113 research outputs found
The partial characterization of the binding of avidin-biotin complex to rat liver plasma membrane
Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4
The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions
Effect of Pyridoxine Deficiency in Young-Rats on High-Affinity Serotonin and Dopamine Receptors
The high-affinity bindings of [3H]-5-hydroxytryptamine to serotonin S-1 receptors,
[3H]-ketanserin to serotonin S-2 receptors in the cerebral cortex, [3H]-
fluphenazine to dopamine D-1 receptors, and [3H]-spiroperidol to dopamine D-2
receptors in the corpus striatum were studied in pyridoxine-deficient rats and
compared to pyridoxine-supplemented controls. There was a significant increase
in the maximal binding (Bmax) of serotonin S-1 and S-2 receptors with a significant
decrease in their binding affinities (Kd). However, there were no significant
changes either in the maximal binding or binding affinity of striatal dopamine D-
1 and D-2 receptors. Receptor sensitivity seems to correlate negatively with the
corresponding neurotransmitter concentrations in the pyridoxine-deficient rats
Chronic catheterization using vascular-access-port in rats: blood sampling with minimal stress for plasma catecholamine determination
Chronic catheterization is illustrated using vascular-access-port model SLA where the port is surgically placed subcutaneously on
the back of the rat. The catheter is tunnelled to the neck and inserted into the jugular vein . Within 24 h rats showed normal blood
pressure and blood samples were collected at intervals with minimal stress to the animals . A comparison of the plasma catecholamine
of blood collected from vascular-access-ports with that obtained from decapitation indicates that there was minimal stress to the rats
when blood was drawn through the vascular-access-port
Enhancement Of High Affinity Y-Aminobutyric Acid Receptor Binding In Cerebellum Of Pyridoxine-Deficient Rat
The high-affinity of [3H]y-aminobutyric acid (GABA) to GABAA receptors and [3H]baclofen to
GABAB receptors were studied in the cerebellum of pyridoxine-deficient rats and compared to
pyridoxine-supplemented controls. There was a significant increase in the maximal binding ( Bmax) of both
GABAA and GABAB receptors with no significant difference in their binding affinities (Kd). The changes
observed suggest a supersensitivity of GABAA and GABAB receptors which seems to correlate negatively
with the concentration of GABA in the cerebellum of pyridoxine-deficient rats
- β¦