Search PubMed⌕ Search

Biomedical subjects

Y Kloog

Publications and source records attributed to Y Kloog.

At least 109 records · Page 6Linked to original sources
PubMed1984

Recognition of the muscarinic receptor by its endogenous neurotransmitter: binding of [3H]acetylcholine and its modulation by transition metal ions and guanine nucleotides.

Agonist binding to the muscarinic receptor in rat cerebral cortex membranes was studied by using the neurotransmitter itself, [3H]acetylcholine [( 3H]AcCho). By using 10 microM atropine or oxotremorine to define specific binding, it was possible to demonstrate specific binding of [3H]AcCho that was sensitive to muscarinic but not to nicotinic ligands. Equilibrium binding experiments with 5-240 nM [3H]AcCho indicated specific binding of the ligand to a saturable population of muscarinic receptors (361 +/- 29 fmol/mg of protein; Kd = 76 +/- 17 nM). This value represented 25% of the available binding sites for a labeled antagonist in the same preparation and corresponds to the proportion of high-affinity agonist binding sites observed previously in competition experiments with labeled antagonists. Inclusion of transition metal ions (e.g., 2 mM Ni2+) in the assay increased the equilibrium binding of [3H]AcCho (628 +/- 38 fmol/mg of protein, Kd = 86 +/- 21 nM) but did not affect equilibrium binding of 3H-labeled antagonists, indicating conversion of low- into high-affinity muscarinic agonist binding sites. The increase developed slowly over 30 min of incubation at 25 degrees C but could be reversed rapidly (approximately equal to 2 min) by the chelating agent EDTA or by guanine nucleotides. These data directly reveal a slow though quickly reversible interconversion of low- into high-affinity muscarinic agonist binding sites.

Acetylcholine↗
PubMed1984

Localization of phencyclidine binding sites on alpha and beta subunits of the nicotinic acetylcholine receptor from Torpedo ocellata electric organ using azido phencyclidine.

A photolabile derivative of phencycliding (PCP), azido phencyclidine (AZ-PCP), was synthesized and used to localize PCP binding sites on the acetylcholine receptor from Torpedo ocellata electric organ. In the dark, the binding of micromolar concentrations of [3H]AZ-PCP to a receptor-enriched membrane preparation fits a single dissociation constant (Kd = 2.65 microM) and is very similar to the binding of [3H]PCP. The agonist carbamylcholine increases the association rate (and the affinity) of these ligands to the receptor, but it does not alter the total number of available binding sites. Following UV irradiation and gel electrophoresis, [3H]AZ-PCP was found to label specifically the alpha and beta subunits of the receptor. The labeling of the alpha subunit band was heavier, and it was inhibited by tetracaine and PCP but not by alpha-bungarotoxin (alpha-Bgt). The addition of carbamylcholine enhanced the labeling of the beta subunit; this effect was diminished by alpha-Bgt. The labeling of the beta subunit was also inhibited by tetracaine and PCP. The effect of carbamylcholine, which binds to the alpha subunit, could be the result of an induced conformational change, which is propagated to the beta subunit and increases its labeling by [3H]AZ-PCP. A simple model which accommodates the binding and photoaffinity labeling data is described. According to the model, the high affinity PCP binding site is located between the alpha and beta receptor subunits, and the drug thus becomes attached simultaneously to both. Hypothetical overlapping recognition sites for PCP on these receptor subunits would allow binding (and labeling) with increased affinity in the presence of carbamylcholine with no increase in the number of available sites.

Affinity Labels↗
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

Protein carboxylmethylation in intact rat posterior pituitary lobes in vitro.

Methylation of proteins was studied in intact rat posterior pituitary lobes in vitro. Posterior pituitary lobes can catalyze the formation of protein carboxylmethyl esters using methionine as a precursor. Methylation inhibitors inhibited formation of protein carboxylmethyl esters, indicating that the reaction proceeded via an enzymatic methylation pathway. At least six proteins were methylated in the intact lobe. Among these are the neurophysins as identified by their characteristic properties: molecular weight, [35S]cysteine labeling, disappearance after salt loading or pituitary stalk section, and low levels in the homozygous Brattleboro rat. Other still unidentified proteins of molecular weight lower than 21,000 were also methylated. Their disappearance after pituitary section and salt loading indicates that, like the neurophysins, these proteins are localized within the hypothalamoneuro-hypophysial axon terminals.

Animals↗
PubMed1983

Protein carboxyl methylation increases in parallel with differentiation of neuroblastoma cells.

Cells of mouse neuroblastoma clone N1E-115 in the confluent phase of growth can catalyze the formation of endogenous protein carboxyl methyl esters, using a protein carboxyl methylase and membrane-bound methyl acceptor proteins. The enzyme is localized predominantly in the cytosol of the cells and has a molecular weight of about 20,000 daltons. Treatment of the cells with dimethylsulfoxide (DMSO) or hexamethylene-bisacetamide (HMBA), agents that induce morphological and electrophysiological differentiation, results in a marked increase in protein carboxyl methylase activity. Maximal levels are reached 6-7 days after exposure to the agents, a time course that closely parallels the development of electrical excitability mechanisms in these cells. Serum deprivation also causes neurite outgrowth but does not enhance electrical excitability or enzyme activity. The capacity of membrane-bound neuroblastoma protein(s) to be carboxyl methylated is increased by the differentiation procedures that have been examined. However, the increase in methyl acceptor proteins induced by DMSO or HMBA is the largest, and its time course parallels electrophysiological differentiation. In contrast, serum deprivation induced a small increase that reached maximal levels within 24 h. The data suggest that increased protein carboxyl methylation is a developmentally regulated property of neuroblastoma cells and that at least two groups of methyl acceptor proteins are induced during differentiation: a minor group related to morphological differentiation, and a major group that may be related to ionic permeability mechanisms of the excitable membrane.

Animals↗
PubMed1983

High-protein carboxymethylase activity and low endogenous methyl acceptor proteins in posterior pituitary lobe of rats lacking neurophysin-vasopressin (Brattleboro rats).

The activity of protein carboxymethylase and the endogenous protein methyl acceptor capacity were examined in the posterior, intermediate, and anterior lobes of the pituitaries of homozygous Brattleboro rats with diabetes insipidus and in heterozygous Brattleboro and Long-Evans control rats. Protein carboxyl methylation is selectively altered in the posterior pituitary lobes of homozygous Brattleboro rats. Protein carboxymethylase activity is higher (+40%) and endogenous methyl acceptor protein capacity is lower (-80%) with respect to heterozygous Brattleboro and Long-Evans control rats. This latter change is correlated with decreased methylation of proteins of a molecular weight of approximately 11K daltons, is selective for the posterior pituitary lobe, since it does not occur in the intermediate and anterior lobes, and probably reflects the absence of vasopressin-associated neurophysin in homozygous Brattleboro rats. Our results support a physiological role of protein carboxyl methylation in the neurosecretory process in the posterior pituitary gland.

Animals↗
PubMed1982

Enzymatic methylation of the membrane-bound nicotinic acetylcholine receptor.

The acetylcholine receptor of the Torpedo electric organ acts as substrate for the enzyme protein carboxyl methyltransferase within receptor-enriched membranes and after it is purified. In right-side-out receptor-enriched vesicles, protein of the membrane. All four receptor subunits were found to be methylated from the inside as well outside of the membrane vesicles. The higher molecular weight subunits were found to be methylated to a greater degree than the lower molecular weight subunits.

Animals↗
PubMed1982

Nonpolar lipid methylation-identification of nonpolar methylated products synthesized by rat basophilic leukemia cells, retina and parotid.

Incorporation of radioactivity from [3H- or 14C-methyl] methionine into nonpolar lipids had been investigated in rat basophilic leukemia (RBL) cells, retina, and rat parotid gland. These nonpolar methylated lipids were extracted into heptane and characterized by thin-layer chromatography, high performance liquid chromatography, gas chromatography, and mass spectrometry. The major methylated nonpolar lipid product in the RBL cells themselves was ubiquinone-9, which accounted for about 90% of the nonpolar lipid and 20-30% of the total radioactive lipid formed. There was a modest increase in the methylation of nonpolar lipids upon stimulation of the RBL cells with IgE and anti-IgE, but the significance of this change is uncertain. In contrast to whole cells, RBL membrane fractions (incubated with [3H-methyl]-S-adenosylmethionine) incorporated radioactivity primarily into fatty acid methyl esters and not ubiquinone. A third product, 2-(methylthio)-benzothiazole, was formed by RBL cells, retina and minced parotid upon incubation in enriched media. This product was formed enzymatically, apparently by the known enzyme S-thiolmethyltransferase, using the thiol substrate which contaminates these media. Evidence suggest that the enzyme may reside, at least in part, on the surface of the cells.

Animals↗
PubMed1981

Nonpolar lipid methylation. Biosynthesis of fatty acid methyl esters by rat lung membranes using S-adenosylmethionine.

Fatty acid methyl esters are the major radioactive lipid products obtained after incubation of rat lung membranes with [methyl-3H or 14C]S-adenosylmethionine. Evidence which suggests an enzymatic transmethylation includes: time and protein dependence, lack of reaction at 0 degrees C or with heat-denatured membranes, an apparent affinity for S-adenosylmethionine of about 1 microM, inhibition by S-adenosylhomocysteine, and lack of inhibition by 0.1% methanol. Activity was highest in microsomes but present in other membranous fractions. Endogenous activity was highest in membranes from parotid, lung, and pancreas. Products were analyzed by organic solvent extraction, thin layer chromatography, column chromatography, high performance liquid chromatography, and gas chromatography. Identification of methylpalmitate, methylstearate, methyloleate, and methyllinoleate was confirmed by mass spectrometry. Presence of the radioactive methyl group was demonstrated by the variation of isotopic ratios with specific activity. Addition of oleate to incubation mixture increased the rate of product formation and preincubation experiments suggested the absence of long lived intermediates. The data suggest an enzymatic transfer of methyl groups from S-adenosylmethionine to free fatty acids.

Animals↗
PubMed1981

Altered ontogenesis of muscarinic cholinergic receptor in mouse brain: effect of L-thyroxine and betamethasone.

The effects of L-T4 and betamethasone treatment of newborn mice on the development of the cholinergic muscarinic receptor in certain brain regions was studied using the potent labeled muscarinic antagonist [3H]4-N-methyl-piperidyl benzilate. Treatment with both L-T4 and betamethasone caused an accelerated accumulation of muscarinic receptors in the cortex 16 days post partum, with a subsequent reduction in level at 30 days. In the cerebellum and caudate putamen, only betamethasone caused a similar early accumulation of muscarinic receptors, while the later effect, namely a reduction in the level at 30 days, was seen with both hormones in these two regions as well as in the hippocampus. The results can explain some behavioral effects observed in other studies after treatment with these hormones.

Aging↗
PubMed1980

Characterization of the presynaptic muscarinic receptor in synaptosomes of Torpedo electric organ by means of kinetic and equilibrium binding studies.

The ligand binding properties of presynaptic muscarinic receptors present in purified synaptosomal fraction isolated from the electric organ of Torpedo have been studied, using the specific tritiated antagonist N-methyl-4-piperidylbenzilate ([3H]-4NMPB). Direct and competition binding studies revealed that antagonists bind with high affinity to the presynaptic receptor, with saturability occurring at very low ligand concentration, in a stereospecific manner, and according to a simple mass action law. The kinetic results for [3H]4NMPB binding could best be fitted by a two-step sequential isomerization model. The affinities of both agonists and antagonists decreased as a function of increasing ionic strength. Analysis of agonist binding was performed according to the two-site model suggested by Birdsall and Hulme. Varying the buffer conditions resulted in changes both in the affinities of the agonist for the two agonist sites and in their relative proportions. For a given buffer composition the proportion of high and low affinity sites is constant, but agonists affinities decrease as a function of increase in ionic strength. The finding that at a given ionic strength the proportion of high and low affinity agonist sites can be manipulated suggests that agonist binding sites in Torpedo receptor may interconvert. The apparent dissociation constants measured in Torpedo physiological buffer for both agonists and antagonists are in the same range as the concentrations affecting synaptosomal ACh release. The affinity of agonists towards the presynaptic muscarinic Torpedo receptor was found to be higher than their affinity towards the postsynaptic muscarinic receptor, e.g. in cat and rabbit irides, mouse cortex, etc., and closely resembles the affinity in mouse medulla pons. The localization of pre- and postsynaptic muscarinic receptors in view of their different binding properties is discussed.

Acetylcholine↗
PubMed1980

Biochemical characterization of muscarinic receptors.

Characteristics of the binding mechanism to specific muscarinic sites were determined by means of high affinity binding of tritium labeled N-methyl-4-piperidylbenzilate (4-NMPB) to homogenate from various regions of mouse brain: cortex, caudate putamen, thalamus, hippocampus, medulla pons and cerebellum. In vivo experiments confirm results obtained when investigating regional distribution of the muscarinic receptors in vitro. Kinetics as well as equilibrium analysis of the binding in the different brain regions to probe the nature of the binding is described. The combined data strongly support the conclusion that mouse brain exhibits functional heterogeneity of muscarinic receptors.

Animals↗
PubMed1980

On the regulation of acetylcholine release: a study utilizing Torpedo synaptosomes and synaptic vesicles.

1. Addition of ATP to isolated highly purified Torpedo synaptic vesicles results in 45Ca2+ uptake. 2. Ca2+ dependent ACh release from Torpedo synaptosomes is accompanied by the phosphorylation of a specific protein with an apparent subunit molecular weight of about 100,000 (band alpha). 3. Activation of the presynaptic muscarinic receptors by an agonist inhibits Ca2+-dependent ACh release from Torpedo synaptosomes. This process seems to be mediated through an interference with the phosphorylation of the band alpha protein, and not by blocking the voltage-dependent presynaptic Ca2+ channel.

Acetylcholine↗