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Biomedical subjects

S Roseman

Publications and source records attributed to S Roseman.

At least 91 records · Page 5Linked to original sources
PubMed1983

Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. I. Evidence for three sugar-specific cell surface receptors.

Dictyostelium discoideum cells appear to be able to recognize particular carbohydrate prosthetic groups at different stages in their life cycle. We therefore used our previously developed model system (consisting of polyacrylamide gels containing putative ligands covalently linked to the polymer) to determine the receptors on these cells capable of recognizing carbohydrates. D. discoideum cells, at different developmental stages from growth phase to late aggregation, were incubated with the derivatized gels, and the number of adherent cells was determined by measuring alanine transaminase after cell lysis. From 70 to 100% of the cells firmly adhered to gels derivatized with glucose, maltose, or cellobiose. The cells were also capable of binding to N-acetylglucosamine and mannose, but both the rate and the extent of binding to these sugars were less than those observed with the glucose derivatives. Furthermore, binding to N-acetylglucosamine decreased to negligible levels during the aggregation stage of development. The cells did not bind to the glucose-derivatized gels in the presence of glucose and a variety of carbohydrates containing glucose at the nonreducing termini, whereas binding was not inhibited by N-acetylglucosamine, mannose, and derivatives of these sugars. Adhesion to all sugars was blocked by 2,4-dinitrophenol. This inhibitor also reversed the binding to gels containing N-acetylglucosamine and mannose, but not to glucose. Differential binding to the three monosaccharides was also observed under conditions affecting the normal amoeboid shape of the cells. In addition, adhesion to N-acetylglucosamine and mannose was trypsin-sensitive, whereas adhesion to glucose was only slightly affected by treating the cells with trypsin (and cycloheximide). These and other results suggest that D. discoideum cell adhesion to derivatized gels is mediated by three different receptors, one highly specific for glucose and two (probably less specific) for N-acetylglucosamine and mannose.

2,4-Dinitrophenol↗
PubMed1983

Sugar transport by the bacterial phosphotransferase system. Preparation of a fluorescein derivative of the glucose-specific phosphocarrier protein IIIGlc and its binding to the phosphocarrier protein HPr.

In diauxic growth, the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) regulates the utilization of certain compounds which are not PTS substrates. It has recently been shown that this PTS regulation is mediated via one of the PTS phosphocarrier proteins, IIIGlc. In the present studies, IIIGlc was derivatized with the fluorescent reagent fluorescein-5-isothiocyanate. One mol of label was incorporated per mol of protein and the label was located at the NH2-terminal amine, as shown by tryptic peptide mapping and one-step Edman-type degradation. The fluorescent moiety was found to be stable and resistant to photodecomposition. The fluorescent IIIGlc was purified and shown to be fully active in its ability to accept phosphate from phospho-HPr (the histidine-containing phosphocarrier protein of the phosphotransferase system), but only 20% active in catalyzing the transfer of the phosphate to methyl alpha-glucoside via the membrane-bound II-BGlc protein. The decay of the fluorescence intensity was dominated by a single component (90%) with a lifetime of 4 ns. The decay of the fluorescence emission anisotropy was determined for excitation in both a negative and positive transition of fluorescein and was best described in terms of a biexponential function, indicating internal motion of the fluorophore and possible anisotropic rotation of the protein as a whole. The formation of a complex between IIIGlc and HPr was demonstrated by using the techniques of time-resolved and steady state fluorescence emission measurements, resonance energy transfer, and equilibrium gel filtration.

Amino Acid Sequence↗
PubMed1983

Kinetic characterization and regulation of phosphoenolpyruvate-dependent methyl alpha-D-glucopyranoside transport by Salmonella typhimurium membrane vesicles.

Membrane vesicles from Salmonella typhimurium SB3507 were used to study the kinetics of methyl alpha-D-glucopyranoside (MeGlc) transport by the phosphoenolpyruvate: glycose phosphotransferase system (PTS). During the first minute of phosphoenolpyruvate-dependent MeGlc transport, two distinct rates were observed; an initial rapid rate, V1 (Vmax, 7.4-8.4 nmol X mg-1 X min-1; Km, 8.2-11.2 X 10(-6)M), followed by a second slower rate, V2 (Vmax, 4-4.6 nmol X mg-1 X min-1; Km, 3.4-6.4 X 10(-6) M). The change in rate occurred when the intravesicular MeGlc phosphate concentration was 0.2 mM or less, depending on the external MeGlc concentration. The rate-limiting component in MeGlc transport was found to be enzyme II-BGlc, not phosphoenolpyruvate uptake or the PTS proteins enzyme I, HPr, and IIIGlc. The change from V1 to V2 thus suggests that the PTS is regulated in intact vesicles. However, this regulation was completely relieved by permeabilizing the vesicles with toluene. That is, the toluene-treated vesicles showed only V1 for MeGlc phosphorylation. Evidence was obtained to show that pyruvate and its metabolic products generated by the vesicles exerted no effect on the rate of MeGlc transport. Furthermore, the result from a dual-label experiment excluded exchange transphosphorylation as the mechanism for regulating MeGlc transport by the vesicles. Possible mechanisms for regulation of the PTS are discussed.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. The glucose receptors of the Salmonella typhimurium phosphotransferase system.

We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and divalent cation, and each is present in membranes isolated from wild type cells. For reasons described in this report, one of the complexes is designated IIGlc and the other IIMan. The IIMan complex has previously been separated into its protein components, II-A and II-B (Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1407-1418), while the accompanying reports describe dissociation of the IIGlc complex into its components, IIIGlc and II-BGlc. Curtis and Epstein (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199) first showed that there are two phosphotransferase systems in whole cells responsible for glucose uptake and obtained the respective mutants, now designated ptsG and ptsM. The present studies provide kinetic conditions for assaying each activity separately (in vivo and in vitro), when both are present in the same membrane preparation. The IIGlc system is responsible for the uptake and phosphorylation of glucose and methyl alpha-glucoside, whereas the IIMan system is less specific and utilizes glucose, mannose, and 2-deoxyglucose. With high sugar concentrations in vitro, IIMan is also capable of phosphorylating methyl alpha-glucoside, fructose, and N-acetylmannosamine, while IIGlc phosphorylates fructose and mannose. The in vivo transport results were qualitatively consistent with the in vitro phosphorylation results, and several of the kinetic parameters also showed good quantitative agreement. The levels of the two activities depended on the growth conditions. In addition, transport studies showed that initial uptake rates of methyl alpha-glucoside and steady state levels of this analogue depended on the energy state of the cells and that these two parameters did not necessarily change in the same direction when metabolic inhibitors were used. A series of E. coli and S. typhimurium mutants were characterized both with respect to their ability to transport the glucose analogues and to phosphorylate them in vitro. The original mutants of Curtis and Epstein, ptsG and ptsM, were found to be defective in II-BGlc and the IIMan complex, respectively.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Studies on the molecular weight and association of enzyme I.

Studies were conducted on the physical properties of Enzyme I, the first protein in the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Since values lower than those previously reported for the monomer molecular weight were obtained, experiments were performed to determine whether Enzyme I had been partially degraded during isolation of homogeneous protein. Crude extracts and partially purified and homogeneous protein preparations exhibited identical behavior in crossed immunoelectrophoresis analyses, indicating that the isolated protein represented native, intact Enzyme I. The monomeric subunit of Enzyme I is globular, with a frictional ratio of about 1. Sedimentation equilibrium experiments provided a monomer molecular weight of 57,700 +/- 3,400, and gel filtration studies under denaturing conditions gave a comparable value of 57,000. The values previously obtained from polyacrylamide gel electrophoresis analyses in the presence of sodium dodecyl sulfate varied with the conditions used, but under one set of conditions agreed with those given above. The sedimentation equilibrium studies were conducted at 8 degrees C, in the absence of substrates and cofactor (phosphoenolpyruvate, pyruvate, Mg2+). Under these conditions Enzyme I self-associates, but the association is weak, favoring primarily monomer. Because of solubility limitations, the sedimentation experiments were performed with Enzyme I at an initial concentration of 0.5 mg/ml, providing a concentration distribution of 0.1 to 2 mg/ml. Computer analysis of the results showed that within this concentration range it was not possible to distinguish between two modes of self-association, monomer-dimer and isodesmic. The physiological significance of the results is discussed.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by enzyme I of Salmonella typhimurium.

The phosphorylation of Enzyme I is the first step in the phosphotransfer reaction sequence catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) from Salmonella typhimurium. The characterization of phospho approximately Enzyme I and the reactions in which it participates are described in this report. About 1 mol of phosphoryl group was incorporated per mol of Enzyme I monomer when the homogeneous enzyme was incubated with [32]phosphoenolpyruvate and Mg2+. The phosphoryl group in phospho approximately Enzyme I is linked at the N-3 position in the imidazole ring of a histidine residue. Phospho approximately Enzyme I donates its phosphoryl group to pyruvate (to form phosphoenolpyruvate (P-enolpyruvate)) and to the histidine-containing phosphocarrier protein of the phosphotransferase system (HPr) (to form phospho approximately HPr). In the presence of HPr and appropriate sugar-specific proteins, the phosphoryl group can be transferred from Enzyme I to methyl alpha-glucoside (to form sugar-phosphate). The phosphorylation of Enzyme I by phosphoenolpyruvate requires divalent cation, but the phosphoryl group is transferred from phospho approximately Enzyme I to HPr in the presence of 20 mM EDTA. Kinetic studies show a biphasic rate for Enzyme I phosphorylation, suggesting that the enzyme is phosphorylated in the associated state. Equilibrium experiments were conducted on the following Reactions A and C. (formula: see text). The apparent K' for Reaction B was calculated from K'A and K'C. K'C was found to be about 11. K'A was studied both at very low and high substrate (P-enolpyruvate and pyruvate) concentrations relative to their respective Km values. At low substrate concentrations, the reaction appeared independent of pH in the range of 6.5 to 8.0, and when analyzed according to the simplest expression that could be written for total species of each component (Reaction A), the apparent average K' was 1.5. At high substrate concentrations, about 50% of the Enzyme I was phosphorylated, and this value changed only slightly with large changes in the P-enolpyruvate to pyruvate ratio. Expressions for K'A are derived which partially explain these results by including enzyme-substrate complexes in the equilibrium expression. The K' values were used to derive apparent standard free energy changes for the hydrolysis of the phosphoproteins of the PTS. Since these are similar to those for the hydrolysis of P-enolpyruvate, the phosphate transfer potentials of the PTS phosphoproteins are among the highest of known biological phosphate derivatives. In addition, unlike the reactions which occur during anaerobic glycolysis and electron transport, the high phosphate transfer potential is conserved in the PTS reaction sequence until the last step, the translocation of the sugar substrate across the membrane concomitant with its phosphorylation. Potential regulation of the PTS, in particular the effect of the intracellular ratio of P-enolpyruvate to pyruvate, is considered.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a phosphocarrier protein HPr from wild type and mutants of Salmonella typhimurium.

HPr, the histidine-containing phosphocarrier protein of the phosphotransferase system, has been isolated and purified from wild type Salmonella typhimurium and from two mutants of this organism. Comparison of the sequences of these three forms of HPr indicates that the two mutants contain substitutions at residue 4 in the polypeptide chain; in place of glutamine in the wild type, one mutant (SB3899) contained serine and the other (SB3093) contained lysine. The substitution of lysine for glutamine resulted in increased positive charge of the molecule which is reflected in an expected decreased mobility on polyacrylamide gel electrophoresis and its behavior on isoelectric focusing. However, these changes had no effect on phosphocarrier activity in the phoshoenolpyruvate:glycose phosphotransferase system as measured by the kinetics of the interaction on the mutant HPr proteins with both Enzyme I and Enzyme II. These results have important implications for potential chemical modification of HPr. The HPr from wild type cells was crystallized.

Amino Acid Sequence↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Primary structure and active site of a general phosphocarrier protein (HPr) from Salmonella typhimurium.

The general histidine-containing phosphocarrier protein (HPr) of the Salmonella phosphotransferase system is required for the phosphorylation of all sugar substrates by this system. The complete amino acid sequence of HPr, consisting of 84 amino acid residues, has been established. The sequence was determined by cleaving the protein with cyanogen bromide, trypsin, and with a protease from Staphylococcus aureus, followed by isolation and amino acid sequence determination of the resulting peptides. The Salmonella typhimurium protein contains two histidine residues, at positions 15 and 75, respectively. The phosphoryl group in phospho-HPr was linked to the His-15 residue. Based on several lines of evidence, the HPr protein from Escherichia coli appears to be identical with the protein from S. typhimurium. The HPr protein from S. aureus has also been isolated in this laboratory and was shown to differ from the HPr proteins described above both with respect to amino acid composition and the inability of the S. aureus and E. coli HPR proteins to substitute for each other in the in vitro sugar phosphorylation assays. The complete amino acid sequence of S. aureus HPr has been reported (Beyreuther, K., Raufuss, H., Schrecker, O., and Hengstenberg, W. (1977) Eur. J. Biochem. 75, 275-286), and its secondary structure has been predicted; this protein contains 70 amino acid residues and only one histidine. In the present studies, three methods were used to predict the secondary structure of S. typhimurium HPr, the results were combined, and a secondary structure for the protein is proposed. Although the amino acid compositions and sequences of the S. typhimurium and S. aureus HPr proteins are quite different, 13 residues are identical in the sequence of the two proteins, and most of these are located near the active site histidine residue. In addition, the predicted secondary structures of the two proteins are quite similar; the additional 14 residues in S. typhimurium, located at the carboxyl terminal end, are predicted to form an alpha-helix.

Amino Acid Sequence↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Nanosecond fluorescence studies of the phosphocarrier protein (HPr) labeled at the NH2-terminal methionine.

HPr is a low molecular weight, phosphocarrier protein of the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system (PTS). This protein was alkylated with the fluorescent reagent (N-iodoacetylaminoethyl)-5-naphthylamino-1-sulfonate under conditions which favor alkylation of the thioether linkage in methionine residues (Link, T. P. and Stark, G. R. (1968) J. Biol. Chem. 243, 1082-1088) to give the corresponding sulfonium derivatives. The isolated fluorescent protein (95-100% pure) was as active as native HPr both as a phosphoryl acceptor protein (phosphoenolpyruvate and Enzyme I of the PTS), and as a phosphocarrier protein in the phosphorylation of methyl alpha-glucoside by the complete PTS. The fluorescent label was shown to be predominantly, possibly exclusively, at the NH2-terminal methionine residue. The decay of the fluorescence intensity could be described in terms of a biexponential function with the time constants tau 1 approximately 7 ns, tau 2 approximately 15 ns, and a ratio of alpha 2/alpha 1 approximately 3 for the pre-exponential factors. The decay of the fluorescence emission anisotropy was found to be consistent with some internal motion of the probe, in addition to the rotation of the protein conjugate as a whole.

Bacterial Proteins↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a glucose-specific phosphocarrier protein (IIIGlc) from Salmonella typhimurium.

The phosphocarrier protein, IIIGlc, of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was purified to homogeneity by two methods. The first method utilized ion exchange and gel filtration chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis, and required several weeks for completion. The second method utilized and antibody affinity column plus two additional steps and could be completed in a few days. By both procedures, two forms of IIIGlc were isolated, which were called IIIGlc Slow and IIIGlc Fast on the basis of their relative mobilities in polyacrylamide gels. IIIGlc Fast is derived from IIIGlc Slow by cleavage of the seven NH2-terminal amino acids from the latter protein. Both IIIGlc Slow and IIIGlc Fast have Mr approximately 20,000; neither protein contains cysteine, tyrosine, or tryptophan. IIIGlc Slow is very stable to heat; only 50% of its sugar phosphorylating activity is lost after 1 h at 100 degrees C. The phosphoryl group in IIIGlc Slow appears to be linked to a histidinyl residue. Direct transfer of the phosphoryl group from HPr (the histidine-containing phosphocarrier protein of the PTS) to IIIGlc slow was demonstrated as well as the reverse reaction. In addition, phospho-IIIGlc Slow served as a phosphoryl donor to methyl alpha-glucoside (or glucose) in the absence of all other PTS components except the partially purified integral membrane protein specific for this sugar, II-BGlc. The loss of the seven amino acids from IIIGlc Slow (giving IIIGlc Fast) leads to a marked alteration in the kinetic properties of the protein in the phosphotransferase system. IIIGlc Slow accepts 1 mol of phosphate from phosphoenolpyruvate via Enzyme I and HPr (the histidine-containing phosphocarrier protein) and participates in the phosphorylation of glucose or methyl alpha-D-glucoside. IIIGlc Fast also accepts 1 mol of phosphate, but phospho-IIIGlc Fast is only 2-3% as active as phospho-IIIGlc Slow in the phosphorylation of sugar. IIIGlc Fast is found only in trace quantities in living cells, and may play a role in the regulation of non-PTS sugar transport systems.

Amino Acid Sequence↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Preparation and characterization of membrane vesicles from mutant and wild type Salmonella typhimurium.

Modifications of published procedures (reviewed by Kaback, H. R. (1974) Science (Wash. D. C.) 186, 882-892) were developed for preparing membrane vesicles from Salmonella typhimurium. The preparations consisted largely of closed, unilamellar structures and contained inner membrane with little to no contamination by outer membrane or cell wall. A variety of cytoplasmic proteins was assayed in the membrane preparations, and they were found to be present at low to trace levels, whereas other proteins known to be associated with membranes were found at high levels (with respect to specific activities) in the vesicle preparations. At least 90% of the vesicles appeared to be oriented right-side-out; we do not know whether the remaining 10% represents closed vesicles oriented inside-out or "leaky" right-side-out vesicles. The vesicle preparations were impermeable to both low and high molecular weight solutes, for example, to both intra- and extravesicular sucrose. In double label experiments, the vesicle volumes were found to be about 6 microliters/mg of protein for preparations isolated from the wild type strain, and about 4.5 microliters/mg of protein for vesicles isolated from a mutant, SB2950, deleted in ptsH, ptsI, and crr genes (proteins HPr, Enzyme I, and IIIGlc, respectively). One advantage of S. typhimurium over Escherichia coli for these studies is that the former can be induced to take up phosphoenolpyruvate. This may be the reason that S. typhimurium vesicles transported methyl alpha-glucoside at 4- to 100-fold the rates reported for vesicles from E. coli, while uptake rates of proline were comparable in the two types of preparations. Vesicles from strain SB2950 were unable to take up methyl alpha-glucoside, but the transport (and phosphorylating) system was reconstituted in the vesicles by trapping the soluble purified proteins inside the vesicles during preparation of the latter. All three proteins were required for reconstruction. Studies with intra- and extravesicular soluble proteins of the phosphoenolpyruvate:glucose phosphotransferase system showed that the IIMan complex, which phosphorylates glucose, 2-deoxyglucose, and other sugars, is symmetrically oriented in the membranes. That is, this complex could phosphorylate 2-deoxyglucose when supplemented with Enzyme I and HPr either inside or outside of the membranes, and the sugar phosphate was found on the same side of the membranes as the soluble phosphotransferase system proteins. The integral membrane protein, II-BGlc, which phosphorylates glucose and methyl alpha-glucoside, showed contrasting behavior. Methyl alpha-glucoside phosphate was formed (intravesicularly) only when the soluble proteins (Enzyme I, HPr, and IIIGlc) were located inside the vesicles. Thus, II-BGlc appears to be asymmetrically oriented in the membranes.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose).

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.

Biological Transport↗
PubMed1982

Studies on the intercellular adhesion of rat and chicken hepatocytes. Tissue-specific adhesion in mixtures of hepatocytes and heart myocytes.

We previously reported that chicken and rat hepatocytes isolated from young adult animals displayed adhesive specificity in that they adhered preferentially to the homologous cell type. However, since we had used cells from two widely divergent species, it was not clear whether the cells were capable of distinguishing their own cell type from cells of other tissues of the same animal. The present experiments were aimed at determining whether, given the choice of adhering to cells obtained from another tissue from the same animal, cells still preferentially adhered to their own cell type, i.e. whether they showed tissue-specific adhesion. An improved collagenase perfusion procedure was developed for preparing single, viable heart myocytes. Cell adhesion experiments were then performed with hepatocytes and myocytes obtained from a single rat or chicken. Marked tissue-specific adhesion was observed under all conditions tested, which included varying the ratio of each cell type (hepatocytes or myocytes), stationary or gyratory conditions, the presence or absence of serum and certain metal ions, etc. The demonstration of tissue-specific adhesion among hepatocytes and myocytes isolated from the same animal is consistent with the idea that the two cell types contain different cell surface components required for cell-cell recognition. Furthermore, that the hepatocytes (and myocytes) can show tissue-specific adhesion validates the use of cells from adult animals for biochemical studies on intercellular adhesion.

Animals↗