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B Sankaran

Publications and source records attributed to B Sankaran.

At least 19 recordsLinked to original sources
PubMed1998

Identification of a structural element in phospholipase C beta2 that interacts with G protein betagamma subunits.

To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.

Adenosine Diphosphate Ribose↗
PubMed1997

Photoaffinity labelling of P-glycoprotein catalytic sites.

Photoaffinity labelling of hamster P-glycoprotein was carried out after trapping of radioactive Mg-8-azido-ADP in the catalytic sites by vanadate or beryllium fluoride. With either trapping agent the same labelled peptide was obtained in homogeneous form, with the sequence -FNEVVFNxPTRPDI-, corresponding to residues 1034-1037 in the C-terminal nucleotide binding site. The missing residue 'x' corresponds to Tyr-1041, which is therefore a primary reaction target of 8-azido-ADP. This tyrosine is conserved in all hamster, mouse and human P-glycoproteins. A second major labelled peptide fraction was also identified. The major sequence in this fraction was -NIHFSxPSR-, corresponding to residues 393-401 of hamster P-glycoprotein, where 'x' corresponds to Tyr-398 in the N-terminal nucleotide binding site. Therefore Tyr-398, which is also conserved in other P-glycoproteins, is also a reaction target for 8-azido-ADP. In sequence alignment of the two nucleotide binding sites, Tyr-398 exactly corresponds to Tyr-1041. The data indicate that these two tyrosines lie close to the adenine ring of bound substrate MgATP in the respective catalytic sites of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Inhibition of P-glycoprotein ATPase activity by beryllium fluoride.

ATPase activity of P-glycoprotein (multidrug-resistance protein) was found to be potently inhibited by beryllium fluoride (BeFx) in combination with MgATP, MgADP, or corresponding Mg-8-azido-nucleotides. Inhibition was due to trapping of nucleoside diphosphate at catalytic sites. Full inhibition was achieved on trapping of 1 mol of nucleotide per mol of Pgp. Reactivation was slow (t(1/2) = 32 min at 37 degrees C), and release of trapped nucleotide correlated with recovery of ATPase. Trapping of 8-azido-ADP followed by UV irradiation yielded permanent inactivation and specific labeling of Pgp in plasma membranes. Both N- and C-terminal nucleotide binding sites were labeled. These findings give strong confirmation of the concepts that in intact Pgp both nucleotide sites are active in MgATP hydrolysis, and that they interact strongly. The characteristics of inhibition by BeFx were similar in general to those seen with vanadate. However, PPi gave strong protection against BeFx inhibition, and in this respect, inhibition by BeFx was clearly different from vanadate inhibition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Inhibition of P-glycoprotein ATPase activity by procedures involving trapping of nucleotide in catalytic sites.

Fluoroaluminate in combination with nucleotide inhibited ATPase activity of P-glycoprotein (Pgp) in plasma membranes and in pure reconstituted form. Low nucleotide concentrations were effective, e.g., half-maximal inhibition was obtained with 10 microM MgATP. With MgATP or MgADP, reactivation occurred with t1/2 = 7 min at 37 degrees C. With 8-azido-ATP, UV irradiation of inhibited Pgp gave specific photolabeling of both nucleotide sites. Fluoroaluminate therefore provides a valuable tool for functional and structural characterization of P-glycoprotein and probably of other ABC transporters. 2-Azido-ATP, in combination with vanadate, fluoroaluminate, or beryllium fluoride, inhibited Pgp ATPase activity. Low concentrations of 2-azido-ATP were effective. However, after UV irradiation of the inhibited Pgp species, in no case was there evidence of covalent labeling of nucleotide sites. Therefore in the Pgp catalytic sites, under conditions of nucleotide trapping, there is no suitable amino acid side chain adjacent to the photoactivated 2-position of bound 2-azido-nucleotide, and 8-azido-ATP is the preferred photolabeling analog.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

A comparison of changes in nucleotide-protein interactions in the striatal, hippocampus and paramedian cortex after cerebral ischemia and reperfusion: correlations to regional vulnerability.

[32P]Azido-purine analogs of ATP and GTP were used to detect changes in phosphorylation and nucleotide binding induced by ischemia and subsequent reperfusion in rat brain striatum, hippocampus and paramedian cortex (PM cortex) tissues. Major changes in phosphorylation were observed for a 130-kDa protein, tentatively identified as the Ca2+ transport ATPase, and calcium/calmodulin-dependent protein kinase II (CaM Kinase II) in all tissues. However, recovery of the phosphorylation of the 130-kDa protein occurred only in the PM cortex on reperfusion. A 200-300% increase in [32P]8N3ATP photoinsertions was observed in the striatum and hippocampus regions for a 43-kDa protein with an isoelectric point of 6.8. This protein was identified as glutamine synthetase (GS) and the increase in binding was found to be due to both increased copy number and activation by Mn2+. An increase in [32P]8N3GTP photoinsertion into a 55-kDa protein, identified as the beta-subunit of tubulin, was found only in the striatum and hippocampus. This indicates the depolymerization of microtubulin in these tissues. These changes correlate to the vulnerability of the striatum and hippocampus to ischemia-induced neuronal death.

Animals↗
PubMed1996

Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase.

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.

Adenine↗
PubMed1995

Both P-glycoprotein nucleotide-binding sites are catalytically active.

The technique of vanadate trapping of nucleotide was used to study catalytic sites of P-glycoprotein (Pgp) in plasma membranes from multidrug-resistant Chinese hamster ovary cells. Vanadate trapping of Mg- or Co-8-azido-nucleotide (1 mol/mol of Pgp) caused complete inhibition of Pgp ATPase activity, with reactivation rates at 37 degrees C of 1.4 x 10(-3) s-1 (t1/2 = 8 min) or 3.3 x 10(-4) s-1 (t1/2 = 35 min), respectively. UV irradiation of the inhibited Pgp yielded permanent inactivation of ATPase activity and specific photolabeling of Pgp. Mild trypsin digestion showed that the two nucleotide sites were labeled in equal proportion. The results show that both nucleotide sites in Pgp are capable of nucleotide hydrolysis, that vanadate trapping of nucleotide at either site completely prevents hydrolysis at both sites, and that vanadate trapping of nucleotide in the N- or C-terminal nucleotide sites occurs non-selectively. A minimal scheme is presented to explain inhibition by vanadate trapping of nucleotide and to describe the normal catalytic pathway. The inhibited Pgp-Mg-nucleotide.vanadate complex is probably an analog of the catalytic transition state, implying that when one nucleotide site assumes the catalytic transition state conformation the other site cannot do so and suggesting that the two sites may alternate in catalysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site.

P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity. The catalytic sites are known to be of low affinity and specificity for nucleotides. From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t 1/2 for reactivation of ATPase activity of 84 min at 37 degrees C and >30 h at 4 degrees C. Reactivation of ATPase correlated with release of trapped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 microM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping. The trapped nucleotide species was ADP. Divalent Cation was required, with magnesium, manganese, and cobalt all effective: cobalt yielded a very stable inhibited species, t1/2 at 37 degrees C = 18 h. No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed. Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp. Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes. Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition at ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Ischemic brain injury selectively alters ATP binding of calcium and calmodulin-dependent protein kinase II.

Calmodulin and ATP affinity and total binding capacity were characterized for CaM kinase II isolated from control and ischemic animals. Ischemic CaM kinase II exhibited equivalent apparent affinity and total binding for calmodulin when compared to control enzyme. However, ischemic CaM kinase II exhibited a significant decrease in apparent affinity for ATP in saturation experiments. ATP binding was characterized using the ATP photoaffinity analog [alpha-32P] Azido-ATP. A significant decrease in total binding and binding affinity for ATP was observed for the alpha (50 kDa) and beta (60 kDa) subunits. The observation that ischemia induced an alteration of ATP binding without affecting calmodulin binding is consistent with the hypothesis that ischemia directly affects the ATP binding of CaM kinase II which results in subsequent inhibition of the enzyme.

Adenosine Triphosphate↗
PubMed1992

Global forebrain ischemia induces a posttranslational modification of multifunctional calcium- and calmodulin-dependent kinase II.

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.

Adenosine Triphosphate↗
PubMed1984

Prosthetics and orthotics in developing countries.

Principles of orthoses and prostheses in developing countries are discussed. Appropriate technological adaptations to suit cultural needs in developing countries have been identified and illustrative examples have been given. In view of the importance of the problem of leprosy in many developing countries, a separate description to cover prosthetic and orthotic appliances including footwear has been attempted. The material is a summary of the excellent publication from Alert in Addis Ababa.

Amputation, Surgical↗