Biomedical subjects

B Sankaran

Publications and source records attributed to B Sankaran.

14 recordsLinked to original sources

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↗

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↗

PubMed1979