Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltranserase (sucrose: 1,6-α-^D-glucan 3-α- and 6-α- glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1–8.4). The molecular weight was estimated to be 151 000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had K_m values of 7.1 μM for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6- α-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

Structural studies of extracellular glucans of Streptococcus mutans by proton magnetic resonance

Proton magnetic resonance spectra at 100 MHz were obtained for water-soluble and water-insoluble glucans from 11 strains of Streptococcus mutans. The percentages of α-D-(1→6) and non-α-D-(1→6)-, namely, α-D-(1→3)-, linkages were calculated from the anomeric-proton resonances in the 4.7-4.8 and 5.0-5.1 p.p.m. range, respectively. The average content of α-D(1→6) linkages in the polymer fractions precipitating from solution during synthesis of the glucans was generally much lower than that of fractions remaining in solution. The frequent appearance of the α-D-(1→3) resonances as doublets in the spectra suggested neighboring-group effects among the possible α-D-(1→3) and α-D-(1→6) linkage-configurations. These effects were confirmed from 100-MHz spectra of products of a dextranase-degraded, water-insoluble glucan, and a 270-MHz spectrum of an undegraded glucan. It was thus possible to assign the doublet resonances to α-D-(1→3), homogeneous, heterogeneous, and branch configurations, although complete differentiation among proportions of each configuration in the glucan chains could not be achieved.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Biotin transport by a biotin-deficient strain of escherichia coli

Biotin uptake has been investigated using an Escherichia coli biotin requiring auxotroph grown under biotin-deficient conditions. This strain accumulated biotin in the free and bound form. In agreement with a previous report by O. Prakash and M.A. Eisenberg (J. Bacteriol. 120 (1974) 785–791), the biotin entry proved to be an active process which depended on an energy source and was inhibited in the presence of uncouplers. The kinetic parameters have been determined (K_M = 0.05 μM, V_max = 7 pmol/min per mg dry weight). The pool of free biotin could be readily exchanged with external biotin and decreased to a very low level in the absence of an energy source. The use of several biotin analogues revealed that this transport system was quite specific for biotin: slight modifications, for instance in the valeric chain. lowered drastically the affinity for the carrier.     

Folded chromosome structure and DNA-binding protein of Streptomyces hygroscopicus

The isolation of the folded chromosomal structure from a Streptomyces strain is described. It is shown that the compact DNA structure of Streptomyces hygroscopicus is stabilized by DNA-RNA interactions. Nucleolytic cleavage decreases the sedimentation rate of the originally isolated (membrane-free) folded Streptomyces chromosome from 1500 S to 800 S or 300 S and finally to completely unfolded DNA. Data on ethidium bromide intercalation suggest a dye-induced relaxation and reintroduction of DNA supertwisting. Like naturally occurring covalently closed circular DNA and like the Escherichia coli chromosome, the folded chromosomal DNA of S. hygroscopicus has about one negative superhelix turn per 200 bp. The results further demonstrate that the isolated Streptomyces nucleoid contains a characteristic S protein which exhibits gel electrophoretic properties of a histone-like component similar to that found in E. coli or Thermoplasma acidophilum. Digestion of the nucleoid with proteinase K at 37°C causes elimination of the S protein and unfolding of the compact structure. The S protein is also present in other species of the genus Streptomyces.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Sucrose transport by the alkaliphilic, thermophilic Bacillus sp. strain TA2.A1 is dependent on a sodium gradient

An alkaliphilic Bacillus designated strain TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Sodium chloride (>5 mM) was an obligate requirement for the growth of strain TA2.A1 on sucrose, and growth on sucrose was inhibited by monensin, an ionophore that collapses the sodium gradient (ΔpNa⁺) across the cell membrane. Sucrose transport by strain TA2.A1 was sodium dependent and was inhibited by monensin. The K_t for sucrose tran-sport was 33 μM and the Eadie–Hofstee plot was linear, suggesting one high-affinity uptake system for sucrose. The affinity for sodium was low (0.5 mM), and the Hill plot had a slope of 1.6, suggesting that sodium binding was noncooperative and that the sucrose transporter had more than one binding site for sodium. Based on these results, Bacillus strain TA2.A1 uses a sodium gradient for sucrose uptake, in addition to the sodium-dependent glutamate uptake system reported previously. Received: March 15, 2000 / Accepted: July 17, 2000     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase (M_r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of $\text{L}\text{-}\text{alanyl}\text{-p-}\text{nitroanilide}$ exhibited a bell-shaped pH dependence for log V_max/K_m(pK₁ = 6.35; pK₂ = 8.50) while the log V_max versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Evidence of multiple branching in the levan elaborated by Streptococcus salivarius strain 51

Enzymic degradation of the levan elaborated by Streptococcus salivarius strain 51 followed by linkage analysis revealed that the levan possesses multiply-branched chains (B-chains). Large segments of the levan are arborescent.     

Solubilization and reconstitution of the glucose transport system from Saccharomyces cerevisiae

The glucose transport system from Saccharomyces cerevisiae was solubilized from isolated plasma membranes by the nonionic detergent, octylglucoside. The transport system was reconstituted into proteoliposomes with removal of detergent from the extract by dialysis, followed by the addition of asolectin liposomes to the dialyzed proteins with a freeze-thaw and brief bath-sonication step. The reconstituted proteoliposomes exhibit specific carrier-mediated facilitated diffusion of d-glucose, including stimulated equilibrium exchange and influx counterflow. Furthermore, the reconstituted facilitated diffusion system shows substrate specificities similar to those of the intact cell d-glucose transport system.     

Electron transport systems of Candida utilis. Purification and properties of the respiratory chain-linked external NADH dehydrogenase+

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 10⁶. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ₆ and CoQ₁ derivatives as acceptors. Rotenone (10^?5 M) and seconal (10^?3 M) do not inhibit enzymatic activity.     

Molecular analysis of thescrA andscrB genes fromKlebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme IIScr of the phosphotransferase system and a sucrose-6-phosphate invertase

TheKlebsiella pneumoniae genesscrA andscrB are indispensable for sucrose (Scr) utilisation. GenescrA codes for an Enzyme II^Scr (II^Scr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), whilescrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscrAB DNA fragment has been cloned fromK. pneumoniae and expressed inEscherichia coli. Its nucleotide sequence was determined and the coding regions forscrA (1371 bp) andscrB (1401 bp) were identified by genetic complementation, enzyme activity tests and radiolabelling of the gene products. In addition, the nucleotide sequence of thescrB gene from the conjugative plasmid pUR400 isolated fromSalmonella typhimurium was also determined and errors in the previously published sequence of thescrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven II^Scr proteins, a hypothetical model of the secondary structure of II^Scr is proposed.     

Asymmetry of glucose transport in the yeast, Kluyveromyces lactis

Uptake and efflux of 6-deoxy-d-[³H]glucose and of 2-deoxy-d-[¹⁴C]glucose by the yeast Kluyveromyces lactis was studied. The tritiated, nonphosphorylatable hexose analogue leaves the cell in the absence and presence of intracellular 2-deoxy-d-glucose 6-phosphate. In energy-rich cells containing pools of hexose 6-phosphate, 2-deoxy-d-glucose is trapped in the cells, for it neither effluxes into glucose-free medium nor exchanges with external, free sugar. In starved, poisoned cells containing negligible amounts of 2-deoxy-d-glucose 6-phosphate, 2-deoxy-d-glucose does leave the cells upon transfer to glucose-free medium. An involvement of analogue structure and availability of metabolites of energy-rich cells in hexose retention is suggested. An internal pool of 6-deoxy-d-glucose does not affect the rate of uptake of 6-deoxy-d-[³H]glucose, nor does internal 2-deoxy-d-[¹⁴C]glucose 6-phosphate influence that rate. Hence, transport of glucose by this yeast is probably not regulated by internal pools of glucose 6-phosphate.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate