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H K Sarkar

Publications and source records attributed to H K Sarkar.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1982

Nature of phototransformation of phytochrome As probed by intrinsic tryptophan residues.

The phototransformation of the photomorphogenic photoreceptor phytochrome was probed by the intrinsic luminescence of the tryptophan (Trp) residues. The red light absorbing form of phytochrome (Pr) showed a decreased tryptophan phosphorescence intensity, compared to that of the far-red light absorbing form of phytochrome (Pfr), and a delayed fluorescence from the chromophore upon excitation of the tryptophan residues with 290-nm light. The tryptophan phosphorescence in both Pr and Pfr showed decreased lifetimes (0.29 and 1.84 s, respectively) compared to that of the free tryptophan (6.00 s). In addition, the decay kinetics of the delayed fluorescence in Pr showed a short-lifetime component (0.24 s), which is similar to the tryptophan phosphorescence lifetime value. This is due to an efficient triplet-singlet (3Trp-1Pr) energy transfer in the Pr from. The increases in the tryptophan phosphorescence quantum yield and lifetime in the Pfr form have been interpreted on the basis of chromophore reorientation on the protein surface as a result of the Pr lead to Pfr phototransformation. The Stern-Volmer plot of the quenching data further confirms preferential exposure of the tryptophan residues in the Pfr form (46% "exposed' tryptophan residues in the Pr form as compared to 72% in the Pfr form). These results provide strong support for the hydrophobic model of Pfr [Hahn, T. R., & Song, P. S. (1981) Biochemistry 20, 2602-2609).

Luminescent Measurements↗
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↗
PubMed1981

Primary photoprocesses of undegraded phytochrome excited with red and blue light at 77 K.

1. Red light irradiation of phytochrome (Pr) at 77 K produces an intermediate absorbing at 696 nm. The photostationary state concentration of this intermediate is rapidly established with that of Pr as the result of spectral overlap between the Qy band of Pr and the Qx band of the intermediate. 2. The 696 nm intermediate reverts back to Pr preferentially without yielding a substantial amount of Pfr upon thawing the 77 K sample to higher temperatures. 3. Blue light irradiation of Pr with or without exogenous FMN at 77 K results in the formation of two intermediates absorbing at 684 nm and 696 nm. The 684 nm intermediate is photochemically converted to the 696 nm intermediate at 77 K. Possibilities for the preferential formation of the 684 nm intermediate with blue light are discussed. 4. At 277 K, blue light irradiation of phytochrome (Pr) containing exogenous FMN increases the rate of phototransformation from Pr to Pfr three times over Pr having no FMN. On the other hand, exogenous FMN has no effect on the rate of transformation of Pr to Pfr by red light. 5. Energy transfer occurs from FMN to Pr at 77 K, initiating the photoprocesses of the Pr. The energy transfer apparently occurs within flavin-phytochrome complexes.

Edible Grain↗