Biomedical subjects

M R Tota

Publications and source records attributed to M R Tota.

22 records · Page 2Linked to original sources

PubMed1990

Partial agonist effects on the interaction between the atrial muscarinic receptor and the inhibitory guanine nucleotide-binding protein in a reconstituted system.

The mechanism of action of the partial muscarinic agonist pilocarpine was analyzed in a reconstituted system consisting of the purified porcine atrial muscarinic receptor and the purified porcine atrial inhibitory guanine nucleotide-binding protein, Gi. When GTPase activity was measured as a function of receptor.agonist complex concentration at saturating concentrations of either the full agonist carbachol or pilocarpine, both ligands gave similar values of kcat (4.3 +/- 0.2 min-1 for carbachol and 5.4 +/- 0.7 min-1 for pilocarpine); however, the observed dissociation constant for the ligand.receptor complex binding to Gi was about 4-fold lower for carbachol (0.81 +/- 0.19 nM) than for pilocarpine (3.02 +/- 0.83 nM). These results suggested that, in this system, the reduced activity of the partial agonist compared with the full agonist was the result of a decrease in affinity of the receptor.ligand complex for Gi, as opposed to differences in their relative abilities to activate the guanine nucleotide-binding protein. Several analogues of oxotremorine were also tested to determine their effects on the GTPase activity of Gi. Results from these studies indicate that the reconstituted system may be useful in determining structure-function relationships for muscarinic agonists with regard to receptor.Gi interactions.

Animals↗

PubMed1987

Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein.

Purified porcine atrial muscarinic receptor (mAcChR) was reconstituted with purified porcine atrial inhibitory guanine nucleotide binding protein (Gi) in a lipid mixture consisting of phosphatidylcholine, phosphatidylserine, and cholesterol (1:1:0.1 w/w). 5'-Guanylyl imidodiphosphate (0.1 mM) had no effect on the binding of the muscarinic antagonist L-quinuclidinyl benzilate but converted high-affinity carbachol binding sites (Kd equal to 1 microM) in the reconstituted preparation to the low-affinity state (Kd equal to about 100 microM). Steady-state kinetic measurements of GTPase activity showed that the turnover number was increased from 0.19 min-1 in the presence of the muscarinic antagonist L-hyoscyamine to 2.11 min-1 for the agonist carbachol. The affinity of Gi for GDP was reduced by about 50-fold upon interaction with the carbachol-mAcChR complex, and the observed rate constant for GDP dissociation was increased by 38-fold from 0.12 to 4.5 min-1. Thus, the increase in steady-state GTPase activity observed for muscarinic agonists is largely, if not exclusively, due to the increase in GDP dissociation from Gi--probably the rate-limiting step in the steady-state mechanism. Carbachol-stimulated GTPase was sensitive to ADP-ribosylation of the reconstituted Gi by pertussis toxin, but the high-affinity agonist binding was uncoupled only when the reconstituted preparation was treated with pertussis toxin in the presence of GTP and the agonist acetylcholine. These results suggest that association with the mAcChR protects Gi from ADP-ribosylation by pertussis toxin.

Adenosine Diphosphate Ribose↗

PubMed1987

Phosphorylation of the porcine atrial muscarinic acetylcholine receptor by cyclic AMP dependent protein kinase.

cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, smooth muscle myosin light-chain kinase, and phosphorylase kinase were examined with respect to their ability to phosphorylate porcine atrial muscarinic receptors (mAcChRs). Experiments were performed both in detergent solution and in a reconstituted system containing the mAcChR alone or in the presence of the purified porcine atrial inhibitor guanine nucleotide binding protein (Gi). Only cAMP-dependent protein kinase was capable of phosphorylating the receptor under any of the experimental conditions examined. Phosphorylation of the mAcChR in the detergent-solubilized state resulted in a loss of ligand binding sites that was reversible upon treatment with calcineurin in the presence of calcium and calmodulin. Upon reconstitution, the apparent stoichiometry of phosphorylation was increased by about 15-fold. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of Gi, suggesting that the large agonist-stimulated increase in phosphorylation observed in vivo [Kwatra, M. M., & Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432] may in part result from a unique receptor conformation that occurs upon association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro. Carbachol-induced stimulation of the GTPase activity of Gi in the reconstituted system was also unaffected by phosphorylation.

Animals↗

PubMedDate not supplied

Purification and reconstitution of a recombinant human neurokinin-1 receptor.

Recombinant human neurokinin-1 receptors expressed in insect cells have been purified to near homogeneity by sequential metal-chelating chromatography and gel filtration chromatography. The purified receptor consists of a single polypeptide with an apparent molecular weight of 50 kD as revealed by SDS gel electrophoresis, and exhibits a specific activity of 19 nmol of L-703,606 bound per mg of protein. Immunoblot experiments further confirm the identity of the stained protein band. The purified receptor binds the antagonist L-703,606 with an affinity similar to that of native human neurokinin-1 receptor, and binds the agonist substance P with an affinity similar to that of the low affinity state of uncoupled native receptor. The purified receptor can be reconstituted with membranes from uninfected insect cells, and the reconstitution results in an increased affinity for substance P, consistent with the reappearance of the high affinity state of the receptor for agonist in the presence of endogenous G proteins. These data indicate that the purified neurokinin-1 receptor is functional with respect to agonist and antagonist binding and G protein coupling.

Amino Acid Sequence↗