Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes

Lowering extravesicular pH stimulated Na⁺-dependent citrate transport in renal brush border membrane vesicles: e.g., at pH_out = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H⁺ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na⁺. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit^1?, Cit^2?) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit^1? and Cit^2?, and over the same pH range [Cit^3?] was inversely related to citrate uptake. A model of the Na⁺-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

The effect on amino acid transport of trypsin treatment of rat renal brush border membranes

Trypsin treatment of isolated rat renal brush border membrane vesicles which preferentially releases l-leucine aminopeptides (EC 3.4.11.2) decreases their ability to take up a variety of amino acids under Na⁺-gradient conditions. Such treatment did not alter the osmotic properties of the vesicles nor affect their fragility. A linear correlation could be demonstrated between the l-leucine aminopeptidase activity of the membranes and the initial rate of uptake of l-leucine and l-proline. Velocity of uptake-concentration dependence studies with these substrates indicate that the major effect of trypsinization is to decrease the maximum velocity (V_max1) of the low-K_m high-affinity system with little effect on the V_max2 of the high-K_m low-affinity transport process and no effect on the apparent Michaelis constants of either. Although the data indicate that l-leucine aminopeptidase activity and uptake of l-leucine and l-proline are affected in parallel, they should not be construed to imply a role of the enzyme in the transport process, especially in view of the global decrease in the uptake of various amino acids and sugars.     

A rapid method for the isolation of kidney brush border membranes

A simple rapid method for the preparation of purified brush border membranes from rabbit kidney proximal tubules is described. The method is based on hypotonic lysis, Ca²⁺ aggregation of contaminants and differential centrifugation. In contrast to most other published methods, the brush border membranes are free of contamination by basolateral membranes.     

Effect of anions on folate binding by isolated brush border membranes from rat kidney

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3′, 5′, 9-³H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70–75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3′,5′,9-³H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroylglutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroylglutamic acid. Inhibition of [3′,5′,9-³H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3′,5′,9-³H]pteroylglutamic acid.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Evidence for a dipeptide transport system in renal brush border membranes from rabbit

Papain treatment of renal brush border vesicles was carried out as a successful first step towards the purification of the membrane components involved in dipeptide transport. The treated vesicles exhibited increased specific transport activity of glycyl-l-proline. In contrast, the specific transport activity of l-alanine in the treated vesicles was less than that in the control vesicles. Papain treatment resulted in the solubilization of 38% of protein, 55% of alkaline phosphatase, 90% of γ-glutamyltransferase and 95% of leucine aminopeptidase. There was no change in the intravesicular volume nor was there any increase in vesicular permeability. Glycyl-l-proline transport was Na⁺-independent in the control and papain-treated vesicles. Diamide reduced the Na⁺-dependent l-alanine transport while glycyl-l-proline transport remained unaffected in the presence of Na⁺. Many dipeptides inhibited glycyl-l-proline transport both in the presence and absence of Na⁺. The inhibition by dipeptides was greater than the inhibition by equivalent concentrations of free amino acids. These data demonstrate that renal brush border vesicles can efficiently handle dipeptides by a mechanism completely different from that of amino acid transport.     

d-Gluconate transport in Arthrobacter pyridinolis. Metabolic trapping of a protonated solute

d-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 5 μM}$). Uptake of d-gluconate was not mediated by a phosphoenolpyruvate: hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, ΔpH, of ?63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, $\text{ΔpH = 0}$. Addition of 8 μM d-gluconate significantly reduced the ΔpH. The transmembrane electrical potential, Δψ, which was ?87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. d-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the Δψ did not energize transport. Rather, the rate of d-gluconate uptake correlated with and appeared to be determined by the rate of d-gluconate metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both d-gluconate uptake and the metabolism of pre-accumulated d-gluconate; (b) the effects of cyanide and azide on d-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on d-gluconate metabolism; (c) extraction and chromatography of intracellular label from d-gluconate uptake revealed that accumulation of unaltered d-gluconate was negligible; (d) a series of mutant strains with lower d-gluconate kinase activities also exhibited low rates of d-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both d-gluconate kinase activity and wild type levels of uptake.     

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [³H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3′-sulfonate (Woodward’s Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H⁺ exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H⁺ antiport mechanism.     

K+- and Na+-gradient-dependent transport of l-phenylalanine by mouse intestinal brush border membrane vesicles

In the presence of an Na⁺- or a K⁺-gradient ($\text{outside > inside}$), l-phenylalanine uptake exhibited an overshoot phenomenon indicating active transport. The amplitudes of the overshoots were increased by increasing either Na⁺ or K⁺ concentrations in the incubation media, indicating that binding alone cannot account for the K⁺ effect. The K⁺-induced overshoot is not due to the presence of a membrane potential alone, as a gradient of choline chloride failed to produce it. Li⁺ could also substitute for Na⁺ though less potent than Na⁺ in inducing an overshoot. Uptake of l-leucine also showed Na⁺- and K⁺-effects and l-leucine and l-alanine could inhibit the Na⁺- and K⁺-overshoots obtained with phenylalanine. These results lead us to postulate the presence of a carrier for neutral amino acids dependent on monovalent cation with higher affinity for Na⁺ in mouse intestine. The Na⁺- and K⁺-driven active transport of l-phenylalanine were shown to be dependent on the presence of a membrane potential, as short-circuiting the membrane with FCCP reduced the amplitude of the overshoots seen with both ions. However, substitution of Cl^? by more lipophilic anions (NO₃^?, SCN^?) produced an inhibition of uptake. A preliminary analysis of the interrelations between Na⁺ and K⁺ for l-phenylalanine uptake showed complex interactions which can be best explained by mutual competition for a common carrier at both sides of the membrane. These results suggest the presence of a new transport system or a variant of an ASC-type system for l-phenylalanine (and neutral amino acids) in the mouse intestine. However, our studies do not rule out the possible involvement of more than one system for neutral amino acid uptake.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Effects of dibutyryl cyclic amp on the transport of α-methyl-d-glucoside and α-aminoisobutyric acid in separated tubules and brush border membranes from rabbit kidney

The effect of dibutyryl cyclic AMP on the transport of α-methyl-d-glucoside and α-aminoisobutyric acid in separated tubules and purified brush border membranes from rabbit kidney was investigated using a rapid filtration procedure. Dibutyryl cyclic AMP stimulated the uptake of α-methyl-d-glucoside and α-aminoisobutyric acid by separated renal tubules in agreement with prior studies utilizing renal slices (Rea, C. and Segal, S. (1973) Biochim. Biophys. Acta 311, 615–624; Weiss, I.W., Morgan, K. and Phang, J.M. (1972) J. Biol. Chem. 247, 760–764). However, in contrast to previous reports, no preincubation of the tissue with dibutyryl cyclic AMP was required for stimulation of transport to be manifest. Dibutyryl cyclic AMP stimulated oxygen consumption by separated tubules suggesting that stimulation of transport may occur by a linkage with renal oxidative metabolism. Dibutyryl cyclic AMP increased the uptake of α-aminoisobutyric acid into purified renal brush border membranes. However the uptakes of α-methyl-d-glucoside, proline, leucine and phosphate into brush border membranes were significantly inhibited.     

Electrogenic transport of 5-oxoproline in rabbit renal brush-border membrane vesicles Effect of intravesicular potassium

The Na⁺-dependent transport of 5-oxoproline into rabbit renal brush-border vesicles was stimulated by a K⁺ diffusion potential (interior-negative) induced by valinomycin. Na⁺ salts of two anions of different epithelial permeabilities also affected 5-oxoproline transport. These results show that the Na⁺-dependent 5-oxoproline transport in renal brush-border vesicles is an electrogenic process which results in a net transfer of positive charge. Maximum transport of 5-oxoproline occurred at an extravesicular pH of 6.0 to 8.0 and over that pH range, 5-oxoproline exists completely as an anion with a negative charge. The simplest stoichiometry consistent with this process is, therefore, the cotransport of one 5-oxoproline anion with two sodium ions. The presence of K⁺ inside the vesicles stimulated the Na⁺-dependent transport of 5-oxoproline. This stimulatory effect was specific for K⁺ and required the presence of Na⁺. The presence of Na⁺ gradient was not mandatory for the K⁺ action. The stimulation by the intravesicular K⁺ was seen in the presence as well as in the absence of a K⁺ gradient. Therefore, the increased influx of 5-oxoproline was not coupled to the simultaneous efflux of K⁺. The presence of K⁺ in the extravesicular medium alone did not affect the Na⁺-dependent transport of 5-oxoproline, showing that the site of K⁺ action was intravesicular. Glutamate did not interact with the Na⁺-dependent 5-oxoproline transport even in the presence of an outward K⁺ gradient.     

Partial purification and characterization of rabbit-kidney brush-border (Ca2+ or Mg2+)-dependent adenosine triphosphatase

The ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca²⁺ and Mg²⁺ and, therefore, should be called (Ca²⁺ or Mg²⁺)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl₂ by CaCl₂ resulted in an ATPase activity of 92% of that with MgCl₂. Using calcium- and magnesium-ATP as substrates, apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of 95 000 and also that of microvillus actin.