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H Bhattacharjee

Publications and source records attributed to H Bhattacharjee.

At least 19 recordsLinked to original sources
PubMed2001

Genomic organization and chromosomal localization of the Asna1 gene, a mouse homologue of a bacterial arsenic-translocating ATPase gene.

The plasmid encoded ArsA ATPase in Escherichia coli is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Arsenite or antimonite allosterically activates the ArsA ATPase activity. In this paper, we report the cloning and characterization of the mouse homologue (Asna1) of the bacterial arsA gene. The Asna1 gene encodes an open reading frame of 348 amino acids and exhibits 27% identity to the bacterial ArsA protein and 99% similarity to its human counterpart (hASNA-1). The Asna1 mRNA is a approximately 1.3 kb transcript and is present at high levels in kidney and testis, moderate levels in brain, liver, lung and skin, and low levels in heart, small intestine, spleen, stomach, and thymus. A negligible amount of Asna1 transcript is detected in skeletal muscle. We have also characterized the genomic structure of the Asna1 gene. The gene spans over 7 kb and consists of seven exons and six introns. All splice sites conform to the GT-AG rule, except for the splice donor site of intron 4 that is GC instead of GT. Fluorescence in situ hybridization indicates that the Asna1 gene is localized in the C3-D1 region of mouse chromosome 8.

Adenosine Triphosphatases↗
PubMed2001

Structure-function analysis of the ArsA ATPase: contribution of histidine residues.

The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.

Antimony↗
PubMed2000

Role of conserved histidine residues in metalloactivation of the ArsA ATPase.

The ArsA ATPase is the catalytic subunit of a pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. Arsenite or antimonite allosterically activates the ArsA ATPase activity. ArsA homologues from eubacteria, archaea and eukarya have a signature sequence (DTAPTGHT) that includes a conserved histidine. The ArsA ATPase has two such conserved motifs, one in the NH2-terminal (Al) half and the other in the COOH-terminal (A2) half of the protein. These sequences have been proposed to be signal transduction domains that transmit the information of metal occupancy at the allosteric to the catalytic site to activate ATP hydrolysis. The role of the conserved residues His148 and His453, which reside in the A1 and A2 signal transduction domains respectively, was investigated by mutagenesis to create H148A, H453A or H148A/H453A ArsAs. Each altered protein exhibited a decrease in the Vmax of metalloid-activated ATP hydrolysis, in the order wild type ArsA>H148A>H453A>H148A/H453A. These results suggest that the histidine residues play a role in transmission of the signal between the catalytic and allosteric sites.

Adenosine Triphosphatases↗
PubMed2000

Structure-function relationships in an anion-translocating ATPase.

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.

Adenosine Triphosphatases↗
PubMed1999

Mechanism of the ArsA ATPase.

The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells. This transport ATPase shares structural and perhaps mechanism features with ABC transporters. The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase. As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump. The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level. Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains. In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change. The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step. We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters.

Adenosine Triphosphatases↗
PubMed1999

Capsulorhexis in intumescent cataract.

This technique of anterior capsulorhexis is for use in eyes with high intralenticular pressure and absence of red reflex as encountered in intumescent cataract. The initial steps of fashioning the anterior capsular flap and lens decompression are done under the microscope's high magnification and noncoaxial oblique illumination. Intralenticular pressure is controlled by filling the anterior chamber with a viscoelastic agent. Once the lens is decompressed, the capsulorhexis is completed with a capsule forceps and an endoilluminator is used as an oblique source of illumination. This technique allows controlled capsulorhexis in eyes with intumescent cataract.

Adult↗
PubMed1998

UDP-galactose 4-epimerase from Kluyveromyces fragilis: reconstitution of holoenzyme structure after dissociation with parachloromercuribenzoate.

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The pathway for the assembly of the holoenzyme structure has been studied after dissociating the native epimerase with p-chloromercuribenzoate into inactive mercurated monomers. The process of dissociation was not associated with unfolding of the molecules. Reconstitution of the functional holoenzyme was done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to monitor maturation of the enzyme from the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary and quarternary structures, Km for the substrate UDP-galactose, reductive inhibition, interaction with the fluorophore 1-anilino 8-naphthalene sulphonic acid (ANS), etc. Reconstitution under low ionic strength buffer (I = 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure. However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I = 0.1). Reactivation was strongly dependent on pH, being most effective at pH 8.1. Kinetic evidence suggested that, at low ionic strength, assembly of NAD over dimeric apoenzyme is the rate-limiting step in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.

Anilino Naphthalenesulfonates↗
PubMed1996

Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase.

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys113, Cys172, and Cys422. To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 A of each other to form a fluorescent adduct. ArsA proteins in which single cysteine residues were altered by site-directed mutagenesis still formed fluorescent adducts. Proteins in which two of the three cysteine residues were substituted did not form fluorescent adducts. These results demonstrate that Cys113, Cys172, and Cys422 are in close proximity of each other. We propose a model in which As(III) or Sb(III) interacts with these three cysteines in a trigonal pyramidal geometry, forming a novel soft metal-thiol cage.

Adenosine Triphosphatases↗
PubMed1995

Role of cysteinyl residues in metalloactivation of the oxyanion-translocating ArsA ATPase.

The ArsA protein, the catalytic subunit of the oxyanion-translocating ATPase responsible for resistance to arsenicals and antimonials in Escherichia coli, is activated by arsenite or antimonite. Activation is associated with dimerization of the ArsA protein. Enzymatic activity was rapidly but reversibly inhibited by the sulfhydryl reagent methyl methanethiosulfonate, suggesting that at least one cysteinyl residue is required for catalytic activity. Each of the four cysteinyl residues in the ArsA protein, Cys26, Cys113, Cys172, and Cys422, were individually changed to seryl residues. The C26S protein had normal properties. Cells expressing the other three mutations lost resistance to arsenite and antimonite. The C113S, C172S, and C422S enzymes each had relatively normal Km values for ATP but reduced affinity for antimonite and arsenite. The Vmax of the activated enzymes ranged from very low for the C113S and C422S enzymes to near normal for the C172S enzyme. These results suggest a mechanism of activation by formation of a tricoordinate complex between Sb(III) or As(III) and the cysteine thiolates 113, 172, and 422.

Adenosine Triphosphatases↗
PubMed1995

Mechanisms of metalloregulation of an anion-translocating ATPase.

The ars (arsenical resistance) operon cloned from R-factor R773 has five genes that encode two repressor proteins, ArsR and ArsD, and three structural proteins, ArsA, ArsB, and ArsC. The ArsA and ArsB proteins form a membrane-bound pump that functions as an oxyanion-translocating ATPase. The substrates of the pump are the oxyanions arsenite or antimonite. The ArsC protein is an arsenate reductase that reduces arsenate to arsenite, which is subsequently pumped out of the cell. This review deals with the mechanism of transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA protein, the catalytic subunit of the pump. The chemical nature of the inducer plays an important role in regulation. In solution arsenite or antimonite exist as oxyanions and reacts with the cysteines in proteins. In both transcriptional regulation by the ArsR repressor and allosteric regulation of the ArsA ATPase, the ability of As(III) and Sb(III) to interact with the cysteines of the proteins, involves their action as effector.

Adenosine Triphosphatases↗
PubMed1992

Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site. Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A. (1980) J. Biol. Chem. 255, 10777-10786). Employing a number of thiol-specific reagents, we now suggest discriminatory and nonidentical roles for these two thiols. Kinetic and statistical analysis of 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide modification reaction of epimerase show that only one thiol is essential for activity. Consecutive modification experiments clearly show that the same active thiol is modified in both cases. However, significant differences are observed when the reactivity of these reagents is monitored in terms of coenzyme fluorescence. Treatment with N-ethylmaleimide leads to a form of inactive enzyme that fully retains its fluorescent properties whereas modification with 5,5'-dithiobis-(2-nitrobenzoic acid), on the other hand, results in the loss of both activity and fluorescence. The closely spaced nonessential second thiol, which is not modified by N-ethylmaleimide is therefore involved in generating and maintaining the coenzyme fluorescence. Modification studies with a series of spin-labeled maleimide shows that only 3-(maleimidomethyl)proxyl causes partial quenching of coenzyme fluorescence. This suggests that the active thiol is situated at a distance of 4.5 A approximately from the coenzyme fluorophore.

Binding Sites↗