The comparative specificity of the inner and outer substrate transfer sites in the choline carrier of human erythrocytes

Summary The substrate specificities on the inner and outer surfaces of the cell membrane have been compared by determining the relative affinities, inside and outside, of a series of choline analogs. The results of two different methods were in agreement: (1) the carrier distribution was determined in the presence of a saturating concentration of an equilibrated analog, using N-ethylmaleimide as a probe for the inward-facing carrier; (2) the degree of competition was measured between an equilibrated analog and choline in the external solution. The carrier sites are found to have markedly different specificities: the outer site is more closely complementary to the structure of choline than is the inner, and even a slight enlargement of either the trimethylammonium or hydroxyethyl group gives rise to preferential binding inside. It is also found that a nonpolar binding region, which is adjacent to the outer site, is absent from the inner site. As the transport mechanism involves the exposure of only one site at a time, first on one surface and then the other, it follows that an extensive reorganization of the structure of the substrate site may occur during the carrier-reorientation step, or alternatively that two distinct sites may be present, only one of which is exposed at a time.     

Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: Evidence for a carrier conformational change on complex formation

Summary The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearlydouble that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1mm N-ethylmaleimide at pH 6.8, 37°C, the constants are 0.57±0.05 min^–1 and 0.33±0.02 min^–1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.     

Substitution of choline by related compounds in the diet of Argyrotaenia velutinana and Heliothis virescens

Argyrotaenia velutinana, the red-banded leaf roller, and Heliothis virescens, the tobacco budworm, both require choline for growth and development when reared on semisynthetic diets. The optimum level for A. velutinana is $\text{50 mg}\text{100 g}$ of diet whereas that for H. virescens exceeds $\text{100 mg}\text{100 g}$ of diet.No choline analog tested can adequately replace choline in the diet. One compound, dimethylethylcholine, will permit some adut emergence but development is slower and mortality is greater than on the corresponding diet containing choline. This is in sharp contrast to a number of Diptera in which mary choline analogs can not only replace choline in the diet but are also incorporated into phospholipids analogous to phosphatidylcholine.In A. velutinana, dimethylisopropylcholine and β-methylcholine, although inadequate as choline replacements, can spare the dietary choline requirement, Isopropylethanolamine is a growth inhibitor for A. velutinana but not for H. virescens.     

Apparent noncompetitive inhibition of choline transport in erythrocytes by inhibitors bound at the substrate site

Summary According to the conventional carrier model, an inhibitor bound at the substrate transfer site inhibits competitively when on the same side of the membrane as the substrate, but noncompetitively when on the opposite side. This prediction was tested with the nonpenetrating choline analog dimethyl-n-pentyl (2-hydroxyethyl) ammonium ion. In zerotrans entry and infinitetrans entry experiments, where the labeled substrate and the inhibitor occupy the same compartment, the inhibition was competitive, but in zerotrans exit it was noncompetitive, in accord with the model. Similar behavior was seen with dimethyl-n-decyl (2-hydroxyethyl) ammonium ion. With this property of the choline transport system established, it becomes possible to estimate the relative affinity inside and outside of inhibitors present on both sides of the membrane. The tertiary amine, dibutylaminoethanol, which enters the cell by simple diffusion, is such an inhibitor. Here the inhibition kinetics were the reverse of those for nonpenetrating inhibitors; zerotrans and infinitetrans exit was inhibited competitively, and zerotrans entry noncompetitively. It follows that dibutylaminoethanol binds predominantly to the inner carrier form.     

Expression of substrate specificity in facilitated transport systems

Summary In facilitated transport systems the carrier reorientation step is shown to be largely independent of the forces of interaction between the substrate and the carrier site, whereas in coupled systems (obligatory exchange or cotransport) reorientation proceeds at the expense of the binding force developed in the transition state. In consequence, the expression of substrate specificity is expected to differ in the two systems. In the facilitated transport of analogs no larger than the normal substrate, the affinity but not the maximum rate of transport can vary widely; with larger analogs, both the affinity and rale can vary if steric constraints are more severe in the translocation step than in binding. In coupled transport, by contrast, the translocation step can be highly sensitive to the structure of the substrate, and binding much less sensitive. The theory agrees with published observations on facilitated systems for choline and glucose in erythrocytes, as well as on Na⁺-coupled systems for the same substrates in other cells. The following mechanism, which could account for the behavior, is proposed. In facilitated systems, the transport site fits the substrate closely and retains its shape as the carrier undergoes reorientation. In coupled systems, the site is initially looser, but during carrier reorientation it contracts around the substrate. In both systems, the carrier encloses the substrate during the translocation step, though for a different reason: in coupled but not in facilitated systems the binding force enormously increases in the enclosed state, through a chelation effect. In both systems, steric interference with enclosure retards the translocation of bulky substrate analogs.     

16α,17α-Cycloalkane Derivatives of Progesterone Intensively Bind to a Rat Serum Protein

The interaction of 6-methyl-[1,2-³H]16,17-cyclohexanoprogesterone with rat serum proteins has been studied. Specific binding of this ligand characterized by K _d = 0.36 ± 0.10 M and concentration of binding sites (B_max) of about 1 M (27.8 ± 12.5 pmol/mg total protein) was found. According to competitive analysis, the affinity of the studied progestins to a protein that differs from transcortin was to some extent correlated with their hydrophobicity. The dissociation kinetics of ³H-ligand–protein complexes were biphasic, the binding sites forming stable and labile complexes with ³H-ligand being eluted in the same region during ion-exchange chromatography. In overall properties, the serum protein differs from the progesterone receptor and the pregna-D"-pentarane-specific protein from rat uterus. It is suggested that the revealed protein may provide high progestagenic activity of 6-methyl-16,17-cyclohexanoprogesterone by prolonging its retention in the bloodstream.     

Regulation of Poly glutamate Production in Bacillus subtilis (natto): Transformation of High PGA Productivity

Bacillus subtilis (natto) produces a considerable amount of polyglutamate (PGA). The genetic character of high PGA productivity of B. subtilis (natto) was transferred by DNA-mediated transformation to B. subtilis Marburg 168 which cannot produce PGA. The enzyme activity of γ-glutamyltranspeptidase (γ-GTP) of the three transformants, 3F1, F1-9 and M5B4, was 124, 233 and 147 mU/ml, which is about 25, 250 and 100% of that of the donor strains, respectively. However, other enzyme activities such as those of alanine racemase or transaminase among the parental strains and representative transformants were almost the same.

These results suggested that γ-GTP activity might mainly participate in the biosynthesis of PGA in B. subtilis (natto).     

Reconstitution of carrier-mediated choline transport in proteoliposomes prepared from presynaptic membranes oftorpedo electric organ,and its internal and external ionic requirements

Summary Proteoliposomes made by a butanol-sonication technique from electric organ presynaptic membranes showed choline transport activity. In contrast to intact nerve terminals, the uptake of choline was dissociated from its conversion to acetylcholine in this preparation. The kinetics of choline uptake by proteoliposomes was best described by two Michaelis-Menten components. At a low concentration of choline, uptake was inhibited by hemicholinium-3 and required external Na⁺ and, thus, closely resembled high-affinity choline uptake by intact cholinergic nerve terminals. Choline transport could be driven by the Na⁺ gradient and by the transmembrane potential (inside negative) but did not directly require ATP. External Cl^–, but not a Cl^– gradient, was needed for choline transport activity. It is suggested that internal K⁺ plays a role in the retention of choline inside the proteoliposome. Proteoliposomes should prove a useful tool for both biochemical and functional studies of the highaffinity choline carrier.Abbreviations ACh acetylcholine - HC-3 hemicholinium-3 - ChAT choline acetyltransferase     

Functional properties of purified and reconstituted mitochondrial metabolite carriers

Eight mitochondrial carrier proteins were solubilized and purified in the authors' laboratories using variations of a general procedure based on hydroxyapatite and Celite chromatography. The molecular mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatography on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties. A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asymmetric orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution. All carriers which have been investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport; i.e., a ternary complex, made up of two substrates and the carrier protein, is involved in the catalytic cycle. The only exception was the carnitine carrier, where a ping-pong mechanism of transport was found. By reaction of particular cysteine residues with mercurial reagents, several carriers could be reversibly converted to a functional state different from the various physiological transport modes. This unphysiological transport mode is characterized by a combination of channel-type and carrier-type properties.     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

Specificity of the Rat Vesicular Acetylcholine Transporter

The protein kinase A–deficient PC12 cell line PC12^A123.7 lacks both choline acetyltransferase and the vesicular acetylcholine transporter. This cell line has been used to establish a stably transfected cell line expressing recombinant rat vesicular acetylcholine transporter that is appropriately trafficked to small synaptic vesicles. Acetylcholine is transported by the rat vesicular acetylcholine transporter at a maximal rate of 1.45 nmol acetylcholine/min/mg protein and exhibits a Km for transport of 2.5 mM. The transporter binds vesamicol with a Kd of 7.5 nM. The ability of structural analogs of acetylcholine to inhibit both acetylcholine uptake and vesamicol binding was measured. The results demonstrate that like Torpedo vesicular acetylcholine transporter, the mammalian transporter can bind a diverse group of acetylcholine analogs.     

Errata

The unidirectional uptake into the trophoblast of l-leucine, l-isoleucine, l-phenylalanine, l-tryptophan, l-methionine and l-tyrosine from either the maternal or fetal circulations of an isolated dually-perfused guinea-pig placenta was studied using a single circulation paired-tracer dilution technique. Significant and equal uptakes were found on both sides. l-Phenylalanine uptake kinetics on the fetal side indicated an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 17.0 mM and a $\text{V}$ of 8.2 μmol/min per g.     

The carrier reorientation step in erythrocyte choline transport: pH effects and the involvement of a carrier ionizing group

Summary Under zero-trans conditions, the facilitated transport of choline across the erythrocyte membrane is limited by the rate of reorientation of the free carrier; as a result the pH dependence of this step can be investigated, independent of other steps in transport. It is found that as the pH declines (between 8.0 and 6.0) the rate of inward movement of the free carrier rises and the rate of outward movements falls, so that the partition of the free carrier increasingly favors the inward-facing form. When the pH of the cell interior and of the medium are varied independently, the partition responds to the internal but not the external pH. The membrane potential, which varies somewhat as the pH is altered, has no effect on the carrier partition. The analysis of the results indicates that the carrier mobility is dependent on an ionizing group of pK_a 6.8, which is exposed on the cytoplasmic surface of the membrane in the inward-facing carrier; in the out-ward-facing carrier the ionizing group appears to be masked, in that its pK_a is shifted downward by more than one unit. The observations can be explained by assuming that an ionizing group is located in the wall of a gated channel connecting the substrate site with the cytoplasmic face of the cell membrane.     

Kinetics of the Reconstituted Tricarboxylate Carrier from Eel Liver Mitochondria

The tricarboxylate carrier from eel liver mitochondria was purified by chromatography on hydroxyapatite and Matrix Gel Blue B and reconstituted into liposomes by removal of the detergent with Amberlite. Optimal transport activity was obtained by using a phospholipid concentration of 11.5 mg/ml, a Triton X-114/phospholipid ratio of 0.9, and ten passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased by cardiolipin and phosphatidylethanolamine and decreased by phosphatidylinositol. The reconstituted tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The maximum transport rate of external [¹⁴C]citrate was 9.0 mmol/min per g of tricarboxylate carrier protein at 25°C and this value was virtually independent of the type of substrate present in the external or internal space of the liposomes. The half-saturation constant (K _m) was 62 M for citrate and 541 M for malate. The activation energy of the citrate/citrate exchange reaction was 74 kJ/mol from 5 to 19°C and 31 kJ/mol from 19 to 35°C. The rate of the exchange had an external pH optimum of 8.     

Kinetic Evidence for the Uniport Mechanism Hypothesis in the Mitochondrial Tricarboxylate Transport System

The kinetics of the transport of citrate by the tricarboxylate transport system located in the inner mitochondrial membrane was studied in proteoliposomes containing the purified carrier protein, in order to verify the previously hypothesized mechanism of uniport (J. Bioenerg. Biomembr. 35, 133–140, 2003) and achieve some information on the kinetic properties of the carrier transport system. For this purpose, a mathematical model has been elaborated and the experimental data were analyzed according to it. The results indicate that the data actually fit with the uniport model, and hence it is confirmed that the carrier has a single binding site for its substrates and can oscillate between the inside and outside form, in both the free and substrate-bound states. The rearrangement of the free form is slower than the bound form in both directions. The dissociation constants for the internal substrate are at least one order of magnitude higher than the one for external citrate. As a consequence of these last two points, the rate of citrate transport by the carrier is much higher when it operates in exchange with another substrate than when it operates in net uniport.     

THE CHEMICAL FORM OF DISSOLVED SI TAKEN UP BY MARINE DIATOMS

Results of past studies of the pH-dependent Si uptake kinetics of Phaeodactylum tricornutum Bohlin suggested that the anion SiO(OH)     is the chemical form of dissolved Si taken up by marine diatoms. We determined the chemical form of Si taken up by three other marine diatom species and P. tricornutum by examining the kinetics of Si use under two dramatically different SiO(OH)     :Si(OH)₄ ratios in seawater by varying pH from ≈8 to ≈9.6. Uptake rates were determined using a precise and sensitive ³²Si tracer methodology. The pH-dependent uptake kinetics obtained for all species except P. tricornutum suggest that marine diatoms transport Si(OH)₄. The half-saturation constant (K _m ) varies strongly as a function of pH for all species when the substrate of transport is assumed to be SiO(OH)     . Kinetic curves for Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, Thalassiosira weissflogii (Grunow) G. Fryxell et Hasle, and Cylindrotheca fusiformis Reimann et Lewin have statistically identical values of K _m at each pH when the substrate for transport is assumed to be Si(OH)₄ ( T. pseudonana and T. weissflogii ) or total dissolved silicon ( C. fusiformis ). In contrast, P. tricornutum exhibits unusual biphasic uptake kinetics: uptake conforms to Michaelis–Menten kinetics up to 15 to 25 μM, above which uptake increases linearly. This enigmatic response may have biased conclusions drawn from past experiments using this species. However, based on the consistency of the results for the three other species, a new model of Si transport in marine diatoms is proposed on the basis of the direct formation of a complex between the Si-transport protein and Si(OH)₄.     

5-Amino-2-pyridyl 1-thioglycosides in synthesis of analogs of glycosyltransferases substrates

We present the synthesis of 1-thioglycosyl derivatives of uridine, which were designed to act as potential donor substrates for glycosyltransferases. We constructed such analogs using 5-amino-2-pyridyl 1-thioglycosides as glycosyl units which were connected to uridine via succinic linker. For preparation of the amide bonds we applied different condensation procedures.     

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k_cat/K_m values >10,000 M^?1 s^?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k_cat/K_m values exhibited against these canonical substrates are well under 10⁵ M^?1 s^?1. By contrast, several Nudix enzymes show much larger k_cat/K_m values (in the range of 10⁵ to >10⁷ M^?1 s^?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Transport of alkali cations through thin lipid membranes by (222)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of K⁺ and Na⁺ transport across the membrane of large unilamellar vesicles (L.U.V.) were compared at two pH's, with two carriers: (222)C ₁₀-cryptand (diaza-1, 10-decyl-5-hexaoxa-4,7,13,16,21,24-bicyclo[8.8.8.]hexacosane) and valinomcyin, i.e. an ionizable macrobicyclic amino polyether and a neutral macrocyclic antibiotic. The rate of cation transport by (222)C₁₀ saturated as cation and carrier concentrations rose. The apparent affinity of (222)C₁₀ for K⁺ was higher and less pH dependent than that for Na⁺ but resembled the affinity of valinomycin for K⁺. The efficiency of (222)C₁₀ transport of K⁺ decreased as the pH fell and the carrier concentration rose, and was about ten times lower than that of valinomycin. Noncompetitive K⁺/Na⁺ transport selectivity of (222)C₁₀ decreased as pH, and cation and carrier concentrations rose, and was lower than that of valinomycin. Transport of alkali cations by (222)C₁₀ and valinomycin was noncooperative. Reaction orders in cationn(S) and carrierm(M) varied with the type of cation and carrier and were almost independent of pH;n(S) andm(M) were not respectively dependent on carrier or cation concentrations. The apparent estimated constants for cation translocation by (222)C₁₀ were higher in the presence of Na⁺ than of K⁺ due to higher carrier saturation by K⁺, and decreased as pH and carrier concentration increased. Equilibrium potential was independent of the nature of carrier and transported cation. Results are discussed in terms of the structural, physicochemical and electrical characteristics of carriers and complexes.