g/L phenol in the culture was reduced from 48 h to 27 h. The model for both responses was found significant and the predicted values were found to be in a good agreement with experimental values and subsequently validated. Increases in phenol degradation rate during Rhodococcus UKMP-5M cultivation corresponded well with increasing phenol hydroxylase activity.     

Inducible expression of erythrocyte band 3protein

A permanent cell line with inducible expression of the humananion exchanger protein 1 (hAE1) was constructed in a derivative ofhuman embryonic kidney cells (HEK-293). In the absence of the inducer,muristerone A, the new cell line had no detectable hAE1 protein byWestern analysis or additional³⁶Cl flux. Increasing dose andincubation time with muristerone A increased the amount of protein(both unglycosylated and glycosylated). The4,4'-dinitrostilbene-2,2'-disulfonate(DNDS)-inhibitable rapid Cl exchange flux was increased up to40-fold in induced cells compared with noninduced cells. There was noDNDS-inhibitable rapid flux component in noninduced cells. This resultdemonstrates inducible expression of a new rapid Cl transport pathwaythat is DNDS sensitive. The additional transport of³⁶Cl and³⁵SO₄had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 µM DNDS,2) activation of³⁶Cl efflux by external Cl with aconcentration producing half-maximal effect of 4.8 mM,3) activation of³⁶Cl efflux by external anionsthat was selective in the orderNO₃ = Cl > Br > I, and4) activation of³⁵SO₄influx by external protons. Under the assumption that the turnovernumbers of hAE1 were the same as in erythrocytes, there was good agreement (±3-fold) between the number of copies ofglycosylated hAE1 and the induced tracer fluxes. This is the firstexpression of hAE1 in a mammalian system to track the kineticcharacteristics of the native protein.

     

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume

In the presence of NH₄Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH₄Cl, while the maximum value is less than that observed in the presence of NH₄Cl. Depending on the cause of swelling, (net uptake of NH₄Cl or decrease in external osmolality) cells show specific responses. The NH₄Cl treatment causes a significant increase in intracellular Na⁺, from 5·14±0·78 to 29·84±0·47 mmoles l⁻¹ cell, while hypotonicity leads to a significant decrease of this cation, to 3·85±0·25 mmoles l⁻¹ cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloride significantly reverses the NH₄Cl effect with respect to intracellular Na⁺. Both treatments cause a significant K⁺ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH₄Cl effect by significantly decreasing their activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Sulphate uptake and metabolism in the chrysomonad,Monochrysis lutheri

The intracellular concentration of inorganic ³⁵SO₄ in Monochrysis lutheri cells exposed to 0.513 mM Na₂ ³⁵SO₄ for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this ³⁵SO₄ with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 μmoles/g cells/hr). The flux of free ³⁵SO₄ to organic ³⁵S was 0.029 μmoles/g cells/hr. Assuming an internal electrical potential in the cells of-70 mV, this intracellular concentration of inorganic ³⁵SO₄ was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a K _m of 3.2×10^-5 M and a V _max of 7.9×10^-5 μmoles sulphate/hr/10⁵ cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the proteins(s) associated with sulphate transport.     

Thiol-dependent K∶Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

Summary The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO₃ or methane sulfonate (CH₃SO₃). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the KCl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983.J. Membrane Biol..73:237–246). Like NEM, diamide activated KCl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L₁, the allo-antibody known to inhibit OR K fluxes.     

Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum

Summary In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of Cl for OH or HCO₃, SO₄ for OH, oxalate for OH, and oxalate for Cl. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO₄OH and oxalate: OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both¹⁴C-oxalate and³⁵SO₄ were equally sensitive tocis-inhibition by unlabeled SO₄ or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate: OH and oxalate: Cl exchanges occur on different carriers because: (i) Cl or SO₄ caused unequalcis-inhibition of these two exchanges; and (ii) as compared to oxalate: Cl exchange, oxalate: OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate: Cl exchange and ClHCO₃ exchange occur on different carriers because: (i) ClHCO₃ exchange was almost completely insensitive tocis-inhibition by oxalate; and (ii) oxalate: Cl exchange was more sensitive to inhibition by DIDS and bumetanide than ClHCO₃ exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: (i) a ClHCO₃ exchanger; (ii) a SO₄OH exchanger, which also transports oxalate; and (iii) an oxalate: Cl exchanger.     

Inactivation of Saccharomyces cerevisiae sulfate transporter Sul2p: use it and lose it

Saccharomyces cerevisiae SO₄⁼ transport is regulated over a wide dynamic range. Sulfur starvation causes ～10,000-fold increase in the ³⁵SO₄⁼ influx mediated by transporters Sul1p and Sul2p; >80% of the influx is via Sul2p. Adding methionine to S-starved cells causes a 50-fold decline (t_1/2 ～5 min) in SUL1 and SUL2 mRNA but a slower decline (t_1/2 ～1 h) in transport. In contrast, SO₄⁼ addition does not affect mRNA but causes a rapid (t_1/2 = 2–4 min) decrease in transport. In met3Δ cells (unable to metabolize SO₄⁼), addition of SO₄⁼ to S-starved cells causes inactivation of ³⁵SO₄⁼ influx over times in which cellular SO₄⁼ contents are nearly constant. The relationship between cellular SO₄⁼ and transport inactivation shows that cellular SO₄⁼ is not the signal for Sul2p inactivation. Instead, the transport inactivation rate has the same dependence on extracellular SO₄⁼ as ³⁵SO₄⁼ influx, indicating that Sul2p exhibits use-dependent inactivation; the transport process itself increases the probability of Sul2p inactivation and degradation. In addition, there is a transient efflux of SO₄⁼ shortly after adding >0.02 mM SO₄⁼ to S-starved met3Δ cells. This transient efflux provides further protection against excessive SO₄⁼ influx and may represent an alternate transport mode of Sul2p.     

Anion Transport in Red Blood Cells II. Kinetics of Reversible Inhibition by Nitroaromatic Sulfonic Acids

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4′-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25°C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was J_A = (0.69 ± 0.11) × 10^-10 moles · min^-1 · cm^-2 and the Michaelis-Menten constants were for the transport site K_S = 41 ± 14 mM and for the modifier site K_S' = 653 ± 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25°C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n ? 1), indicating a single site of inhibition for the two probes. The kinetics of sul- fate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate trans- port site was the target for the action of the inhibitors. The in- hibitory constants (Ki j for the transport sites were 0.45 ± 0.10 PM for DNDS and 0.21 ± 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus oper- andi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion trans- port sites are also the sites of inhibition and of labeling of co- valent binding analogs of BS and DS.     

EFFECTS OF LIGHT AND TEMPERATURE ON NET PHOTOSYNTHESIS AND DARK RESPIRATION OF GAMETOPHYTES AND EMBRYONIC SPOROPHYTES OF MACROCYSTIS PYRIFERA1

The rates of net photosynthesis as a function of irradiance and temperature were determined for gametophytes and embryonic sporophytes of the kelp, Macrocystis pyrifera (L.) C. Ag. Gametophytes exhibited higher net photosynthetic rates based on oxygen and pH measurements than their derived embryonic sporophytes, but reached light saturation at comparable irradiance levels. The net photosynthesis of gametophytes reached a maximum of 66.4 mg O₂ g dry wt^?1 h^?1 (86.5 mg CO₂ g dry wt^?1 h^?1), a value approximately seven times the rate reported previously for the adult sporophyte blades. Gametophytes were light saturated at 70 μE m^?2 s^?1 and exhibited a significant decline in photosynthetic performance at irradiances 140 μE m^?1 s^?1. Embryonic sporophytes revealed a maximum photosynthetic capacity of 20.6 mg O₂ g dry wt^?1 h^?1 (25.3 mg CO₂ g dry wt^?1 h^?1), a rate about twice that reported for adult sporophyte blades. Embryonic sporophytes also became light saturated at 70 μE m^?2 s^?1, but unlike their parental gametophytes, failed to exhibit lesser photosynthetic rates at the highest irradiance levels studied; light compensation occurred at 2.8 μE m^?2 s^?1. Light-saturated net photosynthetic rates of gametophytes and embryonic sporophytes varied significantly with temperature. Gametophytes exhibited maximal photosynthesis at 15° to 20° C, whereas embryonic sporophytes maintained comparable rates between 10° and 20° C. Both gametophytes and embryonic sporophytes declined in photosynthetic capacity at 30° C. Dark respiration of gametophytes was uniform from 10° to 25° C, but increased six-fold at 30° C; the rates for embryonic sporophytes were comparable over the entire range of temperatures examined. The broader light and temperature tolerances of the embryonic sporophytes suggest that this stage in the life history of M. pyrifera is well suited for the subtidal benthic environment and for the conditions in the upper levels of the water column.