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M Sokolovsky

Publications and source records attributed to M Sokolovsky.

At least 127 records · Page 7Linked to original sources
PubMed1983

Species differences determine azido phencyclidine labeling pattern in desensitized nicotinic acetylcholine receptors.

Acetylcholine receptor enriched membranes from Torpedo ocellata, Torpedo marmorata and Torpedo californica were studied using [3H] azido-phencyclidine (AZ-PCP). [3H]-PCP binding to receptors from all three species revealed marked similarities. Photoaffinity labeling by [3H]-AZ-PCP resulted in the tagging of mainly alpha, beta and delta subunits in all species. When carbamylcholine was added, it enhanced the labeling of beta subunits in T. ocellata, delta in T. marmorata and alpha in T. californica, suggesting species differences in the photolabeling pattern. Multiple homologous binding sites for PCP between the receptor subunits would allow small variations in receptor structure to be manifested in labeling by AZ-PCP, with no differences in binding and functional properties of the receptors.

Affinity Labels↗
PubMed1983

Characterization of the interaction of phencyclidine and its derivatives with the ionic channel of the nicotinic receptor.

(3H)-Phencyclidine (PCP) binds specifically to the cholinergic ionophore in synaptic membranes prepared from Torpedo electric organ. Experiments performed by the centrifugation method establish that the binding is saturable, reversible and selective and can be characterized by a single dissociation constant (3.6 +/- 1.8 microM). The maximal binding capacity is 600 +/- 150 pmol/mg of membrane protein. Bound (3H)-PCP can be displaced by unlabelled PCP and a series of its derivatives. The reactivity of PCP derivatives in binding to (3H)-PCP binding sites, as related to structural changes at the phenyl, piperidyl and cyclohexyl moieties, is discussed.

Animals↗
PubMed1983

Metal ion effects on target sites of modification in metallocarboxypeptidase B.

Native carboxypeptidase B and its Co2+-substituted derivative were oxidized by the active-site-directed agent m-chloroperbenzoic acid. The following results were obtained a) In the cobalt enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Peptide or ester pseudo-substrates protected the cobalt enzyme but not the zinc enzyme against inactivation. b) Upon oxidation and formation of Co3+, cleavage of peptide bonds occurred in the cobalt enzyme but not in the zinc enzyme. Both enzymes retained their original metal content. c) Following oxidation of the enzymes, amino acid analysis revealed a modification of a methionyl residue in the zinc enzyme only; the cobalt enzyme, on the other hand, showed a modification of a histidyl residue. d) Peptide mapping of the enzymes after cleavage by cyanogen bromide indicated that two methionyl peptides were missing in the oxidized zinc enzyme. These peptides point to Met-64 as the site of modification. The peptide map of the oxidized cobalt enzyme was similar to that of the unmodified native (i.e., zinc) enzyme. These studies indicate that the specific metal ion present in the enzyme imposes certain structural and functional differences on the active site, leading to differing reactivities of specific amino acid residues and to a different alignment of the active-site-directed reagent in the two enzymes.

Amino Acids↗
PubMed1983

Oligomeric structure of muscarinic receptors is shown by photoaffinity labeling: subunit assembly may explain high- and low-affinity agonist states.

The potent muscarinic photoaffinity reagent N-methyl-4-piperidyl p-azidobenzilate (azido-4NMPB) was used to covalently label specific muscarinic binding sites in various brain regions and in the heart. In the cortex and hippocampus, a single specifically labeled protein with an apparent molecular mass of 86,000 daltons was detected by gel electrophoresis. In the medulla pons, cerebellum, and cardiac atria, there was a 160,000-dalton band in addition to the 86,000-dalton polypeptide. Under certain conditions, alkali or hydroxylamine treatment dissociated both macromolecules into a single 40,000-dalton polypeptide. These results suggest that the muscarinic receptor exists in oligomeric forms and that a dimer and tetramer of a basic 40,000-dalton peptide may exist as interconvertible species. We propose a model to explain the biological architecture of the muscarinic receptors and suggest a possible correlation between the azido-4NMPB-labeled polypeptides and the two states of the receptor observed in agonist binding experiments.

Affinity Labels↗
PubMed1983

Muscarinic antagonists induce different receptor conformations in rat adenohypophysis.

We have employed a method based on ligand competition experiments, which is capable of detecting interactions among ligand-occupied binding sites, to study the interactions between rat adenohypophysis muscarinic receptors occupied by several muscarinic antagonists. In this method, one examines the binding of a labeled ligand (the primary ligand) in the absence and presence of a competing ligand. The inhibition of binding of the primary ligand by the competing ligand shows significant deviations from that expected assuming a population of noninteracting, heterogeneous binding sites. The deviations seen in the case of competition between N-methyl-4-piperidyl benzilate (4NMPB) and (-)-N-methyl scopolamine (a benzilate and tropate) are more pronounced than in the case of 4NMPB and (-)-3-quinuclidinyl benzilate (two benzilate derivatives). The occurrence of such deviations suggests the existence of site-site interactions among rat adenohypophysis muscarinic receptors. On the other hand, no deviations were observed in competition experiments in homogenates of rat cortex and medulla-pons. This finding correlates with the linear Scatchard plots (with no indications for site-site interactions or heterogeneity) obtained for the binding of muscarinic antagonists in these brain regions. A mathematical analysis demonstrates that the deviations from the expectations of the site-heterogeneity model observed in the rat adenohypophysis system (which shows similar binding patterns for all ligands employed) can occur only if the primary and competing ligands induce different conformational transitions upon binding to the receptor. It is concluded that different muscarinic antagonists can lead to different isomerization states of the receptor in the system.

Allosteric Regulation↗
PubMed1982

Affinity labeling of muscarinic receptors in rat cerebral cortex with a photolabile antagonist.

Highly potent photoaffinity probes for muscarinic binding sites were prepared by the incorporation of an azido group into the benzilic acid moiety in two compound, 3-quinuclidinyl benzilate (3QNB) and N-methyl-4-piperidyl benzilate (4NMPB). Inactivation of muscarinic sites in rat cortex depends on the formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes. During photolysis, radiolabeled azido-4NMPB interacted specifically and with high affinity (Kd = 1.06 nM) with the muscarinic receptors, and the ligand could be covalently incorporated into a macromolecule of about 86,000 Mr, presumably the muscarinic receptor. The incorporation was almost stoichiometric when compared to determination of receptor density by reversible ligands. Atropine (10 microM) afforded specific protection (greater than 83%) of the receptor against inactivation by azido-[3H]4NMPB. This compound and the other ligands described here (i.e., amino-4NMPB, amino-3QNB, and azido-3QNB) represent powerful potential probes for the biochemical isolation and characterization of muscarinic receptors.

Affinity Labels↗
PubMed1982

Mechanistic implications of cyanide binding to carboxypeptidase B.

The putative metal coordinating ligand cyanide was used to study the effects of modifications of the metal coordination sphere on the spectral properties and catalytic activity of cobalt and zinc carboxypeptidases. The absorption spectra of Co2+-carboxypeptidase B in the presence of cyanide pointed to a direct interaction of the ligands with the metal. Gel-filtration experiments showed that the binding of one mole of ligand per mole of enzyme metal ion resulted in maximal spectral effects. Binding of cyanide to the metal ion as measured by absorption spectroscopy was inhibited by acetyl-L-arginine, a peptide pseudosubstrate, and by acetyl-D-arginine, a competitive peptide inhibitor. Addition of acetyl arginine to the enzyme-cyanide complex caused displacement of the ligand, as evidenced by the spectral parameters. Cyanide inhibited peptide hydrolysis in a partially noncompetitive manner, i.e. it did not prevent binding of the substrate to the enzyme but the enzyme-substrate-cyanide complex was hydrolyzed at a slower rate than the enzyme-substrate complex. The dissociation constant evaluated from kinetic studies for the binding of cyanide to Co2+-carboxypeptidase B was in good agreement with that obtained from spectral measurements. Hydrolysis of the ester analog of the basis peptide substrate was not affected by cyanide. Based on these data a model is proposed in which the peptide carboxyl group displaces the water molecule from the metal coordination sphere during catalysis without increasing the coordination number.

Animals↗
PubMed1982

Tyrosyl interactions at the active site of carboxypeptidase B.

The phosphorescence emission spectra of native carboxypeptidase B and of chemically modified carboxypeptidase B (at arginyl residues) was measured in the presence and absence of peptide and ester substrates (acetyl-L-arginine and its hydroxy ester analog: acetyl-L-argininic acid). Ester binding did not affect the state of the tyrosyl residue as compared with its state in the substrate-free enzyme. In the modified enzyme, which is devoid of peptidase activity, binding of the peptide pseudosubstrate did not perturb the state of the tyrosyl residue. The luminescence spectra of Zn2+- and Co2+-carboxypeptidase B in the presence of the metal coordinating ligand cyanide, used to displace the water from the metal coordination sphere, is also described. Cyanide did not affect the luminescence spectra of the active-site tyrosyl residue in either Zn2+- or Co2+-carboxypeptidase, indicating that the tyrosyl residue was not interacting directly with the metal bound water. Hence, the effect of peptide on tyrosyl phosphorescence is not caused by the displacement of the tyrosyl from the coordination sphere, but rather by direct interaction of the peptide bond. The data are consistent with the proposition that the tyrosyl residue participates as a proton donor in amide but not in ester hydrolysis.

Animals↗
PubMed1982

Muscarinic mechanisms and sex hormone secretion in rat adenohypophysis and preoptic area.

The highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate [(3H)-4NMPB] was used in direct binding and in competition experiments with the muscarinic agonist oxotremorine to investigate the influence of in vivo endocrine manipulations involving estrogen on muscarinic receptors in the adenohypophysis and hypothalamus. In the adenohypophysis the characteristics of antagonist binding in ovariectomized female rats, like those in androgenized females, resembled those in normal male rats; this trend was reversed in ovariectomized females after implantation of 17 beta-estradiol capsules, with the characteristics of antagonist binding now resembling those in normal female rats at estrus. Agonist binding characteristics also showed some distinct differences between treated and normal female rats: the proportion of high affinity binding sites decreased both in ovariectomized and androgenized rats, while the affinity of these sites became greater as compared to control animals. The results clearly show that changes in the levels of sex steroids, as a result of the above-mentioned endocrine manipulations, influence the behavior of the muscarinic receptors in both areas, and suggest muscarinic participation in the regulation of gonadotropin release in the hypothalamus-pituitary-ovary axis.

Animals↗
PubMed1982

Cooperativity pattern in the interaction of the antiestrogen drug clomiphene with the Muscarinic receptors.

The possible interaction between the muscarinic receptors and the antiestrogenic drug clomiphene citrate was investigated in competition experiments using the highly specific tritiated antagonist N-methyl-4-piperidyl benzilate (4-NMPB) in various regions of rat brain. It was found that clomiphene can displace muscarinic antagonists from their receptor-ligand complexes. Binding analyses as well as determinations of the kinetics of dissociation of [3H]4-NMPB-receptor complexes indicate the binding of more than one molecule of clomiphene in a positively cooperative pattern. These findings suggest that the nonsteroidal antiestrogenic drugs, e.g., clomiphene, might exert their effects not only through the specific estrogen receptor but also in conjunction with the muscarinic system.

Allosteric Regulation↗