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.     

Interactions between Na+-dependent uptake of d-glucose,phosphate and l-alanine in rat renal brush border membrane vesicles

d-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, d-glucose and l-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na⁺-dependent phosphate transport was inhibited in the presence of either d-glucose (39 mM) or l-alanine (2.4 mM). In this model, with d-glucose or with l-alanine the $\text{V}$ value of the phosphate uptake was decreased, whereas the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of l-glucose, d-alanine or d-glucose after phlorizin preincubation. A 30% Na⁺-dependent l-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. d-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, d-glucose decreased the l-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of d-glucose by l-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na⁺ gradient insofar as the rate of the Na⁺ uptake was increased with d-glucose, l-alanine or phosphate and since the absence of variations in membrane potential did not suppress these inhibitions.     

Na+-dependent,potential-sensitive l-ascorbate transport across brush border membrane vesicles from kidney cortex

l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na⁺-dependent and potential-sensitive transport process. This uptake shows saturation ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>:0.1–0.3\; mM</mtext>}}$) and is strongly stimulated by low concentrations of N₃^?. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter.     

Morphology and structure of γ-benzyl-l-glutamate copolymerized with l-phenylalanine, l-valine and l-alanine

Observation of random copolypeptides of γ-benzyl-l-glutamate with l-phenylalanine, l-valine and l-alanine was carried out in an electron microscope with samples cast from dilute solution. The relationship between the morphology and the molecular conformation in solution was studied with mixed solvents composed of chloroform and trifluoroacetic acid; these show a preference for α-helix and random coil, respectively. From the solutions in which molecules take α-helical conformation, fibrous films of nematic structure were formed. From random coil solutions discrete precipitates with folded molecules such as lamellar single crystals, piles of lamellae and structureless particles were formed. A copolypeptide containing l-valine in sufficiently large quantity to form β-structure also showed a variation in morphology with solvent, from films to discrete precipitates. It is suggested that the change in stiffness of the molecules contributes to the morphological variation.     

Na+-dependent,electroneutral l-ascorbate transport across brush border membrane vesicles from guinea pig small intestine

In brush border vesicles from guinea pig small intestine l-ascorbate transport is Na⁺-dependent and electroneutral (in the presence of Na⁺, as shown by its lack of response to either positive or negative Δψ across the membrane).l-Ascorbate transporter has the kinetic characteristics of a mobile carrier (K_m for l-ascorbate, 0.3 mM). d-Isoascorbate (erythorbate) seems to be another, but poorer, substrate of the same transporter.l-Ascorbate transport is subjected to heterologous inhibition by d-glucose.     

Silk fibroin model,poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-seryglycine)

l-Alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine and its pentachlorophenyl ester methanesulphonate have been synthesized as monomers for the preparation of silk fibroin model polypeptide. The former octapeptide was polymerized with diphenylphosphorylazide (DPPA) and triethylamine in DMSO or in HMPA—pyridine, and the latter octapeptide pentachlorophenylester was polymerized by adding triethylamine in DMSO to give poly(l-alanylglycyl-l-alanylglycyl-l-alanylglycyl-l-serylglycine). This sequential polypeptide gave a similar i.r. pattern to the crystalline part of Bombyx mori silk fibroin, which indicated antiparallel β-conformation. Dialysis of the solution of this polymer in 60%, aqueous LiBr against water gave mainly the polymer of α-form. O.r.d. measurements suggest that this polypeptide exists as a random structure in dichloroacetic acid on in 60% aqueous LiBr.     

Mechanism of neomycin stimulation of d-glucose uptake in rabbit intestinal brush border membrane

In order to study the effect of the antibiotic neomycin on the intestinal epithelium, d-glucose was used as a probe molecule and its transport into rabbit brush border membrane vesicles was measured by a rapid filtration method. Treatment of the epithelium with neomycin sulfate prior to the preparation of the brush border membrane enhanced the d-glucose uptake, whereas neutral N-acetylated neomycin did not. This action of neomycin was related to its polycationic character and not to its bactericidal action. No significant difference could be demonstrated between the protein content or disaccharidase-specific activities of the brush border fractions from treated or non-treated intestines. Electrophoretic protein patterns of SDS-solubilized membrane were not significantly different after neomycin treatment. To gain more information on the mechanism involved in the stimulation of d-glucose transport, experiments were conducted on phosphatidyl glycerol artificial membranes and the results compared with those obtained with brush border membrane. At a concentration of 10^?7 M, neomycin decreased the nonactin-induced K⁺ conductance by a factor of approx. 100. The membrane conductance was linearly dependent on the neomycin concentration and the conductance in 10^?2 M KCl was 10 times that in 10^?3 M KCl. The valence of neomycin was estimated, from the slope of these curves, to be between 6 and 4. In contrast, acetylated neomycin had no effect on the nonactin-induced K⁺ membrane conductance. Therefore, the effect of neomycin on artificial membrane is related to its 4 to 6 positive charges. It is proposed that the stimulation of sugar transport in brush border membrane is related to screening of the membrane negative charges by the positively-charged neomycin. Accumulation of anions at the membrane surface then occurs and their diffusion into the intravesicular space would increase the transmembrane potential which, in turn, stimulates the entry of d-glucose.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.     

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.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

l-Glutamate effects on electrical potentials of synaptic plasma membrane vesicles

The electrogenic nature of the l-glutamate-stimulated Na⁺ flux was examined by measuring the distribution of the lipophilic anion [³⁵S]thiocyanate (SCN^?) into synaptic membrane vesicles that were incubated in a NaCl medium. Concentrations of l-glutamate from 10^?7 to 10^?4 M added to the incubation medium caused an enhanced intravesicular accumulation of SCN^?. Based on the SCN^? distribution in synaptic membrane vesicles it was calculated that 10 μM l-glutamate induced an average change in the membrane potential of + 13 mV. l-Glutamate enhanced both the Na⁺ and K⁺ conductance of these membranes as determined by increases in SCN^? influx. Other neuroexcitatory amino acids and amino acid analogs (d-glutamate, l-aspartate, l-cysteine sulfinate, kainate, ibotenate, quisqualate, $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{aspartate}$, and dl-homocysteate) also increased SCN^? accumulation in synaptic membrane vesicles. These observations are indicative of the activation by l-glutamate and some of its analogs of excitatory amino acid receptor ion channel complexes in synaptic membranes.     

Pyrrolizidine alkaloid biosynthesis: Derivation of retronecine from l-arginine and l-ornithine

The relative retention of ³H and ¹⁴C on incorporation of d-, l- and dl-isomers of [¹⁴C]arginine and [¹⁴C]ornithine into retrorsine using L-[5-³H]arginine as an internal standard has been measured. The retronecine portion of the pyrrolizidine alkaloid retrorsine, present in Senecio isatideus plants, is shown to be derived from l-arginine and l-ornithine.     

A new fluorescence assay for dipeptidyleptidase IV using tripeptide l-prolyl-l-prolyl-l-alanine as substrate

We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, l-prolyl-l-prolyl-l-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolosis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 μl of human serum and in crevicular fluid obtained from human gingival sulcus. The K_m value for the tripeptide was 1.7 · 10^?5 M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Single crystals of a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine

Lamellar single crystals were formed from a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine at the ratio of 4 to 1. The copolypeptide takes the αhelical structure. The crystals were formed by casting dilute solutions at room temperature from a solvent consisting of a 1 to 1 mixture of chloroform and trifluoroacetic acid and were observed by electron microscopy. The average crystal thickness was 670 a in the as-polymerized sample, and 580 a in a fractionated sample. The thickness was decreased by annealing at temperatures above 110 C. A hexagonal form, a group of three orthorhombic forms (group 1), and a group of an orthorhombic form and two monoclinic forms (group II) were observed by electron diffraction. The diversity of the crystal structures is suggested to be caused by a variation in crystallization conditions during evaporation of the solvent. The hexagonal form and the structures of group I are changed into the structures of group II by annealing. The crystal structures other than the hexagonal form indicate on ordered arrangements of side chains in the crystals.     

Transport of l-ascorbic acid and dehydro-l-ascorbic acid across renal cortical basolateral membrane vesicles

The uptake of l-ascorbic acid and dehydro-l-ascorbic acid into renal cortical basolateral membrane vesicles has been characterized. The uptake systems for both solutes demonstrate saturation kinetics. The presence of structural analogs of l-ascorbic acid and dehydro-l-ascorbic acid results in cis-inhibition and trans-stimulation. Uptake of each substrate is Na⁺-independent, proceeding to an endpoint of substrate equilibrium across the vesicular membrane. The transport mechanism(s) for l-ascorbic acid and dehydro-l-ascorbic acid appears to be facilitated diffusion.     

Characteristics of dipeptide transport in normal and papain-treated brush border membrane vesicles from mouse intestine I. Uptake of glycyl-l-phenylalanine

Papain treatment of isolated brush border membrane vesicles was carried out to study peptide transport in the absence of hydrolytic events associated with the brush border membrane. Such a treatment allowed a 70% decrease in the activity of membrane-associated oligopeptidases and the study of peptide transport in the complete absence of free amino acids up to 1 min of incubation. A comparison between the time course curves of glycyl-l-phenylalanine uptake by normal and papain-treated vesicles showed that the overshoots seen in the presence of Na⁺ and K⁺ gradients (extravesicular intravesicular) when using normal vesicles were no longer evident after papain treatment. This result, together with the demonstration of uptake into an osmotically reactive intravesicular space and the analysis of uptake of free phenylalanine, allowed the coclusion that peptide transport was the result of two complementary mechanisms, uptake of free amino acids following hydrolysis by the membrane-bound oligopeptidases, and intact peptide transport down a concentration gradient by a non-Na⁺ (and non-K⁺)-dependent process. These results also showed the non-involvement of γ-glutamyltransferase and the γ-glutamyl cycle in peptide absorption. A linear relationship has been established between initial dipeptide uptake and glycyl-l-phenylalanine concentration for the intact peptide transport process. However, this process can be inhibited to various extents by other di- and tripeptides but the inhibition never exceeded 43%. These results are consistent with both passive and facilitated diffusion mechanisms of intact peptide transport, the latter occuring by either a low affinity-high capacity or a high affinity-low capacity system.     

Separate binding sites in rat brain synaptic membranes for l-cysteine sulfinate and for l-glutamate

Binding of l-[³H]cysteine sulfinic acid (CSA) and l-[³H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites.The specific l-[³H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[³H]glutamate binding. The ratio of l-[³H]cysteine sulfinate binding/l-[³H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[³H]cysteine sulfinate and l-[³H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident.In sum, these data revealed the complexity of l-[³H]cysteine sulfinate and l-[³H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system.