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H K Sarkar

Publications and source records attributed to H K Sarkar.

At least 19 recordsLinked to original sources
PubMed1992

Melibiose permease of Escherichia coli: mutation of histidine-94 alters expression and stability rather than catalytic activity.

Previous studies utilizing site-directed mutagenesis [Pourcher et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 468-472] indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important. The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg. Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane. Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type. Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C. Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature. The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se. Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.

Base Sequence↗
PubMed1990

The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties.

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

Amino Acid Sequence↗
PubMed1990

Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system.

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].

Animals↗
PubMed1990

Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli.

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced with Arg. Replacement of His-213, His-442, or His-456 has no significant effect on permease activity, while permease with Arg in place of His-198, His-318, or His-357 retains more than 70% of wild-type activity. In striking contrast, replacement of His-94 with Arg causes a complete loss of sugar binding and transport, although the cells contain a normal complement of permease molecules. Thus, as shown previously with lac permease, only a single His residue is important for activity, but, in the case of mel permease, the critical His residue is present in the 3rd putative transmembrane helix rather than the 10th.

Arginine↗
PubMed1989

Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues.

Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity [Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765], sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗
PubMed1989

A five-residue sequence near the carboxyl terminus of the polytopic membrane protein lac permease is required for stability within the membrane.

The lac permease (lacY gene product) of Escherichia coli contains 417 amino acid residues and is predicted to have a short hydrophilic amino terminus on the inner surface of the cytoplasmic membrane, multiple transmembrane hydrophobic segments in alpha-helical conformation, and a 17-amino acid residue hydrophilic carboxyl-terminal tail on the inner surface of the membrane. To assess the importance of the carboxyl terminus, the properties of several truncation mutants were studied. The mutants were constructed by site-directed mutagenesis such that stop codons were placed at specified positions, and the altered lacY genes were expressed at a relatively low rate from plasmid pACYC184. Permease truncated at position 407 or 401 retains full activity, and a normal complement of molecules is present in the membrane, as judged by immunoblot analyses. Thus, it is apparent that the carboxyl-terminal tail plays no direct role in membrane insertion of the permease, its stability, or in the mechanism of lactose/H+ symport. In marked contrast, when truncations are made at residues 396 (i.e., 4 amino acid residues from the carboxyl terminus of putative helix XII), 389, 372, or 346, the permease is no longer found in the membrane. Remarkably, however, when each of the mutated lacY genes is expressed at a high rate by means of the T7 RNA polymerase system [Tabor, S. & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1079], all of the truncated permeases are present in the membrane, as indicated by [35S]methionine incorporation studies; however, permease truncated at residue 396, 389, 372, or 346 is defective with respect to lactose/H+ symport. Finally, pulse-chase experiments indicate that wild-type permease or permease truncated at residue 401 is stable, whereas permease truncated at or prior to residue 396 is degraded at a significant rate. The results are consistent with the notion that residues 396-401 in putative helix XII are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding.

Amino Acid Sequence↗
PubMed1988

Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells.

The well-characterized erythrocyte glucose transporter is also expressed in brain, adipocytes, kidney, muscle, and certain transformed cells, but not in liver, intestine, or the islets of Langerhans. Using as probe a cDNA encoding the rat brain glucose transporter, we isolated from a rat liver cDNA library a clone encoding a protein 55% identical in sequence to the rat brain transporter, and with a superimpossible hydropathy plot. We expressed this protein in an E. coli mutant defective in glucose uptake; the protein was incorporated into the bacterial membrane and functioned as a glucose transporter. This new transporter is expressed in liver, intestine, kidney, and the islets of Langerhans; immunofluorescence analysis showed that it is present in the plasma membrane of the insulin-producing beta cells. Insulinoma cells express, inappropriately, the erythrocyte glucose transporter, and we suggest that this may be related to their inability to secrete insulin in response to elevations in glucose.

Amino Acid Sequence↗
PubMed1988

Expression of the human erythrocyte glucose transporter in Escherichia coli.

The gene encoding the human erythrocyte glucose transporter, cloned from HepG2 hepatoma cells, was expressed in Escherichia coli by introducing a prokaryote-type ribosome binding site, subcloning the gene into the T7 promoter/T7 polymerase expression system, and transforming a strain that is defective in glucose transport. Cells bearing plasmids with the transporter gene take up 2-deoxy-D-glucose and D-glucose, unlike cells bearing plasmids without the transporter gene. Moreover, 2-deoxy-D-glucose uptake is inhibited by unlabeled D-glucose, cytochalasin B, or mercuric chloride but not by L-glucose. The glucose transport protein is inserted into the membrane of E. coli, as evidenced by immunoblotting experiments with two site-directed polyclonal antibodies, one directed against the COOH terminus of the glucose transporter and the other directed against a synthetic peptide containing amino acid residues 225-238. As detected with both antibodies, the protein migrates with apparent molecular mass of 34 kDa in sodium dodecyl sulfate/12% polyacrylamide, a size similar to that of the unglycosylated glucose-transport protein synthesized in vitro.

Base Sequence↗
PubMed1986

lac permease of Escherichia coli: histidine-205 and histidine-322 play different roles in lactose/H+ symport.

The lac permease of Escherichia coli was modified by site-directed mutagenesis such that His-205 or His-322 is replaced with either Asn or Gln. Permease with Asn or Gln in place of His-205 exhibits normal activity, while permease with Asn or Gln in place of His-322 exhibits no activity. The results are consistent with the interpretation that His-205 and His-322 play different roles in lactose/H+ symport, the former involving hydrogen bonding of the imidazole nitrogens and the latter requiring positive charge in the imidazole ring. In addition, it is demonstrated that permease with Arg in place of His-322 does not catalyze efflux, exchange, or counterflow. The observations, in conjunction with those in the accompanying paper [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (following paper in this issue)], suggest that His-322 plays an important role in H+ translocation, possibly as a component of a charge-relay system with Glu-325, a neighboring residue in helix 10.

Base Sequence↗
PubMed1986

Site-specific mutagenesis of cysteine 148 to serine in the lac permease of Escherichia coli.

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the lac Y gene of Escherichia coli such that Cys148 in the lac permease is converted to Ser. A mutagenesis protocol is used that significantly improves the efficiency of mutant recovery by in vitro methylation of closed-circular heteroduplex DNA containing the mutation, followed by nicking with HindIII in the presence of ethidium bromide and heat denaturation prior to transfection. In contrast to Gly148 permease (Trumble, W.R., Viitanen, P.V., Sarkar, H.K., Poonian, M.S., and Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860-867), permease containing Ser at position 148 catalyzes active lactose transport at a rate comparable to wild-type permease. Like Gly148 permease, however, transport activity is less sensitive to inactivation by N-ethylmaleimide, and galactosyl-1-thio-beta-D-galactopyranoside affords no protection against inactivation. The observations provide strong support for the contention that Cys148 is obligatory for substrate protection against inactivation by sulfhydryl reagents, but does not play an essential role in lactose:H+ symport.

Cysteine↗
PubMed1986

Substitution of glutamine-60 with glutamic acid causes the lac permease of Escherichia coli to become temperature sensitive.

The lac Y gene of Escherichia coli was modified by oligonucleotide-directed, site-specific mutagenesis so that Gln-60 is replaced with Glu. Although the replacement introduces a negative charge into a putative hydrophobic, transmembrane alpha-helical segment of the lac permease, lactose/H+ symport is unimpaired. However, the modified permease is more susceptible to heat inactivation. That is, upon incubation at 45 degrees C, Glu-60 permease loses activity with a t1/2 of 20 min relative to a t1/2 of 50 min with wild-type permease.

Base Sequence↗
PubMed1985

Site-directed mutagenesis of cysteine-148 in the lac permease of Escherichia coli: effect on transport, binding, and sulfhydryl inactivation.

By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue [Trumble, W. R., Viitanen, P. V., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860]. Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E. coli T184. Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl alpha-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease. In contrast, the Vmax for lactose transport in vesicles containing mutant permease is significantly decreased. Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl alpha-D-galactopyranoside with high affinity. Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity. Importantly, beta-galactosyl 1-thio-beta-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant. The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease. Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl alpha-D-galactopyranoside from wild-type permease, does not affect binding in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

cys154 Is important for lac permease activity in Escherichia coli.

The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport.

Base Sequence↗
PubMed1985

Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a normal complement of permease molecules, as determined by immunoadsorption assays. Interestingly, although substitution of histidine-205 or histidine-322 by arginine results in the loss of ability to catalyze active lactose transport, permease molecules with arginine at residue 322 appear to facilitate downhill lactose movements at high concentrations of the disaccharide. The results provide strong support for the contention that histidine residues in the lac permease play an important role in the coupling between lactose and proton translocation.

Arginine↗
PubMed1984

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli.

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

DNA, Single-Stranded↗
PubMed1981

Phototransformation and dark reversion of phytochrome in deuterium oxide.

The photostationary equilibrium between the Pr and Pfr forms of phytochrome shows a strong solvent deuterium isotope effect. Phytochrome transformation from the Pr to the Pfr form exhibits a small deuterium isotope effect, in Tris-D2O upon irradiation with red light, only after a photocycling of the phytochrome. In contrast, both the photoreversion and dark reversion of Pfr show an enhanced rate in D2O. In addition to the shift in the photostationary equilibrium in D2O, another pronounced effect of D2O on phytochrome is reflected in a significant enhancement of the fluorescence quantum yield of phytochrome (Pr). This result is interpreted in terms of the primary reaction involving an intramolecular proton transfer and its consequence in the phototransformation of phytochrome. It is further proposed that a tyrosyl residue acts as a general acid catalyst in the Pr to Pfr phototransformation, which is slower in D2O than in H2O. The D2O solvent isotope effect on the photoreversion and dark reversion of Pfr is explained on the basis of acid catalysis, probably a specific acid catalysis by deuteronium ion.

Darkness↗