Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.     

The relationship between glucose transport and the production of succinoglucan exopolysaccharide by Agrobacterium radiobacter

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-D-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.     

Molecular weights of nitrogenase components from Azotobacter vinelandii.

Meniscus depletion sedimentation equilibrium ultracentrifuge experiments were performed on purified MoFe and Fe proteins of $\text{Azotobacter vinelandii}$. The MoFe protein was found to have a molecular weight of 245,000, using an experimentally confirmed partial specific volume of 0.73. The MoFe protein formed one band on sodium dodecyl sulfate gel electrophoresis and had a subunit molecular weight of 56,000. The subunit molecular weight from ultracentrifuge experiments in 8 M urea was 61,000. The molecular weight of the Fe protein was calculated to be 60,500 in meniscus depletion experiments. Similar experiments in 8 M urea solvent indicated a subunit molecular weight of 30,000. A subunit molecular weight of 33,000 was obtained from sodium dodecyl sulfate gel electrophoresis experiments.     

Isolation and immunological characterization of a glucose-regulated fibroblast cell surface glycoprotein and its nonglycosylated precursor.

We have isolated and immunochemically characterized a major membrane glycoprotein of mouse 3T3 cells. This GRP (glucose/glycosylation-regulated protein) is labeled by lactoperoxidase-mediated iodination and by ¹⁴C-glucosamine, binds concanavalin A and has an apparent molecular weight in SDS-polyacrylamide gels of 92,000 daltons (or 97,000 daltons in a discontinuous gel system). Glycosylated GRP was isolated from plasma membranes using Triton X-100 extraction, affinity chromatography on concanavalin A-Sepharose and preparative SDS gel electrophoresis.Antibody against this glycosylated GRP stains the external surfaces of mouse cells and induces patches and caps. Immunofluorescence and immunoprecipitation studies indicate that this glycoprotein can exist in the membrane in two molecular forms, either as a glycosylated or as a nonglycosylated protein. The nonglycosylated form is induced under conditions of limited glycosylation or glucose deprivation. This nonglycosylated GRP remains accessible to antibodies on the exterior of cells, but becomes inaccessible to lactoperoxidase.The immunoprecipitation of the 92K GRP with its specific antibody is always associated with the precipitation of a small fraction of the other major GRP of molecular weight 75,000 daltons. We suggest that both GRP (92K and 75K) may function in close association in the membrane.     

Isolation ofn-ethylmaleimide-labelled phlorizin-sensitived-glucose binding protein of brush border membrane from rat kidney cortex

A glucose receptor with high affinity for phlorizin from isolated brush border of rat kidney was labelled specifically withN-[¹⁴C]ethylmaleimide and then extracted from the membranes.After the solubilization of the brush borders with sodium dodecyl sulphate theN-[¹⁴C]ethylmaleimide-labelled receptor protein was isolated and was found to have a molecular weight of approximately 30 000 as determined by sodium dodecyl sulphate-polyacrylamide gel disc electrophoresis. The receptor protein eluted from the sodium dodecyl sulphate-containing gels migrates as a single band on sodium dodecyl sulphate-free polyacrylamide gels.The receptor protein can also be released from the brush borders with low concentrations of sodium deoxycholate. Under these conditions the molecular weight of theN-[¹⁴C]ethylmaleimide-labelled receptor protein is approximately 60 000 in contrast to the protein component solubilized with sodium dodecyl sulphate. Since this detergent is known to dissociate the brush border membrane into its protein components, our results suggest that the phlorizin- sensitive glucose receptor protein has a molecular weight of about 30 000.     

Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture

Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.     

Isolation of dicarboxylic acid- and glucose-binding proteins from Pseudomonas aeruginosa.

Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates.     

Molecular characteristics of rat brain glucose transporter: a novel species with Mr 45,000

The glucose transporter of rat brain was examined by the use of cytochalasin B, a potent inhibitor. The dissociation constants (Kd) of D-glucose-inhibitable cytochalasin B binding in various membrane fractions were about 100 nM. Solubilization and partial purification of glucose transporter were carried out by procedures of DE 52 column chromatography, Bio Gel HT column chromatography and Sepharose CL-6B column chromatography from postnuclear membrane fraction. Purified transporter, reconstituted in lipid vesicles, showed D-glucose-specific transport activity with a Michaelis constant (Km) of 7 mM. The molecular weight was estimated to be about 200K by gel filtration in the presence of 0.1% Triton X-100. The subunit molecular weight was estimated to be 45K by SDS-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a covalent probe, indicating that rat brain glucose transporter is a tetramer.     

Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane-Bound Polysomes

Abstract: Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[³⁵S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α¹, α², β¹, and α²) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to α-tubulin. Only trace amounts of α- and β-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and α- and β-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α- and β-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to α-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.     

Purification and properties of maize polyamine oxidase: a flavoprotein

Polyamine oxidase was purified from maize shoots to homogeneityby the criteria of polyacrylamide gel electrophoresis and ultracentrifugation.The purified yellow enzyme showed absorption maxima at 278,380 and 460 nm. The molecular weight estimated by gel filtrationwas about 65,000 and the sedimentation coefficient was 5.95S. Sodium dodecylsulfate gel electrophoresis yielded a singleband at a molecular weight of 65,000. The enzyme contained 1mole of FAD per mole of enzyme. Amino acid composition and kineticproperties of the enzyme are presented. (Received April 30, 1980; )     

Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin

A gastrin binding protein (GBP) has been identified in detergent extracts of porcine gastric mucosal membranes by covalent cross-linking to 125I-[Nle15]gastrin with disuccinimidyl suberate. The apparent molecular weight of the cross-linked complex (80,000) is uneffected by reduction suggesting that the GBP is not composed of disulfide-bonded subunits. Subtraction of the molecular weight of 125I-gastrin indicates that the molecular weight of the GBP is 78,000. A similar molecular weight has been observed previously for the gastrin receptor (74,000) on intact canine parietal cells and plasma membranes therefrom, and for the receptor for the related hormone cholecystokinin (76,000-85,000) on pancreatic acinar membranes under reducing conditions. The similarity in molecular weight between the gastrin receptor and the solubilized GBP suggests that the latter protein is probably the gastrin receptor. However, the concentration (2 microM) of [Nle15]gastrin required for 50% inhibition of cross-linking of gastrin to the GBP solubilized in 0.1% Triton X-100 is 200-fold greater than the value (10 nM) observed for the gastrin receptor on isolated canine gastric parietal cells. A lower concentration (0.3 microM) of [Nle15]gastrin was required to inhibit cross-linking in a milder detergent (0.4% digitonin, 0.08% cholate). Thus, the reduced affinity for gastrin of the putative solubilized form of the gastrin receptor appears to be a result of detergent extraction.     

Purification and properties of the thermostable acid protease of Penicillium duponti.

An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme.     

Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).     

A bacterial high-affinity GABA binding protein: isolation and characterization

A gamma-aminobutyric acid (GABA) binding protein (GBP) was isolated from a bacterial mutant which has high-affinity GABA binding characteristics comparable with the GABA(A) brain receptor in mammals. The GBP was partially purified and characterized and was shown to be a periplasmic protein of approximately 42,000 molecular weight. To determine the molecular weight, a bacterial GABA binding assay was used with SDS-PAGE. This procedure did not require large amounts or complete purification of protein and may be useful as a simple method in estimating the molecular weight of other bacterial binding proteins.     

Superior Appetite Hormone Profile After Equivalent Weight Loss by Gastric Bypass Compared to Gastric Banding

The goal of this study was to understand the mechanisms of greater weight loss by gastric bypass (GBP) compared to gastric banding (GB) surgery. Obese weight‐ and age‐matched subjects were studied before (T0), after a 12 kg weight loss (T1) by GBP (n = 11) or GB (n = 9), and at 1 year after surgery (T2). peptide YY_3–36 (PYY_3–36), ghrelin, glucagon‐like peptide‐1 (GLP‐1), leptin, and amylin were measured after an oral glucose challenge. At T1, glucose‐stimulated GLP‐1 and PYY levels increased significantly after GBP but not GB. Ghrelin levels did not change significantly after either surgery. In spite of equivalent weight loss, leptin and amylin decreased after GBP, but not after GB. At T2, weight loss was greater after GBP than GB (P = 0.003). GLP‐1, PYY, and amylin levels did not significantly change from T1 to T2; leptin levels continued to decrease after GBP, but not after GB at T2. Surprisingly, ghrelin area under the curve (AUC) increased 1 year after GBP (P = 0.03). These data show that, at equivalent weight loss, favorable GLP‐1 and PYY changes occur after GBP, but not GB, and could explain the difference in weight loss at 1 year. Mechanisms other than weight loss may explain changes of leptin and amylin after GBP.     

Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

Binding-protein-dependent glucose transport by Agrobacterium radiobacter grown in glucose-limited continuous culture

Agrobacterium radiobacter NCIB 11883 was grown in glucose-limited continuous culture at low dilution rate. Whole cells transported glucose using an energy-dependent mechanism which exhibited an accumulation ratio greater than 2000. Three major periplasmic proteins were purified and their potential role as glucose-binding proteins (GBP) were investigated using equilibrium dialysis. Two of these, GBP1 (Mr 36,500) and GBP2 (Mr 33,500), bound D-glucose with high affinity (KD 0.23 and 0.07 microM respectively), whereas the third protein (Mr 30,500) showed no binding ability. Competition experiments using various analogues showed that those which differed from glucose at C-6 (e.g. 6-chloro-6-deoxy-D-glucose and 6-deoxy-D-glucose) variably decreased the binding of glucose to both GBP1 and GBP2, whereas those which differed at C-4 (e.g. D-galactose) were only effective with GBP1. The rate of glucose uptake and the concentration of the glucose-binding proteins increased in parallel during prolonged growth under glucose-limitation due to the emergence of new strains in which GBP1 (e.g. strain AR18) or GBP2 (e.g. strain AR9), but not both, was hyperproduced and accounted for at least 27% of the total cell protein. It is concluded that A. radiobacter synthesizes two distinct periplasmic binding proteins which are involved in glucose transport, and that these proteins are maximally derepressed during growth under glucose limitation.     

Human lymphocyte membrane proteins treated with neuraminidase

Human peripheral blood lymphocytes were surface-iodinated, treated with neuraminidase from Vibrio cholerae and lysed with non-ionic detergent. In addition, surface membrane fractions were isolated from surface-iodinated cells in the absence of detergents and treated with neuraminidase after membrane isolation. The effect of neuraminidase treatment on the membrane proteins was studied by two-dimensional gel electrophoresis. One surface-labelled protein of 45 000 molecular weight which is characterized by its association with the detergent-resistant matrix of the cells and by its specific enrichment in an isolated membrane fraction, was found to be particularly sensitive to neuraminidase treatment both of intact cells and isolated membranes. A prominent labelled protein of apparent molecular weight of 60 000 is observed in the soluble fraction after neuraminidase treatment of intact cells. The analogous protein is detected when isolated membrane fractions are treated with neuraminidase.     

Purification and properties of extracellular carboxyl proteases of acid-tolerant bacteria, isolated from compost

Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55 degrees C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.