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At least 577 records · Page 32Linked to original sources
PubMed1987

Affinity labels for beta-adrenoceptors: preparation and properties of alkylating beta-blockers derived from indole.

New alkylating ligands derived from indole with high affinity for beta-adrenoceptors were synthesized and their properties examined. N8-(Bromoacetyl)-N1-[3-(4-indolyloxy)-2-hydroxypropyl]-(Z)-1,8-dia mino-p- menthane (8) and its N1,N8 isomer (9) were prepared by the reaction of bromoacetyl bromide with a product of the condensation of 4-indolyl glycidyl ether with (Z)-1,8-diamino-p-menthane. A similar reaction employing 2-cyano-4-indolyl glycidyl ether yielded the respective cyano derivatives 10 and 11. Apparent affinities (Ki, M) for beta-adrenoceptors on membrane preparations from rat heart and lung were 4.6 X 10(-10) and 1.34 X 10(-9) for 8, 2.3 X 10(-8) and 4.5 X 10(-9) for 9, 6.1 X 10(-10) and 1.49 X 10(-9) for 10, and 1.83 X 10(-9) and 2.78 X 10(-9) for 11, respectively. When membranes were preincubated with the above ligands (1 X 10(-8) M, 30 min, 30 degrees C) and then washed extensively, reduction in the concentration of specific binding sites of [3H]dihydroalprenolol ranged from 7% to 76% and there was no change in KD of the remaining binding sites. (+/-)-Alprenolol and (-)-isoproterenol, but not (+)-isoproterenol, when included with the alkylating ligands in the preincubation mixtures, prevented the reduction in concentration of [3H]dihydroalprenolol binding sites. Compounds 8-11 alone did not stimulate adenylate cyclase activity in rat heart homogenates. However, these compounds inhibited (-)-isoproterenol-stimulated adenylate cyclase activity with Ki values ranging between 5 X 10(-9) and 60 X 10(-9) M. These results suggest that high-affinity irreversible beta-adrenergic antagonists were obtained that may be useful for in vivo studies of beta-adrenoceptors.

Adenylyl Cyclases↗
PubMed1991

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.

Affinity Labels↗
PubMedDate not supplied

[Adenosine- and ethenoadenosine-5'-trimetaphosphates: the effect of covalent bond formation on the state of the affinity label in the complex with phenylalanyl-tRNA-synthetase].

epsilon ATP is a substrate of phenylalanyl-tRNA synthetase and epsilon Ado is a competitive inhibitor of ATP in the reaction of tRNA aminoacylation (Ki = 1.6 mM). The association of phenylalanyl-tRNA synthetase with ATP or Ado results in synergistic binding of phenylalaninol and phenylalanine, respectively. However neither epsilon ATP nor epsilon Ado exhibit synergism. Adenosine- and ethenoadenosine-5'-trimethaphosphates are shown to be similar affinity reagents of phenylalanyl-tRNA synthetase. ATP being covalently bound to the enzyme shows essentially lower synergistic effect in comparison with free ATP. epsilon ATP-label is practically insensitive to the ligands namely ATP, Phe, phenylalaninol and is highly accessible for I- ions. The scheme of behaviour of affinity labels is assumed to be as follows: a) the formation of specific reagent-enzyme complex, b) the covalent attachment of the reagent to the enzyme, c) the covalent binding induced disruption of the specific complex formed before.

Adenosine Triphosphate↗
PubMed1990

Structural arrangement of the decoding site of Escherichia coli ribosomes as revealed from the data on affinity labelling of ribosomes by analogs of mRNA--derivatives of oligoribonucleotides.

Using derivatives of oligoribonucleotides bearing an active group at the 5'- or 3'-end, the affinity modification of Escherichia coli ribosomes has been investigated in model complexes imitating various steps of initiation and elongation with a different extent of approximation to the real protein-synthesizing system. The protein environment of the ribosome decoding site is determined. The S3, S4, S9, L2, L7/L12 proteins belong to the 5'-region of the decoding site, and the S5, S7, S9, L1, L16 proteins to the 3'-region. In the process of translation the template moves along the external side of the 30 S subunit, from the L1 ridge to the L7/L12 stalk. The structural arrangement of the decoding site or its nearest environment depends on the functional state of ribosomes in the process of translation.

Affinity Labels↗
PubMed1986

Affinity-labeling steroids as biologically active probes of antiglucocorticoid hormone action.

The role of the glucocorticoid receptor in the expression of antiglucocorticoid action has been investigated with a chemically-reactive derivative of three glucocorticoid steroids with differing biological potencies, i.e. the C-21 mesylates of cortisol, dexamethasone and deacylcortivazol. Dexamethasone 21-mesylate (Dex-Mes) was the most useful derivative due to its favorable balance of high receptor affinity and predominantly irreversible antiglucocorticoid activity. A number of criteria have been used to conclude that [3H]Dex-Mes covalently labels glucocorticoid receptors in the steroid-binding cavity. The available data indicate that covalent Dex-Mes-labeled receptors (mol. wt approximately equal to 98,000) are responsible for the irreversible antiglucocorticoid activity while the partial agonist activity of Dex-Mes is due to non-covalent Dex-Mes-bound receptors. Further support for this hypothesis comes from the observations that deacylcortivazol 21-mesylate was a full glucocorticoid and did not affinity label receptors (and marginally labeled cytosol proteins) although it was capable of covalently-labeling bovine serum albumin. Several mechanisms for the expression of irreversible antiglucocorticoid activity by covalent Dex-Mes-labeled receptors were examined and can be eliminated. Covalent receptor-Dex-Mes complexes formed in whole HTC cells were found to have a decreased capacity for nuclear binding. This decreased nuclear-binding capacity could be responsible for the whole-cell irreversible antiglucocorticoid activity of Dex-Mes.

Affinity Labels↗
PubMed1993

Affinity labeling of bovine brain protein kinase C by tosyl lysyl chloromethane. A kinetic study.

The kinetics of inactivation of bovine brain protein kinase C (PKC) by N alpha-p-tosyl L-lysyl chloromethane (TosLysCH2Cl) were investigated. In absence of activators PKC gave non-linear semilog inactivation plots. At each reagent concentration a plateau of residual activity was reached after some time; its value was inversely proportional to TosLysCH2Cl concentration but the plateau was not due to inactivator depletion. On the other hand, in the presence of Ca2+, phosphatidylserine and phorbol 12-myristate 13-acetate, the activity loss followed saturation kinetics, with k(inact) = 0.6 x 10(-3) s-1 and Kinact = 1.9 mM. The study of protection effects by ATP Mg2+ and histone required the presence of 50% glycerol in the incubation mixtures, otherwise the controls (kinase in the presence of activators and ATP Mg2+ or histone) rapidly lost activity. In the presence of 50% glycerol, the inactivation parameters were somewhat altered (k(inact) = 0.3 x 10(-3) s-1 and K(inact) = 0.2 mM); ATP Mg2+ proved to afford a mixed competitive-non competitive protection effect, while histone protected in a competitive manner with a Kp of 0.06 microgram/ml. In the presence and the absence of glycerol, plots of log k(obs) versus log inactivator concentration yielded straight lines with slopes of 0.7-0.9, indicating that 1 mol of reagent is sufficient for inactivation. The results described in this paper suggest that the reagent TosLysCH2Cl hits the catalytic domain of activated PKC at the active site, which is not available in resting PKC; in non-activated PKC, the labeling site would be different.

Adenosine Triphosphate↗