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T H Wilson

Publications and source records attributed to T H Wilson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Alteration of Na(+)-coupled transport in site-directed mutants of the melibiose carrier of Escherichia coli.

Asn-58 of the Escherichia coli melibiose carrier was replaced by Ala, Leu, Ser, and Gln. Trp-54 was replaced by Leu and a double mutant Leu-54/Ala-58 was constructed using site-directed mutagenesis. Cation/sugar cotransport and sugar-induced cation uptake were studied for each mutant. The change of Asn-58 to Ala results in a nearly complete loss of Na(+)-stimulated galactoside transport as well as sugar-stimulated Na+ uptake. Substitutions of Leu, Gln, and Ser for Asn-58 were also defective in Na(+)-stimulated sugar transport. The Trp-54 to Leu mutant shows moderate sugar accumulation with cation selectivity similar to wild-type. The double mutant Leu-54/Ala-58 shows elevated H(+)-melibiose cotransport as well as reduced Na(+)-stimulated melibiose cotransport. These results suggest that Asn-58 is important for Na+ recognition.

Amino Acid Sequence↗
PubMed1996

Chemostat selection of an Escherichia coli mutant containing permease with enhanced lactose affinity.

Chemostats supplied with limited lactose were used to ask whether it was possible to generate and isolate any mutant of Escherichia coli lactose permease which allowed cells to grow faster. The permease and beta-galactosidase activities of the chemostat culture initially rose together to reach a plateau. After 30 days, the former underwent a second increase alone. From this culture, a faster-growing mutant was isolated. Its permease gene was cloned, sequenced, and found to have a single base pair changed. Thymine at position 199 was changed to guanine, resulting in serine 67 being substituted by alanine. Cells bearing this mutant in the plasmid could grow faster than parents in 10 microM lactose. The Km of the mutant permease toward lactose was 1.4 mM, about half of the wild-type value. Thus, a mutant with higher affinity for substrate could be selected from the chemostat.

Bacteriological Techniques↗
PubMed1996

Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli.

Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 --> Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.

Base Sequence↗
PubMed1995

GLY113-->ASP can restore activity to the ASP51-->SER mutant in the melibiose carrier of Escherichia coli.

ASP51 in the putative membrane-spanning helix 2 of the melibiose carrier of Escherichia coli was replaced by SER. This mutation caused failure of the cell to transport melibiose and failure to ferment melibiose on indicator plates. A melibiose-positive revertant was isolated from these plates and was found to have two additional mutations, GLY113-->ASP (in helix 4) and PHE16-->LEU (in helix 1). The double mutant ASP51-->SER/GLY113-->ASP was constructed and showed accumulation of melibiose. On the other hand ASP51-->SER/PHE16-->LEU showed no activity. It is concluded that the new carboxyl group at position 113 compensates for the loss of the carboxyl group at position 51.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of the gene for the lactose carrier of Citrobacter freundii.

The gene coding for the lactose carrier of Citrobacter freundii was cloned into the plasmid pBR322. The gene was sequenced and the amino acid sequence was found to be 70% identical to the lactose carrier of E. coli. All of the charged residues in the membrane spanning region were conserved. The sugar specificity is somewhat different from that of E. coli. The C. freundii carrier has less activity for lactose and more activity for o-nitrophenyl-galactose (ONPG) than the carrier of E. coli.

Amino Acid Sequence↗
PubMed1994

Characterization and sequencing of an uncoupled lactose carrier mutant of Escherichia coli.

A lactose carrier mutant of Escherichia coli (ML308-22) showed a severe defect in thiomethylgalactoside accumulation but a faster than normal entry of o-nitrophenyl-galactoside. Sequencing of the mutant lacY gene revealed a point mutation resulting in the substitution of glycine-159 by a cysteine residue. The mutant showed an increased sensitivity to sulfhydryl reagents, a property that is consistent with the view that the Cysteine-159 is in or near the sugar recognition site and the energy coupling region of the carrier.

Amino Acid Sequence↗
PubMed1994

Transport properties of Asp-51-->Glu and Asp-120-->Glu mutants of the melibiose carrier of Escherichia coli.

Asp-51-->Glu and Asp-120-->Glu mutants of the melibiose carrier of Escherichia coli were investigated for their cation/sugar cotransport properties. The carrier containing Glu-51 showed proton/melibiose cotransport but was extremely defective in Na+ or Li+ stimulation of sugar accumulation. On the other hand, the carrier containing Glu-120 had lost the ability to couple protons with melibiose uptake while retaining considerable Na+ or Li+ cotransport with melibiose (40-fold accumulation versus 90-fold for the wild type in the presence of Na+). It is concluded that both Asp-51 and Asp-120 are important for cation recognition.

Biological Transport↗
PubMed1994

Replacement of alanine 58 by asparagine enables the melibiose carrier of Klebsiella pneumoniae to couple sugar transport to Na+.

The melibiose carrier of Klebsiella pneumoniae couples sugar transport to H+ and Li+, while that of Escherichia coli uses Na+ besides the other two cation species (Hama and Wilson, 1992). We have shown that the K. pneumoniae melibiose carrier is capable of recognizing Na+ when the amino-terminal 81 residues are replaced by the corresponding region of the E. coli melibiose carrier (Hama and Wilson, 1993). In this amino-terminal region there are 5 residues that are not conserved between the two carriers. In this study, we changed each of the 5 residues of the K. pneumoniae carrier to the one in the E. coli carrier. The substitutions are Ile-36-->Val, Val-43-->Leu, Leu-54-->Trp, Ala-58-->Asn, and Cys-68-->Ala. With four of the five mutants, Ile-36-->Val, Val-43-->Leu, Leu-54-->Trp, and Cys-68-->Ala, sugar accumulation was not affected by Na+. In striking contrast, melibiose and methyl-1-thio-beta-D-galactopyranoside accumulation was greatly stimulated by Na+ with the Ala-58-->Asn mutant. Furthermore, Na+ uptake coupled to downhill melibiose transport was observed with the Ala-58-->Asn mutant. These results indicate that the Ala-58-->Asn substitution enables the K. pneumoniae melibiose carrier to couple sugar transport to Na+. It is clear that the Asn-58 residue (Asn-54 in the E. coli carrier) is involved in Na+ recognition.

Base Sequence↗
PubMed1994

Characterization of the lactose transport system in Citrobacter freundii.

The lactose transport system of Citrobacter freundii was characterized. Both the lactose transport system and beta-galactosidase were induced with either lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG), the latter being the better inducer. The Km values for methyl-beta-D-thiogalactopyranoside (TMG) transport and lactose transport were 0.61 mM and 1.1 mM, respectively, and the Vmax values were 53 nmol/min/mg cell protein and 12 nmol/min/mg cell protein, respectively. Thus, TMG is a better substrate than lactose. Thiogalactopyranoside (TDG) was a very potent competitive inhibitor. Neither Na+ nor Li+ had a significant effect on the TMG transport or the lactose transport. Proton/substrate cotransport (symport) via this system was observed.

Citrobacter freundii↗
PubMed1993

Lysine 319 interacts with both glutamic acid 269 and aspartic acid 240 in the lactose carrier of Escherichia coli.

It is believed that there are several charged amino acid residues in membrane-spanning alpha-helices of the lactose carrier of Escherichia coli. Evidence has previously been presented for two different salt bridges in membrane-spanning regions of the lactose carrier. One of these involves an interaction between Asp-237 and Lys-358; another involves interaction between Asp-240 and Lys-319. Additional studies of Lys-319 suggest that it may interact with Glu-269 as well as Asp-240. A cell containing the LacY gene with the mutation Lys-319-->Asn failed to ferment melibiose and after several days melibiose-positive mutants arose on indicator plates. These revertants showed second site mutations which replaced Asp-240 by neutral amino acids (Val or Gly). In addition, a second site mutation showed Glu-269 changed to Asn. Cells containing the mutation Lys-319-->Leu also failed to ferment melibiose and melibiose-positive revertants showed Asp-240-->Ala and Asp-240-->Tyr as well as Tyr-236-->Phe and His-322-->Arg. Second site revertants were also sought from the mutant Glu-269-->Asn which grew poorly on melibiose minimal plates. Melibiose-positive revertants included the double mutant Gln-269/Asn-319. All of the Glu-269-->Asn mutants were extremely defective in transport. It was concluded that Lys-319 interacts with Glu-269 and Asp-240 probably as salt bridges.

Amino Acid Sequence↗
PubMed1993

Cation-coupling in chimeric melibiose carriers derived from Escherichia coli and Klebsiella pneumoniae. The amino-terminal portion is crucial for Na+ recognition in melibiose transport.

The melibiose carrier of Escherichia coli couples sugar transport to H+, Na+, and Li+, while that of Klebsiella pneumoniae utilizes only H+ and Li+. We made five chimeric carriers derived from the two carriers to identify the region(s) involved in Na+ recognition. The chimeric carriers E2K10, E4K8, E6K6, E8K4, and E10K2 have the amino-terminal 77, 144, 197, 298, and 349 amino acid residues derived from E. coli and the rest derived from K. pneumoniae, respectively. Melibiose accumulation through the chimeric carriers E2K10, E4K8, and E6K6 was strongly stimulated by Na+ and Li+ as is the case with the E. coli carrier. On the other hand, there was very little stimulation with the carriers E8K4 and E10K2. These results suggest that, 1) the amino-terminal 77 amino acids of the E. coli carrier, which has 5 different and 4 fewer amino acids than the K. pneumoniae carrier, have a crucial role in Na+ recognition in melibiose transport and 2) the carboxyl-terminal half of the carrier also forms a part of the Na+ recognition site which may be distorted in chimeric structures. In contrast with melibiose accumulation, there was very little Na+ stimulation of TMG (methyl-1-thio-beta-D-galactopyranoside) transport and no Na+ stimulation was observed in lactose transport with any of the chimeric carriers, whereas in E. coli Na+ stimulates TMG and lactose transport. These results suggest that there is no universal Na+ recognition site for all the sugar substrates. Instead different parts of the carrier seem to participate in cation recognition for different sugar substrates.

Base Sequence↗
PubMed1993

Impact of multiple risk factors and ranitidine prophylaxis on the development of stress-related upper gastrointestinal bleeding: a prospective, multicenter, double-blind, randomized trial. The Ranitidine Head Injury Study Group.

OBJECTIVES: To evaluate the impact of risk factors on the development of stress-related upper gastrointestinal bleeding in severe head injury patients randomized to treatment with a 6.25 mg/hr continuous ranitidine infusion or placebo. DESIGN: Prospective, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. SETTING: Ten intensive care units in the United States. PATIENTS: Patients with severe head injury, defined as having a Glasgow Coma Score of < or = 10, were eligible for enrollment. INTERVENTIONS: Ranitidine 6.25 mg/hr or saline placebo was administered by continuous infusion for a maximum of 5 days. MEASUREMENTS AND MAIN RESULTS: Patients were evaluated every 8 hrs for the presence of stress-related upper gastrointestinal bleeding. Bleeding developed in 15 (19%) of 81 placebo-treated patients vs. three (3%) of 86 ranitidine-treated patients (p = .002). None of the individual risk factors had a significant effect on bleeding frequency. No bleeding occurred in the four patients with one risk factor. Placebo bleeding rates in patients with 2, 3 to 5, and > 5 risk factors were 20%, 20%, and 18%, respectively. For the ranitidine-treated patients, bleeding was reported in 0%, 5%, and 0% in the 2, 3 to 5, and > 5 risk factor subgroups, respectively. Pneumonia occurred in 19% of the placebo-treated patients vs. 14% in the ranitidine treatment group. CONCLUSIONS: The full risk to develop stress-related upper gastrointestinal bleeding was realized when two risk factors were present concomitantly. The presence of additional risk factors did not increase the occurrence of bleeding. A continuous infusion of ranitidine at 6.25 mg/hr provided significant protection from bleeding, regardless of the number of risk factors present.

Adult↗
PubMed1993

Ranitidine is effective therapy for erosive esophagitis.

Two ranitidine dosages were compared for the treatment of erosive esophagitis in a multicenter, double-blind, randomized, parallel-group, placebo-controlled study. Adults with endoscopically verified erosive esophagitis were treated with either ranitidine 150 mg four times daily (n = 106), ranitidine 300 mg four times daily (n = 106), or placebo (n = 116) for up to 12 wk. Patients were also encouraged to adhere to lifestyle modifications (e.g., to elevate the head of bed, etc). Erosive esophagitis healing, determined by endoscopy, was achieved in 69% and 62% of ranitidine-treated patients by 8 wk and in 79% and 74% by 12 wk (150 mg and 300 mg, respectively) compared with 28% of placebo-treated patients by 8 wk and 40% by 12 wk (p < 0.001 ranitidine vs. placebo). Onset of heartburn relief occurred within 24 h of initiating either ranitidine dosage, and relief was maintained throughout the 12-wk study. Both ranitidine dosages displayed safety profiles similar to that of placebo. We conclude that ranitidine 150 mg or 300 mg administered four times daily is effective for healing erosive esophagitis and relieving its symptoms.

Double-Blind Method↗
PubMed1992

Possible salt bridges between transmembrane alpha-helices of the lactose carrier of Escherichia coli.

Although it is energetically extremely unfavorable to have charged amino acid residues of a polypeptide in the hydrophobic environment of the membrane phospholipid bilayer, a few such charged residues are found in membrane-spanning regions of membrane proteins. Ion pairs (salt bridges) would be much more stable in low dielectric media than single ionized residues. This paper provides indirect evidence for a salt bridge between Asp-240 and Lys-319 in the lactose carrier of Escherichia coli. When Asp-240 was changed to alanine by site-directed mutagenesis, there was a loss of the ability to accumulate methyl-beta-D-thiogalactopyranoside (TMG), melibiose, or lactose. Fast-growing revertants were isolated on melibiose minimal agar plates. Two second-site revertants were isolated: Asp-240-->Ala plus Gly-268-->Val and Asp-240-->Ala plus Lys-319-->Gln. These revertants showed extremely poor accumulation of TMG, melibiose, and lactose, but showed significant "downhill" lactose entry into beta-galactosidase-containing cells with sugar concentrations of 2 and 5 mM. It is concluded that there is some important interaction between Asp-240 and Lys-319, possibly a salt bridge.

Amino Acid Sequence↗
PubMed1992

Primary structure and characteristics of the melibiose carrier of Klebsiella pneumoniae.

The melB gene coding for the melibiose carrier of Klebsiella pneumoniae was cloned and sequenced. There were two potential translation initiation sites. It was predicted that the melibiose carrier consists of 471 (or 467) amino acid residues. Seventy-eight percent of the 471 amino acids were identical to the Escherichia coli melibiose carrier. Sugar transport characteristics were studied using an E. coli mel- mutant expressing cloned K. pneumoniae melB gene. Accumulation of melibiose via the K. pneumoniae melibiose carrier was not stimulated by adding NaCl or LiCl which stimulates melibiose accumulation via the E. coli melibiose carrier. Lactose was accumulated only in the presence of LiCl. TMG (methyl-1-thio-beta-D-galactopyranoside) was accumulated in the absence of added NaCl or LiCl. The accumulation was stimulated by LiCl but not by NaCl. Rapid H+ uptake was observed when melibiose or TMG was added to cell suspensions. These results suggest that the preferred cation couplings via K. pneumoniae melibiose carrier are H(+)-melibiose, Li(+)-lactose, and H+/Li(+)-TMG. This coupling spectrum is quite different from that of the E. coli melibiose carrier. It is of special interest that the K. pneumoniae melibiose carrier seems to be lacking the ability to recognize Na+ which is a preferred coupling cation of the E. coli melibiose carrier for all known sugar substrates. Further investigation of these two carriers may give us insight into the Na+ recognition site.

Amino Acid Sequence↗
PubMed1992

Membrane topology of the melibiose carrier of Escherichia coli.

The minimum structural information necessary to formulate and assess mechanistic models of integral membrane protein function is that of membrane topology. This paper characterizes the topological structure of the melibiose carrier of Escherichia coli based on constraints provided by genetic fusions to the compartment-specific reporter protein alkaline phosphatase. Twenty-eight unique chimeras exhibiting either low alkaline phosphatase activity (cytoplasmic location of the fusion joint) or high alkaline phosphatase activity (periplasmic location of the fusion joint) were characterized and used in conjunction with Goldman-Engelman-Steitz hydropathy analysis to model topological structure. The melibiose carrier is predicted to have a cytoplasmic amino terminus, two sets of six transmembrane domains separated by an unusually large cytoplasmic loop ("six-loop-six" arrangement), and a 45-residue cytoplasmic carboxyl tail. Remarkably, the identical six-loop-six arrangement is predicted from the hydrophobicity plots of the H(+)-coupled lactose, arabinose, xylose, and citrate cotransporters of E. coli, the glucose transporter from rat brain, the family of glucose transporters isolated from various human tissues and cell lines, and the human, mouse, and hamster multidrug resistance transporters (Henderson, P.J.F. (1990) Res. Microbiol. 141, 316-328; Maloney, P.C. (1990) Res. Microbiol. 141, 374-383). Such a broad degree of conservation (or convergence) suggests a distinct structural and/or mechanistic advantage associated with the six-loop-six motif. The nature of this advantage is as yet unknown.

Alkaline Phosphatase↗
PubMed1992

Amino acid substitution in the lactose carrier protein with the use of amber suppressors.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.

Amino Acid Sequence↗