Radiation inactivation studies of the renal brush-border membrane phlorizin-binding protein

Renal brush-border membrane vesicles were irradiated in the frozen state with a high energy electron beam. The integral membrane proteins, alkaline phosphatase and 5'-nucleotidase, each showed a single exponential loss of activity with radiation dose, indicating target sizes of 67,000 and 58,000 daltons, respectively. Inactivation of sodium-dependent phlorizin binding to the brush-border membrane D-glucose transporter was more complex. One-half of the phlorizin binding sites were lost after even the smallest doses of radiation suggestive of large functional units (greater than 4 X 10(6) daltons) for a subpopulation of phlorizin binding proteins. The remaining sites behaved as a single radiation target of 110,000 +/- 8,000 daltons and retained the kinetic characteristics commonly associated with phlorizin binding to the glucose transporter. Thus, the data are consistent with the assignment of a molecular weight of 110,000 to the phlorizin binding moiety of the brush-border membrane D-glucose transport protein.     

Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP ¹H­¹⁵N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Responses of the ant Lasius niger to various compounds perceived as sweet in humans: a structure-activity relationship study

A behavioural study on the ant Lasius niger was performed by observing its feeding responses to 85 compounds presented in a two-choice situation (tested compound versus water control or sucrose solution). Among these compounds, only 21 were phagostimulating: six monosaccharides (D-glucose, 6-deoxy-D-glucose, L-galactose, L-fucose, D-fructose, L-sorbose), four derivatives of D-glucose (methyl alpha-D-glucoside, D-gluconolactone and 6-chloro- and 6-fluoro-deoxy-D-glucose), five disaccharides (sucrose, maltose, palatinose, turanose and isomaltose), one polyol glycoside (maltitol), three trisaccharides (melezitose, raffinose and maltotriose) and two polyols (sorbitol and L-iditol). None of the 16 non-carbohydrate non-polyol compounds tested, although perceived as sweet in humans, was found to be active in ants. The molar order of effectiveness of the major naturally occuring compounds (melezitose > sucrose = raffinose > D-glucose > D-fructose = maltose = sorbitol) is basically different from the molar order of their sweetness potency in humans (sucrose > D-fructose > melezitose > maltose > D-glucose = raffinose = sorbitol). On a molar basis melezitose is in L. niger about twice as effective as sucrose or raffinose, while D-glucose and D-fructose are three and four times less effective, respectively, than sucrose or raffinose. From a structure-activity relationship study it was inferred that the active monosaccharides and polyols should interact with the ant receptor through only one type of receptor, through the same binding pocket and the same binding residues, via a six-point interaction. The high effectiveness of melezitose in L. niger mirrors the feeding habits of these ants, which attend homopterans and are heavy feeders on their honeydew, which is very rich in this carbohydrate.     

The human erythrocyte sugar transporter presents two sugar import sites

The human erythrocyte sugar transporter presents sugar import (e2) and sugar export (e1) sites simultaneously. This study asks whether the sugar transporter exposes only one or multiple import sites. We approached this question by analysis of cytochalasin B binding to the human erythrocyte sugar export site in the presence of sugars that bind to the sugar import site. Extracellular maltose does not enter human erythrocytes. High concentrations of maltose (1-100 mM) inhibit cytochalasin B binding to human red cells. Low concentrations (25-500 microM) increase the level of erythrocyte cytochalasin B binding. Maltose modulation of cytochalasin B binding is mediated by altered affinity of sugar export sites for cytochalasin B. Similar results are obtained with other cell-impermeant inhibitors of sugar uptake. Extracellular D-glucose (a transported sugar) stimulates cytochalasin B binding at low D-glucose concentrations (10-250 microM), but this effect is lost at higher concentrations. Intracellular D-glucose inhibits cytochalasin B binding. Low concentrations of extracellular maltose and other nontransported inhibitors stimulate 3-O-methylglucose uptake in erythrocytes. Higher sugar concentrations (1-100 mM) inhibit transport. These data support the hypothesis that the erythrocyte sugar transporter presents two sugar import sites and at least one sugar export site. This conclusion is consistent with the proposed oligomeric structure of the sugar transporter, a complex of four GluT1 proteins in which each subunit presents a translocation pathway.     

Synthetic reaction of Cellvibrio gilvus cellobiose phosphorylase.

The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with beta-D-glucose than with the alpha-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. beta-D-Cellobiose was also phosphorolyzed faster than the alpha-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with alpha-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.     

Gentamicin inhibits Na+-dependent D-glucose transport in rabbit kidney brush-border membrane vesicles

We studied the effect of gentamicin on Na+-dependent D-glucose transport into brush-border membrane vesicles isolated from rabbit kidney outer cortex (early proximal tubule) and outer medulla (late proximal tubule) in vitro. We found the same osmotically active space and nonspecific binding between control and gentamicin-treated brush-border membrane vesicles. There was no difference in the passive permeability properties between control and gentamicin-treated brush-border membrane vesicles. Kinetic analyses of D-glucose transport into 1 mM gentamicin-treated brush-border membrane vesicles demonstrated that gentamicin decreased Vmax in the outer cortical preparation, while it did not affect Vmax in the outer medullary preparation. With regard to Km, there was no effect of gentamicin in any vesicle preparation. When brush-border membrane vesicles were incubated with higher concentrations of gentamicin, Na+-dependent D-glucose transport was inhibited dose-dependently in both outer cortical and outer medullary preparations. Dixon plots yield inhibition constant Ki = 4 mM in the outer cortical preparation and Ki = 7 mM in the outer medullary preparation. These results indicate that the Na+-dependent D-glucose transport system in early proximal tubule is more vulnerable to gentamicin toxicity than that in late proximal tubule.     

Photoaffinity labeling of the K562 cell membrane D-glucose transporter with cytochalasin B

D-glucose carrier protein in K562 cell membrane was studied by photoaffinity labeling with cytochalasin B. The saturable cytochalasin B binding in purified K562 cell membranes was 90 pmol/mg and 200 pmol/mg protein in the presence of D-glucose and D-sorbitol, respectively. More than half of the total cytochalasin B binding could be depressed by D-glucose. The results of SDS-PAGE analysis of K562 cell membranes after photoaffinity labeling at 0.1 microM cytochalasin B showed that the main peak of covalently bound [3H]-cytochalasin B was in the Mr range of 46-65 KDa. The label found in the peak was reduced by more than 50% in the presence of 0.5 M D-glucose, the inhibition similar being to that obtained in the binding experiment. This polypeptide has a slightly higher molecular weight than that of the human erythrocyte cell membrane.     

Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein

D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.     

A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.     

Stereospecific D-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific D-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of D-glucose proceeds without chemical alteration. The rate of stereospecific uptake of D-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific D-glucose uptake appears linear for 0.3 min, reaches a maximum at 2--5 min, and declines to zero by 5 h as L-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B (Ki = 0.13 microM) and the structural analogues of D-glucose : D-mannose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-galactose and maltose, but not by sucrose of L-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11 degrees C to 18.1 kcal/mol above 11 degrees C. Stereospecific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific D-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.     

Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism.     

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.     

Enhancement of glucose transport in small intestinal brush border membrane of retinol administered rat

Retinylpalmitate (200 IU/kg body weight) was administered intraperitoneally to rats once daily for 4 days. Brush border membrane vesicles (BBMVs) were prepared from small intestinal epithelium cells from along the crypt-villus axis. D-glucose uptake by BBMVs was examined under the inwardly directed Na+ gradient. The D-glucose uptake by BBMVs from the villus-tip and mid-villus cells of retinylpalmitate treated rats was significantly larger than that of control (corn oil treated) rats, respectively. Thus, retinol treatment of rats promoted the D-glucose transport in small intestinal brush border membrane. Interestingly, the enhancement of D-glucose transport was more prominent in villus-tip and mid-villus than in lower villus.     

Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach

Background

In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.

Results

To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.

Conclusion

Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.     

Partial purification and reconstitution of the Na+-D-glucose cotransport protein from pig renal proximal tubules

Brush border membranes from renal proximal tubules were solubilized with deoxycholate, and the proteins were incorporated into liposomes formed from cholesterol and phosphatidylserine by a freeze-thaw procedure. In the proteoliposomes Na+-D-glucose cotransport was demonstrated by showing that the D-glucose concentration in the liposomes increased far above the equilibrium value if a Na+ gradient was applied. The initial D-glucose uptake rate, stimulated by an inside directed gradient of 89 mM Na+, was 4 pmol/mg of protein-1 s-1. High affinity phlorizin binding could not be measured. After two precipitation steps with the solubilized membrane proteins, a protein fraction was obtained in which significantly high affinity phlorizin binding was detected. After reconstitution, proteoliposomes were formed in which more than 70% of the protein was represented by two polypeptides with molecular weights of 94,000 and 52,000. An initial Na+ gradient-dependent D-glucose uptake rate of 118 pmol/mg of protein-1 s-1 was obtained. In these liposomes, the D-glucose uptake rate could be inhibited by phlorizin (Ki = 0.3 microM), and 55-pmol phlorizin-binding sites per mg of protein (KD = 0.5 microM) were measured. In different liposomal preparations a correlation between Na+ gradient-dependent D-glucose uptake rate and the amount of 52,000 molecular weight polypeptide was observed.     

Increased Na(+)-dependent D-glucose transport and altered lipid composition in renal cortical brush-border membrane vesicles from bile duct-ligated rats.

Erythrocyte membranes of patients with liver disease are characteristically enriched in cholesterol, a change known to impair several carrier-mediated membrane transport functions. In the present study we have assessed whether experimental liver disease can affect the membrane lipid composition and transport function of kidney epithelial cells. Small (about 5%) but significant (P less than 0.01) increases were found in the cholesterol-to-phospholipid molar ratio (C/PL) of rat renal cortical brush-border membrane (BBM) vesicles 3, 8, and 15 days after bile duct ligation which correlated closely with increased fluorescence polarization, i.e., decreased membrane fluidity (r = 0.75, P less than 0.001; n = 27). A lipoprotein-mediated pathogenesis was suggested by the close relationship between BBM C/PL and plasma C/PL (r = 0.69, P less than 0.001). The mean high-affinity Na(+)-coupled D-glucose uptake by BBM vesicles was higher 1, 3, 8, and 15 days after ligation than in non-operated rats, significantly so at 3 and 8 days (611 +/- 37 and 593 +/- 22 vs. 507 +/- 21 pmol/mg protein per 4 sec; P less than 0.05), and was positively correlated with BBM C/PL (r = 0.58, P less than 0.01) and fluorescence polarization (r = 0.41, P less than 0.05). Brief incubation of BBM vesicles from normal rats with cholesterol-rich phospholipid liposomes simultaneously increased BBM C/PL and Na(+)-dependent D-glucose uptake. Stimulation of BBM Na(+)-glucose cotransport in ligated rats was not due to delayed dissipation of the Na+ gradient or to a more rapid development of membrane potential. High-affinity Na(+)-dependent D-glucose uptake kinetics in 3-day bile duct-ligated rats showed a lower Kt, without an alteration in maximum velocity, Vmax, compared to sham-operated animals (0.298 +/- 0.015 vs. 0.382 +/- 0.029 mM; P less than 0.05), whilst the binding dissociation constant, Kd of high-affinity phlorizin binding sites was reduced by ligation (0.453 +/- 0.013 vs. 0.560 +/- 0.015 microM; P less than 0.001). We conclude that an early effect of bile duct ligation is to enrich renal cortical brush-border membranes in cholesterol, thereby decreasing membrane fluidity and stimulating Na(+)-dependent D-glucose uptake by increasing the affinity of the carrier.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

Insulin regulates Na+/glucose cotransporter activity in rat small intestine

In order to examine the involvement of insulin in the activity of Na+/glucose cotransporter in rat small intestine, we compared Na(+)-dependent uptake of D-glucose by brush-border membrane vesicles prepared from control, streptozotocin-induced diabetic, insulin-treated diabetic and starved diabetic rats. In four groups, the uptake of D-glucose showed a transient overshoot in the presence of Na+ gradient between medium and vesicles (medium greater than vesicles). The overshoot magnitude was increased (1.8-fold of controls) in diabetic brush border membrane vesicles and recovered to the control level by the treatment of diabetic rats with insulin. In contrast, increased uptake of D-glucose in diabetic rats was not recovered by the starvation of diabetic rats although the blood glucose level was the same as that of controls. Furthermore, we attempted to examine phlorizin binding activities among four groups. Scatchard analysis indicated that phlorizin binding to diabetic brush border membrane vesicles was increased (1.6-fold of controls) without a change of the affinity for phlorizin as compared with controls. Increased binding of phlorizin to diabetic brush border membrane vesicles was also recovered to the control level by the treatment of diabetic rats with insulin, but not by starvation. These results suggested that the increased activity of Na+/glucose cotransporter in diabetic rats was due to the increase of the number of cotransporter and that intestinal cotransporter was physiologically controlled by insulin, but not by blood glucose levels.