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G R Jacobson

Publications and source records attributed to G R Jacobson.

At least 37 records · Page 2Linked to original sources
PubMed1989

Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer.

The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1989

Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions.

Column chromatography of the Escherichia coli mannitol permease (mannitol-specific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEP-dependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.

Biological Transport↗
PubMed1989

Characterization and sequence analysis of the scrA gene encoding enzyme IIScr of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system.

The Streptococcus mutans GS-5 scrA gene coding for enzyme IIScr of the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS) was localized upstream from the scrB gene coding for sucrose-6-phosphate hydrolase activity after Mu dE transposon mutagenesis of plasmid pMH613. The cloned scrA gene product was identified as a 68-kilodalton protein by minicell analysis after isolation of the gene in plasmid pD4. In addition, the membrane fraction from Escherichia coli cells containing pD4 exhibited sucrose PTS activity upon complementation with enzyme I and HPr from strain GS-5. The nucleotide sequence of the scrA region revealed that this gene was located immediately upstream from the scrB gene and divergently transcribed from the opposite DNA strand. The scrA gene was preceded by potential Shine-Dalgarno and promoterlike sequences and was followed by a transcription terminator-like sequence. The scrA gene coded for an enzyme IIScr protein of 664 amino acid residues with a calculated molecular weight of 69,983. This enzyme IIScr protein was larger than the comparable proteins from Bacillus subtilis and E. coli containing sucrose-metabolizing plasmid pUR400. The 491-amino-acid N-terminal sequence of the S. mutans enzyme IIScr was homologous with the B. subtilis and E. coli sequences, and the 173-amino-acid C-terminal sequence of the S. mutans protein was also homologous with the Salmonella typhimurium enzyme IIIGlc and the 162-amino-acid C terminus of E. coli enzyme IIBgl. These results suggest that the sucrose PTS system of S. mutans is enzyme III independent.

Amino Acid Sequence↗
PubMed1989

Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities.

We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtlA gene, which encodes the mannitol-specific enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtlA were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N- and C-terminal domains.

Binding Sites↗
PubMed1988

Starvation-induced stimulation of sugar uptake in Streptococcus mutans is due to an effect on the activities of preexisting proteins of the phosphotransferase system.

We examined the effects of sugar concentration in the medium on sugar uptake and phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) activities in Streptococcus mutants GS-5. Kinetic analyses of sucrose uptake in cells harvested under conditions of sucrose excess or sucrose limitation showed that increased uptake under the latter condition was almost completely due to an increase in the Vmax of the high-affinity PTS. In a series of experiments in which cells growing under conditions of sucrose or glucose excess were shifted to a medium lacking sugar, starvation resulted in a stimulation of sugar uptake and a parallel increase in PTS activity. These starvation-induced increases in PTS-mediated uptake were not affected by the presence of either chloramphenicol or rifampin during the starvation period, indicating that neither protein nor RNA synthesis was necessary for the stimulation. In vivo labeling experiments with 32Pi revealed that uptake stimulation during starvation was accompanied by a loss of acid-stable phosphate covalently bound to the phosphocarrier protein HPr of the PTS. We conclude, therefore, that stimulation of PTS-mediated uptake of sucrose and glucose during sugar limitation in S. mutans GS-5 is at least partially the result of increased activities of preexisting PTS proteins and that this may be due, at least in part, to dephosphorylation of a previously identified site in S. mutans HPr that can be phosphorylated by an ATP-dependent kinase.

Biological Transport, Active↗
PubMed1987

ATP-dependent protein kinase activities in the oral pathogen Streptococcus mutans.

ATP-dependent protein kinase activities were detected in both membrane and cytoplasmic fractions from the oral pathogen Streptococcus mutans. Different polypeptides were phosphorylated by endogenous kinase(s) in the two fractions. In membranes, five phosphoproteins were detected with apparent masses of 82, 37, 22, 12, and 10 kilodaltons (KD). In cytoplasm, two major acid-stable phosphoproteins were found. One was identified as HPr of the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), while the other had an apparent mass of 61 KD. Both of these proteins were phosphorylated on a seryl residue. Fructose 1,6-bisphosphate stimulated phosphorylation of HPr by the kinase and inhibited phosphorylation of the 61-KD protein. In contrast, fructose 1-phosphate, 2-phosphoglycerate, 3-phosphoglycerate, and dihydroxyacetone phosphate inhibited phosphorylation of HPr and stimulated phosphorylation of the 61-KD protein. Several other glycolytic intermediates as well as inorganic phosphate inhibited phosphorylation of either or both proteins. Preincubation of cytoplasm with PEP prior to incubation with ATP reduced the amount of phospho-(seryl)-HPr formed, but not that of the 61-KD phosphoprotein. The latter protein has not yet been identified but has properties that suggest that it may be the protein kinase itself. These results provide evidence for one or more soluble ATP-dependent protein kinases in S mutans that are regulated by glycolytic intermediates and that may play a role in the modulation of carbohydrate uptake and metabolism in this organism. A model for feedback regulation of sugar transport in S mutans, mediated by an allosterically regulated kinase, is presented.

Bacterial Proteins↗
PubMed1986

Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains.

Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1986

Subunit interactions of the Escherichia coli mannitol permease: correlation with enzymic activities.

A fraction of the phosphorylated form of the Escherichia coli mannitol permease (enzyme IIMtl) of the sugar phosphotransferase system can be extracted from the membrane in a dimeric form [Roossien, F.F., & Robillard, G.T. (1984) Biochemistry 23, 5682-5685]. Using E. coli minicells in which this protein can be specifically labeled with [35S]methionine, we show in this paper that part of the unphosphorylated form of enzyme IIMtl can also be extracted from the membrane as a dimer. We further demonstrate that both phosphoenolpyruvate-dependent phosphorylation of the permease and conditions promoting turnover of the enzyme decrease the amount of extractable dimer. Thus, the dimer of these forms of the enzyme appears to be less stable than that of the unmodified form, at least in detergent solution. In contrast, inorganic phosphate, which activates the permease-catalyzed phospho exchange between mannitol 1-phosphate and mannitol ("transphosphorylation"), stabilizes the dimer. These results support the hypothesis that the mannitol permease dimer is more active in transphosphorylation than the monomer. Treatment of minicell membranes with oxidizing agents produced heat-stable, high molecular weight aggregates of the permease on dodecyl sulfate gels, but no heat-stable dimer could be detected. The nonionic detergent Lubrol PX decreased the amount of dimer extractable at 30 degrees C with a concomitant increase in the monomeric form. These results suggest that the dimer depends predominantly on hydrophobic interactions for its stability and is not covalently cross-linked in that form by oxidizing agents.

Enzyme Activation↗
PubMed1985

31P-NMR studies of the oral pathogen Streptococcus mutans: observation of lipoteichoic acid.

We have used 31P-nuclear magnetic resonance spectroscopy to identify phosphorus-containing compounds in whole cells of two serotype c strains of the oral pathogen Streptococcus mutans. The major resonance, centered at 0 ppm in whole cells, was attributed to lipoteichoic acid on the basis of its chemical shift, insensitivity to pH changes, cellular localization and a comparison with spectra obtained with purified lipoteichoic acid from S. mutans. The linewidths of resonances observed for intact cells and purified lipoteichoic acid were moderately narrowed by increasing the ionic strength, and substantially broadened in the presence of the lectin concanavalin A. Experiments with purified lipoteichoic acid suggest that this compound in whole cells is complexed with divalent cations such as Mg2+. Intracellular pools of other phosphorus-containing metabolites were found to be low when compared to the lipoteichoic acid concentration in both starved and glycolyzing cells.

Cell Membrane↗
PubMed1984

Effects of insulin receptor down-regulation on hexose transport in human erythrocytes.

D-Glucose and D-galactose influx and efflux rates in human erythrocytes were studied using infinite-cis and zero-trans assay methods. It was found that insulin decreased the infinite-cis Km for both D-glucose and D-galactose influx by 44 and 56%, respectively, while the Vmax was unchanged. The Km for D-glucose efflux in the presence of insulin decreased by 47% when compared to controls, and the change in Vmax was statistically insignificant. If insulin receptors were first down regulated, and then influx and efflux assays were performed, decreases in the infinite-cis and zero-trans Km values were also observed in the absence of exogenous insulin. These affinity changes were not due to persistent surface insulin receptor occupation by the insulin which was used to induce down-regulation. These affinity changes were comparable to those observed in non-down-regulated cells in the presence of insulin.

Biological Transport, Active↗
PubMed1984

Resolution of the phosphotransferase enzymes of Streptococcus mutans: purification and preliminary characterization of a heat-stable phosphocarrier protein.

The sucrose phosphotransferase system of Streptococcus mutans catalyzes the phosphorylation of sucrose to sucrose-6-phosphate with concomitant translocation of this disaccharide across the cytoplasmic membrane in reactions requiring intracellular phosphoenolpyruvate. Soluble proteins released by vigorous homogenization of cells with glass beads are shown to be necessary for the phosphoenolpyruvate-dependent phosphorylation of sucrose in combination with one or more proteins that remain tightly associated with the membrane fraction. We have partially purified phosphotransferase enzyme I and have purified a heat-stable phosphocarrier protein (HPr) to apparent homogeneity, by gel filtration and ion-exchange chromatography from the soluble fraction. HPr from S. mutans has an apparent molecular weight larger than that of Escherichia coli HPr but has properties similar to those of Staphylococcus aureus HPr. Furthermore, it appears to be partially complexed with a heat-stable enzyme III-like protein in cell-free fractions from S. mutans, and we also report the purification of this complex. Enzyme I from S. mutans is a protein (native Mr greater than 100,000) that cross-complements enzyme I from S. aureus. Preliminary characterizations of homogeneous HPr and its complex with the putative enzyme III are also presented.

Amino Acids↗
PubMed1984

Identification and properties of distinct sucrose and glucose phosphotransferase enzyme II activities in Streptococcus mutans 6715g.

We investigated phosphoenolpyruvate-dependent phosphotransferase system enzyme II activities for sucrose and glucose in Streptococcus mutans 6715g. Two integral membrane proteins, enzyme IIscr and enzyme IIglc, each specific for its sugar substrate, sucrose or glucose, were identified by their abilities to catalyze specific sugar:sugar-phosphate exchange reactions. Some of the properties of these two transport proteins are also presented.

Cell Membrane↗
PubMed1983

Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease.

The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.

Cross Reactions↗
PubMed1983

The intramembrane topography of the mannitol-specific enzyme II of the Escherichia coli phosphotransferase system.

The D-mannitol-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is an integral cytoplasmic membrane protein responsible for concomitant transport and phosphorylation of this hexitol. We have investigated the intramembrane topography of this enzyme/permease using proteases, membrane-impermeable reagents, and antibodies against the purified protein. The results of these experiments suggest that this protein spans the membrane in a single orientation with a sizeable proportion of its mass extending into the cytoplasm, but with little of the polypeptide exposed at the outside surface of the membrane. Such an orientation is consistent with the reception and transport roles of the mannitol Enzyme II in E. coli.

Cell Membrane↗
PubMed1983

Substrate and phospholipid specificity of the purified mannitol permease of Escherichia coli.

D-Mannitol is transported and phosphorylated by a specific enzyme II of the phosphotransferase system of Escherichia coli. This protein was purified previously in detergent solution and has been partially characterized. As one approach in understanding the structure and mechanism of this enzyme/permease, we have tested a number of sugar alcohols and their derivatives as substrates and/or inhibitors of this protein. Our results show that the mannitol permease is highly, but not absolutely, specific for D-mannitol. Compounds accepted by the enzyme include those with substitutions in the C-2(= C-5) position of the carbon backbone of the natural substrate as well as D-mannonic acid, one heptitol and one pentitol. All of these compounds were both inhibitors and substrates for the mannitol permease except for D-mannoheptitol, which was an inhibitor but was not phosphorylated by the enzyme. No compound examined, however, exhibited an affinity for the enzyme as high as that for its natural substrate. We have also investigated the phospholipid requirements of the mannitol permease using phospholipids purified from E coli. The purified protein was significantly activated by phosphatidylethanolamine, but little activation was observed with phosphatidylglycerol or cardiolipin. These observations partially delineate requirements for interaction of sugar alcohols and phospholipids with the mannitol permease. They suggest approaches for the design of specific active site probes for the protein, and strategies for stabilizing the enzyme's activity in vitro.

Escherichia coli↗
PubMed1983

Detection, assessment, and diagnosis of alcoholism. Current techniques.

The need for a continuing evolution in methods of detection, assessment, and diagnosis of alcohol abuse and alcohol dependence disorders is emphasized as a necessary step toward altering the stagnating effects of several static and outdated constructs and practices which, despite their humanitarian benefits, may actually be impediments to progress in prevention and treatment. Distinctions are drawn among the purposes and techniques of detection, assessment, and diagnosis; although all three activities are vital, it is the last that must provide a logical basis for understanding causes, courses, and treatment for health problems. Thus far, there has been little genuine progress beyond simply naming the problems we attempt to treat. To illustrate the promising directions that progress might take and to recommend research advances, recent developments in detection, assessment, and diagnosis over the past 5 years are reviewed and critiqued. Special attention is paid to the NCA Diagnostic Criteria and its newest modifications, the Michigan Alcoholism Screening Test and its derivatives, the MacAndrew Scale, the Mortimer-Filkins test, the Essential-Reactive Alcoholism Interview Schedule, and the Alcohol Use Inventory. All of these approaches have demonstrated their utility, but none has yet fulfilled its potential. Their relative advantages are discussed, their flaws are identified, and suggestions are given as to how researchers and clinicians might better use the available tools.

Alcoholism↗