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A Levitzki

Publications and source records attributed to A Levitzki.

At least 163 records · Page 9Linked to original sources
PubMed1983

Reconstitution of a functional beta-adrenergic receptor using cholate and a novel method for its functional assay.

Solubilization of purified turkey erythrocyte membranes at increasing cholate to protein ratios and in the presence of salt, extracts up to 20% of the beta-adrenergic receptor together with the GTP stimulatory protein (Ns) of adenylate cyclase. Upon removal of the cholate, by active absorption on Bio-beads, the functional interaction between the beta-receptor and the GTP regulatory protein Ns is quantitatively restored. The receptor (R) in the presence of l-isoproterenol and p[NH]ppG is able to catalyze the activation of Ns to its permanently active state, N's p[NH]ppG, with a rate constant (kon) identical to that of the native membrane. Reconstitution of the R/Ns mixture using poly(ethyleneglycol)-6000 restores the receptor binding properties as effectively as SM-2 Bio-beads. Unlike SM-2 Bio-beads, however, poly(ethyleneglycol) is not as efficient in restoring the R to Ns functional coupling. In this communication we also report on the ability to monitor quantitatively N's . p[NH]ppG, using native turkey erythrocyte membranes in the presence of Lubrol-PX as the source of the catalytic unit (c) of adenylate cyclase. The latter method is as efficient as using S49 AC- lymphoma cell membranes but much less expensive. Using this technique, we also demonstrate that when the Ns to C interaction is nullified, employing treatment with N-ethylmaleimide, the parameters which characterize R to Ns coupling remain unchanged.

Adenylyl Cyclases↗
PubMed1982

The interaction of Mn2+ with turkey erythrocyte adenylate cyclase.

Mn2+ at concentrations below 0.1 mM supports the activation of turkey erythrocyte adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by beta-agonists or NaF, similarly to Mg2+ at 5.0 mM. At higher concentrations, Mn2+ is strongly inhibitory, as is Mg2+ above 6 mM. Also, Mn2+ with GTP, but in the absence of beta-agonist, is very potent in reversing the Gpp(NH)p permanently active state to the basal state. beta-Receptor (R) to guanyl nucleotide regulatory protein (N) coupling still occurs at inhibitory Mn2+ concentrations, since the intrinsic kinetic parameters which characterize the R to N coupling interrelationship are unaffected by Mn2+ at a wide concentration range. It is suggested that the inhibitory effect of Mn2+ is due to the impairment of the guanyl nucleotide regulatory protein (N) to the catalytic subunit (C) interaction.

Adenylyl Cyclase Inhibitors↗
PubMed1982

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes.

High affinity binding sites for [3H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [3H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2',5'-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [3H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.

Adenosine↗
PubMed1982

Kinetics of interaction between beta-receptors, GTP protein, and the catalytic unit of turkey erythrocyte adenylate cyclase.

The kinetics of turkey erythrocyte membrane adenylate cyclase activation by beta-agonists and guanyl-5'-yl imidodiphosphate is explored as a function of the concentration of the GTP regulatory protein and of the catalytic unit. It was found that the overall kinetics of activation is first order and is independent of the concentration of the GTP regulatory unit N, the catalytic unit C, and of hormone over a very wide concentration range. It was established that the rate-limiting step does not involve GDP dissociation from the inactive N unit or the association between activated N' and C. Also, it was found that guanyl-5'-yl imidodiphosphate binding occurs in a random fashion and is not hormone dependent. These results enable us to exclude models of the sequential type in which N in its inactive form is bound to receptor R, is released in an active form N' upon hormone activation, and then binds to C, activating the latter. An acceptable model that accounts for all of the data conforms to the original formulation of "collision coupling" in which N is tightly associated to C at all times.

Adenylyl Cyclases↗
PubMed1982

Prostaglandin-stimulated GTP hydrolysis associated with activation of adenylate cyclase in human platelet membranes.

In membranes purified from human blood platelets, basal guanosine triphosphate (GTP) hydrolysis is reduced by a factor of approximately 6 by exposure to N-ethylmaleimide (10 mM). This decreased background enables the detection of an additional GTP hydrolysis in the presence of prostaglandin E1 (PGE1). The PGE1-stimulated GTPase has several properties correlated with PGE1-stimulated adenylate cyclase in this preparation. The two enzymes have similar dose-response relationships (half-maximal stimulation at 0.1 microM PGE1). Exposure to cholera toxin blocks the PGE1-stimulated GTPase and activates adenylate cyclase. Both enzymes are activated by submicromolar concentrations of GTP, although the Km for the GTPase is about 10 times greater than that for the adenylate cyclase. The data are discussed in relation to the hypothesis that hormone-stimulated adenylate cyclase (i) is activated as a regulatory component binds a molecular of GTP and (ii) is deactivated as this molecule is hydrolyzed.

Adenylyl Cyclases↗
PubMed1982

Mechanism of control of the turkey erythrocyte beta-adrenoceptor dependent adenylate cyclase by guanyl nucleotides: a minimum model.

Treatment of native turkey erythrocyte membranes with GMP and epinephrine produces a highly active but metastable form of adenylate cyclase which decays slowly to basal native state. The decay process is greatly facilitated by GTP and GDP beta S1, and is further enhanced by 1-epinephrine. This decay process is prevented reversibly by GMP. GppNHp, like GMP, prevents the decay process first reversibly, but with time stabilizes the highly active state in a persistently active state. The expression of the catalytic activity of the enzyme in the metastable state can also be inhibited reversibly by GTP, GDP beta S and GMP at all times during the decay process. The GppNHp stabilized form is not susceptible to nucleotide inhibition. Thus, two forms of the guanyl nucleotide unit are postulated to exist: an "open" and a "closed" form. In the presence of hormone and GTP, the enzyme shuttles between these two forms continuously. GMP and GppNHp favor the complete conversion to the "open" form in the presence of beta-agonist. Evidence is also presented for the existence of two GTP dependent processes which exhibit different apparent affinities towards the nucleotide: A high affinity GTP binding process is essential for the fruitful coupling between receptor and enzyme, and a low affinity GTPase site which is responsible for the termination of the hormonal signal.

Adenylyl Cyclases↗
PubMed1982

Activation of adenylate cyclase from purified platelet membranes by prostaglandin E1 and its inhibition by L-epinephrine: mechanistic effects.

Activation and inhibition of adenylate cyclase in purified human platelet membranes by hormones and guanyl nucleotides was studied. The rate constant of enzyme activation (kon) was measured using the GTP analog guanylimidodiphosphate (Gpp(NH)p), and the rate constant of enzyme deactivation (koff) was determined using the guanosine diphosphate analog guanosine 5'-0-(2-thio)-diphosphate (GDP beta S). PGE1 which was found previously (15) to accelerate kon has been found to accelerate also koff, from 0.1 sec-1 (6 min-1) to 0.2 sec-1 (12 min-1). The alpha 2-adrenergic inhibitory hormone 1-epinephrine did not alter kon, whether measured in the absence or presence of GTP, and also did not alter koff, whether measured under basal or PGE1-dependent GTP-ase activity at any concentration of GTP tested. These results suggest that the alpha 2-adrenergic receptor exerts its inhibitory effect on adenylate cyclase by a mechanism which does not involve its direct interaction with the GTP regulatory protein that is associated with the stimulatory hormone. These results support the view that the inhibitor involves the interaction of the inhibitory receptor with a GTP binding unit separate from the one mediating hormonal stimulation. This regulatory unit attenuates the adenylate cyclase activity either by interacting directly with the catalytic moiety or by modulating the interaction of the stimulatory GTP regulatory protein with the catalytic moiety.

Adenylyl Cyclase Inhibitors↗
PubMed1981

The reversal of the Gpp(NH)p-activated state of adenylate cyclase by GTP and hormone is by the "collision coupling" mechanism.

The guanylyl imidodiphosphate-activated state of turkey erythrocyte cyclase can be reversed to the basal state by the simultaneous action of beta-agonists and GTP. The rate of reversal diminishes progressively with decreasing concentration of beta-adrenergic receptors on the membrane surface, whereas the extent of reversal is always maximal. The rate of reversal is found to be linearly dependent on the concentration of beta-adrenergic receptors within the membranes. This result supports the notion that the interaction of the enzyme unit and the beta-adrenergic receptor is catalytic and therefore of the "collision coupling" type. The dependence of the rate of reversal reaction on epinephrine concentration is noncooperative with an apparent dissociation constant of KD = 3.0 X 10(-6) M. The fraction of guanylyl imidodiphosphate-activated cyclase system which can be reversed by GTP and a beta-agonist strongly depends on temperature and reveals a sharp transition at 24 degrees C which is the freezing temperature of the inner monolayer. It is suggested that the GTP regulatory unit is inactive when the inner monolayer is frozen.

Adenylyl Cyclases↗
PubMed1981

The beta-adrenergic receptor and its mode of coupling to adenylate cyclase.

The article first includes a discussion on the classification of catecholamine receptors followed by a discussion on the binding studies of beta-receptors and their affinity labeling. Next a brief discussion on the solubilization and the current attempts to purify the receptor is presented. A large section is then devoted to the mode of coupling between beta-receptors and cyclase where much space is devoted to the role of GTP and of the membrane matrix. The review ends with a discussion on beta-receptor desensitization, supersensitivity, and the "spare receptor" concept.

Adenylyl Cyclases↗
PubMed1981

Outward pressure within erythrocytes.

The osmotic pressure of human erythrocyte contents was measured and found to be higher by 35 mosmol/liter, or 0.79 atm, than that of the surrounding plasma. The pressure difference also exists under hypotonic conditions, although it is smaller than under isotonic conditions. Binding capability of the concentrated intracellular protein for additional ions was shown. It is suggested that the osmotic pressure difference is balanced by the inward pressure exerted by the membrane together with the osmotic pressure of the surrounding plasma.

Erythrocyte Membrane↗