Na-dependent <Emphasis Type="SmallCaps">D</Emphasis>-Glucose Transport by Intestinal Brush Border Membrane Vesicles from Gilthead Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

Brush border membrane vesicles (BBMV) enriched in sucrase, maltase and alkaline phosphatase, and impoverished in Na⁺-K⁺-ATPase, were isolated from proximal and distal intestine of the gilthead sea bream (Sparus aurata) by a MgCl₂ precipitation method. Vesicles were suitable for the study of the characteristics of D-glucose apical transport. Only one D-glucose carrier was found in vesicles from each intestinal segment. In both cases, the D-glucose transport system was sodium-dependent, phlorizin-sensitive, significantly inhibited by D-glucose, D-galactose, α-methyl-D-glucose, 3-O-methyl-D-glucose and 2-deoxy-D-glucose, and showed stereospecificity. Apparent affinity constants of D-glucose transport (K_t) were 0.24 ± 0.03 mM in proximal and 0.18 ± 0.03 mM in distal intestine. Maximal rate of influx (Jmax) was 47.3 ± 2.2 pmols. mg⁻¹ protein for proximal and 27.3 ± 3.6 pmols. mg⁻¹ protein for distal intestine. Specific phlorizin binding and relative abundance of an anti-SGLT1 reactive protein were significantly higher in proximal than in distal BBMV. These results suggest the presence of the same D-glucose transporter along the intestine, with a higher density in the proximal portion. This transporter is compatible with the sodium-dependent D-glucose carrier described for other fish and with the SGLT1 of higher vertebrates.This revised version was published online in June 2005 with a corrected cover date.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Mechanism of interaction of the cyanine dye DiS−C3-(5) with renal brush-border vesicles

Summary The equilibrium binding mechanism and kinetics of binding of diS–C₃-(5) (3,3-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS–C₃-(5) exists as a monomer at concentrations <5 m with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropyr=0.102, and lifetime =1.2 nsec (23°C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K⁺/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS–C₃-(5) rotation is unhindered with a rotational rateR=0.57 nsec^–1 while membrane monomer is hindered with steady-state anisotropyr=0.190, lifetime =2.1 nsec,R=0.58 nsec^–1 and limiting anisotropyr =0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant,K _d=[M]²/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS–C₃-(5). There is rapid binding of diS–C₃-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Kinetics of the Reverse Mode of the Na<Superscript>+</Superscript>/Glucose Cotransporter

This study investigates the reverse mode of the Na⁺/glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na⁺- and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The current-voltage relationship saturated at positive membrane voltages (30–50 mV), and approached zero at −150 mV. The half-maximal concentration for αMDG-evoked outward current (K_0.5^αMDG) was 35 mM (at 0 mV). In comparison, K_0.5^αMDG for forward sugar transport was 0.15 mM (at 0 mV). K_0.5^Na was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-O-methyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na⁺/sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.     

Pulmonary diffusing capacity in reptiles (Relations to temperature and O2-uptake)

Summary Pulmonary CO-diffusing capacity (D l _CO), lung volume, pulmonary perfusion and O₂-uptake were measured by non-invasive techniques in the lizardsVaranus exanthematicus andTupinambis teguixin (mean body weight 2.2 kg for both species).The CO-diffusing capacity was at 25–27°C 0.059 mlstpd·kg^–1·min^–1·Torr^–1 inVaranus, which is 47% greater than the value of 0.040 mlstpd·kg^–1·min^–1·Torr^–1 inTupinambis. The lung volume ofVaranus was 36 ml·kg^–1 and that ofTupinambis 20 ml·kg^–1. At 35–37°C the diffusing capacity of lizard lungs are about 25% of those for mammals of comparable size.InVaranus pulmonary CO-diffusing capacity increased with temperature from 0.027 mlstpd·kg^–1·min^–1·Torr^–1 at 17–19 °C to 0.075 mlstpd·kg^–1·min^–1·Torr^–1 at 35–37 °C. This change closely matched a concomitant increase of O₂-uptake. Pulmonary perfusion increased from 27 ml·kg^–1·min^–1 to 55 ml·kg^–1·min^–1 within this temperature range.The study emphasizes that pulmonary diffusing capacity cannot be fully evaluated without information on pulmonary perfusion and O₂-uptake. In reptiles and other ectotherms diffusing capacity must be reported at specified body temperature.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R₃₃ of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K _T=0.22 mmol/L) and the glucose carrier (K _T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H⁺ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H⁺ symporter.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Phosphate transport in intestinal brush-border membrane

In the small intestine of the rabbit the process of Na⁺-dependent uptake of phosphate occurs only at the brush-border of duodenal enterocytes. Li⁺ can replace Na⁺. The process is activated when either K⁺, Cs⁺, Rb⁺, or choline is present in the intravesicular space. The presence of membrane-permeable anions is essential for maximum rates of phosphate transport. We conclude that the mechanism of the phosphate carrier is electrogenic at pH 6–8, probably two Na⁺ moving with each H₂PO ₄ ^– . This. will lead to the development of a positive charge within the vesicle. The variation of theK _m for H₂PO ₄ ^– with pH is thought to be the consequence of the affinity of the carrier protein for H₂PO ₄ ^– increasing as the pH increases. Polyclonal antibodies against membrane vesicles isolated from rabbit duodenum, jejunum, and ileum were prepared. The antibodies raised against the ileum and jejunum both activated the phosphate transport process, while the anti-duodenum antibody preparation inhibited phosphate transport.     

Kleine Beiträge zur Bestimmung des IEP von Protoplasten

Zusammenfassung Neben einer Würdigung des mikro-kataphoretischen Verfahrens für die gestellte Aufgabe wird zuerst eine Übersicht über die Untersuchungsapparatur, über die zu beachtenden Fehlerquellen und über die benutzten Untersuchungsobjekte und ihre Behandlungsweise gegeben. Das Verfahren von L.Michaelis wird hinsichtlich der Elektroden durch Vorschläge von J.Gicklhobn und K.Umrath, hinsichtlich der Mikrokammer durch Verwendung des E.Busch'schen Durchfluß-Objektträgers zu verbessern gesucht.Die praktischen Versuche ergeben für die nackten Protoplasten vonSolanum nigrum den IEP bei einer C_H von 1,6·10^–5 oder wenig darunter, für jene vonVitis vinifera in der Zone zwischen 6,3·10^–5 und 2,5·10^–5. Der gesuchte Wert wird für die Stachelkugeln der beidenNitella- Arten nach zwei verschiedenen Untersuchungsweisen übereinstimmend zu 1,6·10^–5 bis 3,2·10^–6 angenommen, während er bei der untersuchten untergärigen Rasse vonSaccharomyces cerevisiae zwischen 1,6·10^–5 und 2,2·10^–5 liegen dürfte.Die Diskussion führt zu einer knappen Übersicht über die möglichen Beladungsursachen der Objekte und untersucht kurz die Anwendbarkeit des Kataphoreseversuches für weitere Ziele der Protoplasmaforschung (analytisch-chemischer Aufbau der Objekte, ihre intrazelluläre C_H, Ladungswert der Protoplasten).Fortsetzung aus Protoplasma,11, 85–96;12, 268–278;14, 83–90, 90–96.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.