Affinity For Sugars Research Articles

Cysteine Scanning Mutagenesis of the Segment between Putative Transmembrane Helices IV and V of the High Affinity Na+/Glucose Cotransporter SGLT1: EVIDENCE THAT THIS REGION PARTICIPATES IN THE Na+AND VOLTAGE DEPENDENCE OF THE TRANSPORTER

Site-directed mutagenesis and chemical modification of specific cysteine amino acid side chains by methanethiosulfonate (MTS) derivatives were combined to elucidate structure/function relationships of the cloned rabbit Na+/glucose cotransporter, SGLT1. Each amino acid in the region (residues 162-173) between putative transmembrane helices IV and V of SGLT1 was replaced individually with Cys. Mutant proteins were expressed in Xenopus laevis oocytes and studied using the two-electrode voltage clamp method. At certain key positions, Cys substitution resulted in 1) a change in the apparent affinity for sugar, 2) an alteration in the voltage dependence of the transient currents, and 3) a sensitivity to inhibition by either the ethylamine (MTSEA) or the ethylsulfonate MTS derivatives. For the three Cys mutants inhibited by MTSEA (F163C, A166C, and L173C), inhibition of steady state transport is related to changes in membrane potential-dependent transitions within the Na+/glucose transport cycle. MTSEA shifted the transient currents of these Cys mutants toward more negative membrane potentials (DeltaV0. 5 = -18 mV for F163C and A166C, -12 mV for L173C). When the mutations were combined to produce double and triple Cys mutants, the degree to which the transient currents were shifted along the membrane potential axis by MTSEA correlated with the number of cysteines. In this way it was possible to manipulate the voltage dependence of the transient currents over a range spanning 91 mV. Examination of the Na+ dependence of the transient currents indicates that a 91-mV shift is equivalent to that caused by a 10-fold reduction in the external Na+ concentration. We conclude that this region has a role in determining the Na+ binding- and voltage-sensing properties of SGLT1 and that it forms an alpha-helix with one surface possibly lining a Na+ pore within SGLT1.

The diversity of lectin-detectable sugar residues on root hair tips of selected legumes correlates with the diversity of their host ranges for rhizobia

Summary. Four legumes and two nonlegumes were investigated for the presence of sugars at the tips of their root hairs, using commercially available lectins which have specific affinities for certain sugars. It was found that while only one lectin (RCA-I, which binds to [3D-galactose) bound to narrow-host-range legumes and one nonlegume, five out of ten lectins tested bound to the root hair tips of the broad-host-range legume siratro (Macroptilium atropurpureum). None of the lectins tested bound to any part of Arabidopsis roots. Binding of lectins (and therefore the presence of sugars) only at the tips of growing root hairs has led us to deduce that the sugars are the glyco moiety of membrane-bound glycoproteins that are recycled at the base of the tip apical dome, along with excess plasma membrane that is known to be recycled there. As many kinds of signal transduction molecules are membrane-bound glycoproteins, we suggest that these sugars may be involved in early interactions with rhizobia, and that the broad-host-range legume siratro has more kinds of sugars to cope with the wide range of rhizobia it is able to accept for symbiotic interactions. As far as we know, this is the first report of multiple sugars at the same surface area of a tip-growing plant cell.

Identification and Characterization of Galectin-9, a Novel β-Galactoside-binding Mammalian Lectin

A 36-kDa beta-galactoside mammalian lectin protein, designated as galectin-9, was isolated from mouse embryonic kidney by using a degenerate primer polymerase chain reaction and cloning strategy. Its deduced amino acid sequence had the characteristic conserved sequence motif of galectins. Endogenous galectin-9, extracted from liver and thymus, as well as recombinant galectin-9 exhibited specific binding activity for the lactosyl group. It had two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, with no homology to any other protein. Galectin-9 had an alternate splicing isoform, exclusively expressed in the small intestine with a 31-amino acid insertion between the N-terminal domain and link peptide. Sequence homology analysis revealed that the C-terminal carbohydrate-binding domain of mouse galectin-9 had extensive similarity to that of monomeric rat galectin-5. The presence of galectin-5 in the mouse could not be demonstrated by polymerase chain reaction or by Northern or Southern blot genomic DNA analyses. Sequence comparison of rat galectin-5 and rat galectin-9 cDNA did not reveal identical nucleotide sequences in the overlapping C-terminal carbohydrate-binding domain, indicating that galectin-9 is not an alternative splicing isoform of galectin-5. However, galectin-9 had a sequence identical with that of its intestinal isoform in the overlapping regions in both species. Southern blot genomic DNA analyses, using the galectin-9 specific probe derived from the N-terminal carbohydrate-binding domain, indicated the presence of a novel gene encoding galectin-9 in both mice and rats. In contrast to galectin-5, which is mainly expressed in erythrocytes, galectin-9 was found to be widely distributed, i.e. in liver, small intestine, thymus > kidney, spleen, lung, cardiac and skeletal muscle > reticulocyte, brain. Collectively, these data indicate that galectin-9 is a new member of the galectin gene family and has a unique intestinal isoform.

Cation Effects on Protein Conformation and Transport in the Na+/Glucose Cotransporter

Cation-driven cotransporters are essential membrane proteins in procaryotes and eucaryotes, which use the energy of the transmembrane electrochemical gradient to drive transport of a substrate against its concentration gradient. Do they share a common mechanism? Cation selectivity of the rabbit isoform of the Na+/glucose cotransporter (SGLT1) was examined using the twoelectrode voltage clamp and the Xenopus oocyte expression system. The effect of H+, Li+, and Na+ on kinetics of SGLT1 was compared to the effects of these cations on the bacterial melibiose. In SGLT1, substitution of H+ or Li+ for Na+ caused a kinetic penalty in that the apparent affinity for sugar (K0.5sugar) decreased by an order of magnitude or more (from 0.2 to 30 mM) depending on the membrane potential and cation. The effect of the cation on the K0.5sugar/V profiles was independent of the sugar for glucose and alpha-methyl-beta-D-glucose; this profile was maintained for galactose in Li+ and Na+, but was 2 orders of magnitude higher in H+, but the Imax for glucose, galactose, and alpha-methyl-beta-D-glucose in a given cation were identical. Li+ supported a lower maximal rate of transport (Imax) than Na+ (approximately 80% of ImaxNa), while the Imax in H+ was higher than Na+ (>/=180% of ImaxNa). Our interpretation of these results and simulations using a six-state mathematical model, are as follows. 1) Binding of the cation causes a conformational change in the sugar binding pocket, the exact conformation being determined by the specific cation. 2) Once the sugar is bound, it is transported at a characteristic rate determined by the cation. 3) Mathematical simulations suggest that the largest contribution to the kinetic variability of both cation and sugar transport is associated with cation binding. Similarity to the effects of cation substitution in MelB suggests that the mechanism of energy coupling has been evolutionarily conserved.

Lectins implicate specific carbohydrate domains in electric field stimulated nerve growth and guidance.

Both endogenous lectins and DC electric fields may control aspects of early nerve growth and nerve guidance. To test whether such endogenous cues interact, lectins of varying sugar affinity and valency were studied for effects on electric field induced growth and reorientation of cultured Xenopus neurites. Concanavalin A (Con A), succinylated concanavalin A (S-Con A), and wheat germ agglutinin all completely inhibited field-induced cathodal reorientation. Lentil and pea lectins, which share the same sugar affinity as Con A/S-Con A, were only partially effective in inhibiting reorientation. Because S-Con A does not alter lateral mobility of membrane receptors, the previously accepted notion that Con A inhibited field-induced reorientation by preventing receptors from translocating and becoming redistributed asymmetrically in the membrane may be oversimplified. There are likely to be additional steric interactions that Con A and S-Con A share that inactivate asymmetrically redistributed receptors and prevent reorientation. Additionally, nerves growing in an applied field branch more commonly toward the cathode. Con A and S-Con A alone prevented this development of asymmetric branching. All the lectins tested prevented the normal field-induced increase in nerve growth rate, while all, except peanut agglutinin, prevented the usual faster growth cathodally than anodally. We suggest that lectin interactions with electric field effects in vitro may involve modulation of neuronal nicotinic acetylcholine receptors, neurotrophin receptors, or voltage-dependent calcium channels. Similar interactions between endogenous lectins and endogenous electric fields are to be expected.

Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

The suggestion that acidic residues in the hydrophobic NH2-terminal domains of Mel permease (Asp-31 in helix I, Asp-51 and Asp-55 in helix II, Asp-120 in helix IV) may be essential components of a coordination network involved in cation recognition (Pourcher, T., Zani, M.L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215) is further analyzed using site-directed mutagenesis. To study whether nearby polar residues also contribute to the cation recognition process, Tyr-24, Tyr-27 and Tyr-28 (aligned with Asp-31) and Tyr-109 and Tyr-116 (aligned with Asp-120) were individually converted into a phenylalanine. The effect of replacing Arg-48 (aligned with Asp-51 and Asp-55) or Asn-83 (in the middle of helix III) by an alanine was also studied. The importance of the position of the carboxylate of the residue at position 31, 51, 55, or 120 was next examined by replacing each Asp by a Glu residue. Sugar binding and/or transport activity measurements indicate that all polar-->apolar or Asp-->Glu mutants use Na+ or Li+ for active sugar transport. Moreover, two groups of mutants could be distinguished. One group, composed of Y27F, Y28F, D31E, and Y109F mutants, retains wild type permease properties. A second group (Y24F, N83A, and Y116F and also D51E, D55E, and D120E) exhibits concomitant reduction of affinity for sodium and sugars and altered sugar specificity but conserves wild type cation selectivity profile. The data reinforce the notion that Asp-51, Asp-55, and Asp-120 residues and the position of their carboxyl side chains are of primary importance for cation recognition. Finally, since Mel permease properties are predominantly modified by mutagenizing residues located in the cytoplasmic half of the permease, we propose that Mel permease has a well-like shape opened toward the periplasmic space and is closed at its cytoplasmic extremity by a gate.

A neutrophil migration-inducing lectin from Artocarpus integrifolia.

A neutrophil migration-inducing protein has been isolated from the saline extract of Artocarpus integrifolia seeds by successive sugar affinity chromatography steps during which the protein was not absorbed by D-galactose resin, and then was absorbed to and eluted from D-mannose resin by 0.1 M D-mannose. Gel filtration on Superdex 75 HR indicated a molecular mass of 52 kDa when 0.1 M D-mannose was present in the elution buffer. A single band of apparent molecular mass of 13 kDa was demonstrable by SDS-PAGE only after heating, both in the presence and absence of reducing agent, suggesting that the molecule is a tetramer formed by the noncovalent association of 13 kDa chains. Isoelectric forms corresponding to isoelectric points of 4.0, 4.2, 5.0, and 5.2 were demonstrable by isoelectric focusing-PAGE, and four active forms having the same isoelectric points were separated by chromatofocusing. The minimal m.w. calculated from amino acid analysis data was 13,193. The protein, denoted KM+, stimulated neutrophil migration in the rat peritoneal cavity assay in a dose-related manner in the range of 1 to 300 micrograms per rat. The dose-response curve of the in vitro chemotactic activity of KM+ was bell shaped and its ascending limb was dose dependent in the range of 1 ng to 10 micrograms/well. D-Mannose (0.1 M) inhibited the in vitro (80%) and in vivo (60%) neutrophil migration-inducing activities of KM+ and also its hemmaglutinating activity. The chemotactic activity was shown to be caused by haptotaxis rather than chemokinesis. The physical and biologic properties of KM+ suggest that this lectin may attract neutrophils by a mechanism involving a haptotactic gradient as has been proposed for IL-8. KM+ might be used as tool to study protein-carbohydrate interactions during neutrophil migration through the extracellular matrix.

Protons drive sugar transport through the Na+/glucose cotransporter (SGLT1).

Na(+)-dependent transporters, such as the Na+/glucose cotransporter (SGLT1) have long been characterized as having an overwhelming preference for Na+ as the essential ion. We have applied electrophysiological measurements to analyze the ability of protons to transport glucose with a cloned transporter, SGLT1, expressed in Xenopus laevis oocytes. Our results show that protons can drive sugar transport through SGLT1 in the absence of Na+ with the following characteristics: 1) the affinity of SGLT1 for H+ is 3 orders of magnitude higher than its affinity for Na+ (3 microM versus 20 mM); 2) H+ supports a higher maximum transport than Na+, suggesting an alteration in rate-limiting processes; and 3) the cation determines the transporter's affinity for sugar (at Vm = -50 mV, the apparent affinity for alpha-methyl-D-glucoside is 0.2 mM in Na+ and 20 mM in H+). The similarity in the kinetics of H(+)- and Na(+)-dependent sugar transport suggests that the transport mechanism for SGLT1 does not depend on the driving ion.

Steady-state and presteady-state kinetics of the H+/hexose cotransporter (STP1) from Arabidopsis thaliana expressed in Xenopus oocytes.

We have investigated the steady-state and presteady-state kinetics of the cloned H+/hexose cotransporter from Arabidopsis thaliana (STP1) expressed in Xenopus oocytes using the two-electrode voltage-clamp method. Steady-state sugar-dependent currents were measured between -150 and +50 mV as a function of external [3-O-methyl-D-glucose] (3OMG) and [H+]. At pH 6.5 (316 nM H+) the maximal current for sugar, i3OMGmax, was voltage-dependent, increasing from 40 nA at -30 mV to 95 nA at -150 mV. The apparent affinity of sugar, K3OMG0.5, at pH 6.5 decreased from 30 microM at -30 mV to 11 microM at -70 mV and was then voltage-independent between -70 and -150 mV. Increasing the extracellular [H+] to 3160 nM (pH 5.5) increased i3OMGmax to 65 nA at -30 mV and 212 nA at -150 mV. K3OMG0.5 at pH 5.5 also increased and was voltage-dependent: 15 microM at -50 mV rising to 25 microM at -150 mV. At pH 6.5 and 5.5, the Hill coefficient n for 3OMG was voltage-independent and averaged 1.2 and 1.4, respectively. At saturating [3OMG] iHmax was voltage-dependent and KH0.5 was voltage-independent, averaging 370 nM (pH 6.4). The Hill coefficient n for H+ was voltage-independent and averaged 1. The sugar specificity of STP1 was D-mannose > or = 2-deoxyglucose > D-galactose > or = 3OMG > D-xylose > D-glucose > D-fucose > D-fructose > L-glucose > L-arabinose > D-arabinose, demonstrating that STP1 has a low substrate specificity. Transient currents recorded after rapid steps in membrane potential relaxed with time constants tau, between 3 and 14 ms. The charge movement Q (the integral of the current transients) fitted to a Boltzmann relation with maximal charge Qmax of 3.4 nanocoulombs and an apparent valence z approximately 1 corresponding to a transporter density of 2 x 10(10)/oocyte. Potential for 50% Qmax (V0.5) was -28 mV. At saturating 3OMG and at low external [H+] (pH 7.5), the transient STP1 currents were eliminated. The presteady-state data indicate that STP1 can bind H+ in the absence of sugar, and the steady-state data suggest that H+/hexose cotransport occurs via a sequential mechanism.

Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli.

Acidic residues which are found on transmembrane segments within the lactose permease may play an important role in H+ and/or sugar recognition. To examine the functional roles of Glu-269 and Glu-325, we have constructed a variety of amino acid substitutions (e.g. aspartate, glycine, alanine, serine, or glutamine) via site-directed mutagenesis. At position 269, all mutations appear to have a detrimental effect on sugar affinity, downhill transport, and counterflow. The Asp-269 mutant was able to accumulate lactose against a concentration gradient, whereas all of the nonionizable substitutions at position 269 were completely defective. Nevertheless, in spite of their inability to actively accumulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H+ upon the addition of galactosides. Mutations at position 325 had a markedly different phenotype. For example, the Asp-325, Gly-325, and Gln-325 mutants exhibited an apparent Km for lactose transport (e.g. 0.21, 0.47, and 0.50 mM, respectively), which was actually lower than that of the wild-type strain (1.44 mM). In counterflow assays, all position 325 mutants also appear to catalyze lactose exchange. Similar to the results obtained at position 269, the Asp-325 mutant exhibited moderate levels of accumulation, whereas none of the nonionizable mutations at position 325 were able to accumulate galactosides against a concentration gradient. However, unlike the position 269 mutants, no H+ transport was observed in the Gly-325, Ala-325, Ser-325, or Gln-325 strains upon the addition of lactose, S-beta-D-galactopyranosyl-(1,1)-beta-thiogalactopyranoside, 1-O-methyl-beta-D-galactopyranoside, or melibiose. Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to delta pH. Overall, these results are consistent with the notion that an acidic residue at position 325 is required for H+ transport via the lactose permease. Alternative hypotheses are also discussed.

Chemical modification and sugar binding properties of two major lectins from Pinhao (Araucaria brasiliensis) seeds

Araucaria brasiliensis lectins, lectin I and lectin II, were subjected to various chemical modifications to detect the amino acid residues present in their carbohydrate binding sites. Modification of tyrosine and arginine residues did not affect binding activities of lectins. However, modification of tryptophan and histidine led to a complete loss. Modification of amino and carboxyl groups of both lectins also abolished completely their hemagglutinating capacity. The carbohydrate moiety of lectins was not involved in maintaining their hemagglutinating activities. Binding of monosaccharides was studied by UV difference spectroscopy which showed that both lectins have two binding sites with different affinities for sugars

Purification and characterisation of d‐glucose oxidase from white‐rot fungus Pleurotus ostreatus

D-Glucose oxidase was purified 27.5-fold to apparent homogeneity with an overall yield of 23.8%, from Pleurotus ostreatus, through a purification procedure of ammonium sulphate precipitation, gel-permeation, anion-exchange and hydrophobic-interaction chromatography. The molecular mass determined by gel filtration was found to be 290 kDa. SDS/PAGE revealed that the enzyme consists of four subunits with a molecular mass of 70 kDa. The absorption spectra of the enzyme exhibit maxima at 280, 360 and 460 nm. The enzyme shows a fluorescence spectrum with an excitation maximum at 470 nm and an emission maximum at 530 nm. These results indicate that the prosthetic group of the enzyme is flavin and that the enzyme contains 4 mol flavin/mol enzyme. The enzyme is optimally active at 50 degrees C and at pH 5.5-6.0. It exhibits broad affinity for various sugars and specificity for D-glucose with Km value of 1.34 mM. 2,6-Dichloroindophenol, Wurster's blue, and 4-benzoquinone can function as electron acceptors but phenazine methosulphate cannot function as an electron acceptor. The enzyme is inhibited completely by mercuric chloride and partially by silver sulphate, sodium azide 8-hydroxyquinoline.

Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress.

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.

On the specificity of the d-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of d-galactose and thiogalactose

The d-galactose-binding lectin (PA-I) from the bacterium Pseudomonas aeruginosa, isolated by affinity chromatography on Sepharose, was examined for its relative affinities for simple sugars and their derivatives using equilibrium dialysis and hemagglutination inhibition tests. The lectin, which was found to bind 0.68 mol of d-galactose per subunit of 12.8 kDa, exhibited an association constant ( K a) of 3.4·10 4 M −1 for d-galactose and higher affinities for hydrophobic and thio derivatives of d-galactose (with highest affinity for the hydrophobic thio derivatives). α-Methyl-galactoside was a stronger inhibitor than the β-methyl derivative and α-lactose was a weak inhibitor but the hydrophobic phenylated derivatives of the β-configuration of d-galactose were more potent inhibitors than the respective α-galactosides.

Isolation and characterization of isolectins from Erythrina variegata seeds

Three isolectins were isolated from seeds of Erythrina variegata (Linn.) var. Orientalis by ion-exchange chromatography, followed by affinity chromatography on lactose-Sepharose 4B and acid-treated Sepharose 4B columns. The purified isolectins (EVLI, EVLII and EVLIII) are all specific for galactopyranosides and N-acetylgalactosamine, and their affinities for simple sugars are EVLIII > EVLII > EVLI. EVLI and EVLIII are homodimers made up of an A-subunit of molecular mass 36 000 and a B-subunit of molecular mass 33 000, whereas EVLII is a heterodimer composed of the A- and B-subunits. Upon treatment with trifluoromethansulphonic acid, the molecular masses of both subunits decreased to 31 000. Rechromatography of EVLII on the acid-treated Sepharose 4B column again produced the homodimeric lectins (EVLI and EVLIII). It is suggested that the constituent subunits of Erythrina variegata isolectins are eschangeable with each other in vitro.

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a Glyser insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.

Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor.

The extracellular portion of the macrophage mannose receptor is composed of several cysteine-rich domains, including a fibronectin type II repeat and eight segments related in sequence to Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of animal lectins. Expression of portions of the receptor in vitro, in fibroblasts and in bacteria, has been used to determine which of the extracellular domains are involved in binding and endocytosis of ligand. The NH2-terminal cysteine-rich domain and the fibronectin type II repeat are not necessary for endocytosis of mannose-terminated glycoproteins. CRDs 1-3 have at most very weak affinity for carbohydrate, so the carbohydrate binding activity of the receptor resides in CRDs 4-8. CRD 4 shows the highest affinity binding and has multispecificity for a variety of monosaccharides. However, CRD 4 alone cannot account for the binding of the receptor to glycoproteins. At least 3 CRDs (4, 5, and 7) are required for high affinity binding and endocytosis of multivalent glycoconjugates. In this respect, the mannose receptor is like other carbohydrate-binding proteins, in which several CRDs, each with weak affinity for single sugars, are clustered to achieve high affinity binding to oligosaccharides. In the mannose receptor, these multiple weak interactions are achieved through several active CRDs in a single polypeptide chain rather than by oligomerization of polypeptides each containing a single CRD.

Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins.

Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated. To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions, corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants. Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the structure and function of this regulatory protein.

The one-site model of human erythrocyte glucose transport: testing its predictions using network thermodynamic computer simulations

Network thermodynamic computer simulations were carried out using parameters experimentally derived by Lowe and Walmsley ((1987) Biochim. Biophys. Acta 903, 547–550) for two tests of the one-site model of human erythrocyte glucose transport. In the temperature-jump experiment, the simulations predicted the amplitude and relaxation time of accelerated uptake, but underestimated the net uptake due to an unexpectedly low measured basal rate. In the maltose-acceleration experiment, the dissociation constant of maltose was assessed at 0°C by measuring the inhibitory effects of maltose on both cytochalasin B binding and on 3- O-methylglucose uptake, and using this value (52 mM) to calculate the dissociation constant (2.9 mM). The simulated experiment then did show a transient acceleration in uptake comparable in magnitude to that observed experimentally, except that the relaxation time was more than 10-fold longer in the simulations. Measurements of the temperature dependence of the inhibition of cytochalasin B binding by maltose and 3- O-methylglucose indicated that apparent sugar affinity is sensitive to carrier orientation at low temperatures, whereas at more physiologic temperatures the intrinsic dissociation constant predominated.

Lektinhistochemische Untersuchungen des retinalen Pigmentepithels bei Netzhautablösung

The retinal pigment epithelium (RPE) exhibits a broad spectrum of morphological changes under pathological conditions. Since RPE cells cannot be immunocytochemically characterized with certainty, lectin histochemical investigations were performed to study the lectin binding pattern of different morphological RPE variants in human globes. Normal RPE with attached retinas was compared to reactive changes in RPE following retinal detachment. Lectin binding sites were visualized by a modified PAP technique performed on paraffin-embedded tissue sections. Eight lectins of different sugar affinity (Con A, WGA, PNA, RCA 1, SBA, UEA 1, DBA, LPA) were tested for binding sites on the RPE. Both normal and reactively changed RPE possess receptors for Con A, WGA, PNA, and RCA 1. Modifications in the lectin binding pattern occurred simultaneously with the morphological changes within the RPE cells. RPE cells which form a monolayer on Bruch's membrane mainly have lectin binding sites in their apical portions. Proliferating and migrating RPE cells, especially RPE macrophages, which have withdrawn from the basal cell layer were found to contain lectin binding sites dispersed over the entire cytoplasm. RPE macrophages exhibited additional binding sites for the lectin SBA. These results indicate that RPE cell variants can be designated by means of their specific combination of lectin binding sites for Con A WGA, PNA, and RCA 1 not found on other cell types in the retina.