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Biomedical subjects

A Levitzki

Publications and source records attributed to A Levitzki.

At least 199 records · Page 11Linked to original sources
PubMed1978

Mode of coupling between the beta-adrenergic receptor and adenylate cyclase in turkey erythrocytes.

The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a proportional decrease in the rate of activation, but no change in the maximum level of activity was attained. Progressive inactivation of the enzyme by p-hydroxymercuribenzoate was found not to change the rate of activation nor the capacity of the receptor to bind hormone. Only the maximal level of activation was found to be decreased. These results are not compatible with the precoupled model of receptor and cyclase nor with floating receptor models in which an intermediate of hormone, receptor, and cyclase is in equilibrium with its reactants. The data strongly suggest that the collision coupling is the mode of coupling between the beta receptor and cyclase coupling in turkey erythrocyte membranes.

Adenylyl Cyclases↗
PubMed1978

Coupling of a single adenylate cyclase to two receptors: adenosine and catecholamine.

A detailed kinetic analysis on the rate of activation of adenylate cyclase by 1-epinephrine and by adenosine, separately and combined, was performed. Both ligands were found to induce the activation of adenylate cyclase to its permanently active state in the presence of guanylyl imidodiphosphate (GppNHp). The activation followed strictly first-order kinetics. On the basis of these experiments, it was found that all of the enzyme pool can be activated by the beta-adrenergic receptor, but only 60 to 70% of the enzyme can also be activated by an adenosine receptor. The remaining 30 to 40% cannot be activated by adenosine. While previous experiments have led us to conclude that the epinephrine receptor is uncoupled from the adenylate cyclase, it seems that the adenosine receptor is either precoupled to the enzyme or forms a long-lived intermediate of adenosing-receptor-enzyme complex. From the pattern of enzyme activation by the two ligands and GppNHp, it may be concluded that the two ligands, adenosine and the beta-agonist, activate the adenylate cyclase through a common guanyl nucleotide regulatory site. This assertion is supported by the finding that both adenosine and 1-epinephrine, in the presence of GTP, induce the reversal of the permanently active state, irrespective by which pathway the enzyme was activated.

Adenosine↗
PubMed1977

The allosteric inhibition by calcium of soluble and partially purified adenylate cyclase from turkey erythrocytes.

Adenylate cyclase from turkey erythrocyte membranes was solubilized in Lubrol-PX and partially purified (22-fold) by molecular sieve chromatography on Biogel A5M. The molecular weight of the enzyme was found to be 316000. The partially purified solubilized enzyme was found to retain all the kinetic and regulatory properties of the native membrane-bound enzyme except its sensitivity to beta-agonists. The enzyme responds to Mg2+ in a positively cooperative fashion, with a Hill coefficient of nH = 2.0. The enzyme is inhibited by Ca2+ in a positively cooperative fashion with a Hill coefficient of nH = 2.0. The calcium effect is only on the kcat of the reaction and not on the binding and kinetic parameters of the enzyme towards the other ligands such as MgATP and Mg2+. The Mn2+-supported adenylate cyclase is not inhibited by Ca2+ as was found for the native membrane-bound enzyme.

Adenylyl Cyclase Inhibitors↗
PubMed1977

Probing of beta-adrenergic receptors by novel fluorescent beta-adrenergic blockers.

The synthesis of two high-affinity fluorescent beta-adrenergic blockers is described: dl-N(1)-[2-hydroxy-3-(1-naphthyloxy)propyl]-N(2)-(9-acridyl)-1,2-propanediamine (9-aminoacridylpropanolol, 9-AAP) and dl-N-[2-hydroxy-3-(1-naphthyloxy)propyl]-N'-dansylethylenediamine (dansyl analogue of propranolol, DAPN). Both 9-AAP and DAPN inhibit competitively the l-epinephrine-dependent adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in turkey erythrocyte membranes without affecting the fluoride-stimulated adenylate cyclase activity. Similarly, 9-AAP and DAPN inhibit in a competitive manner the binding of [(125)I]-iodohydroxybenzylpindolol to these beta-adrenergic receptors. The two fluorescent beta-adrenergic blockers 9-AAP and DAPN probe specifically beta-adrenergic receptors in the central nervous system as well as in other organs when injected into rats. The fluorescence pattern can be monitored by fluorescence microscopy performed on cryostat slices of these organs. The appearance of the characteristic fluorescence pattern can be blocked in a stereospecific fashion by a prior injection of l-propranolol and not by a prior injection of d-propranolol. These compounds therefore offer a powerful means to map beta-adrenergic receptors in vivo. The stereospecific displacement of 9-AAP from the beta-adrenergic receptors of turkey erythrocyte membranes by l-propranolol and by l-epinephrine can be detected in vitro using front-face fluorescence. The potential use of these compounds to probe beta-receptors in vitro and in vivo is discussed.

Adenylyl Cyclase Inhibitors↗
PubMed1976

An analysis on the slope of Scatchard plots.

The Scatchard plot, [X]b/[E]t[X] versus [X]b/[EP]t where [X]b denotes the concentration of bound ligand, [E]t the total concentration of the binding protein and [X] the free ligand concentration, was designed originally for plotting data of ligand binding to a macromolecule possessing identical non-interacting ligand binding sites. However, the plot is used for describing cooperative binding processes. In such cases, the slope of the Scatchard plot is not then equal to minus the intrinsic association constant any more. The meaning of the slope in such cases is a complex function of the binding parameters and its exact interpretation depends on the particular model used to analyze the binding data. In this communication, the meaning of the slope of the Scatchard plot is analyzed in terms of the different allosteric models.

Carrier Proteins↗
PubMed1976

The refolding of lactate dehydrogenase subunits and their assembly to the functional tetramer.

The renaturation process of different lactate dehydrogenase isozymes (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from their unfolded subunits was investigated using a number of techniques. (a) kinetics of activity regain, (b) the kinetics of fluorescence change of fluoresecence change of the protein tryptophans, (c) kinetics of regain of the fluorescence properties of a covalently attached fluorescence probe (fluorescein) and (d) the kinetics of assembly, by following the intermediate oligomeric species appearing in the assembly pathway from monomers to tetramers. The results indicate that the unfolded polypeptide is converted to the active oligomeric species by the following scheme: Denatured subunit I leads to partially refolded subunit II leads to folded subunit III leads to dimer IV leads to tetramer. Step I and step II are first-order where step II is rate limiting. The ligands NAD+ and NADH accelerate step II, thus converting step I to the rate-limiting process. The fact that partially folded lactate dehydrogenase subunits are capable of co-enzyme binding may indicate the possible role of these ligands in the assembly of lactate dehydrogenase in vivo. Steps III and IV were found to be fast. The intermediate formation of an enzyme dimer which then dimerizes to the tetrameric species is found to be the major assembly pathway. Only a small portion of the lactate dehydrogenase tetramer is formed through the intermediate formation of a trimer intermediate.

Animals↗