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Biochemical, immunological and functional studies of an α-bungarotoxin receptor purified from chick optic lobe
Cell Biology international
P249
REGULATION INDEPENDENT
Reports, Vol. 14, Abstracts Supplement
OF GP IIB/IIIA EXPOSURE OF SIGNAL GENERATION.
Jan-Willem N. Akkerman and Gijsbert van Willigen, Dept. of Haematology, Univ. Hosp. Utrecht, The Netherlands. When human platelets are stimulated with Platelet Activating Factor, ADP or epinephrine in the absence of fibrinogen and 1a51-fibrinogen is added at different time fall in exposed intervals thereafter, an exponential fibrinogen bindingsites is seen. Thus, these agonists IIB/IIIA exposure of glycoprotein induce a transient bindingsites for complex. In contrast, thrombin-exposed (coagulation prevented by fibronectin and fibrinogen PPACK) remain stable for as long as 30 min (22°C). even when thrombin is removed from its receptor 15 set later, with an excess of hirudin and signal generation via are terminated and Phospholipase-C and Ca 2+-mobiliration (30uM formation is abolished thromboxane-A, However, exposed sites close rapidly indomethacin). following addition of a Protein Kinase-C (PK-C) inhibitor (JuM staurosporin) or PGI, (IOng/ml), which raises that maintenance of exposed binCAMP, indicating and a low CAMP dingsites requires an active PK-C comparison between PC&-induced content. A different bindingsites exposed by disappearance of agonists reflects the degree of coupling between the different receptors and Gi and G,, the GTP-binding proteins that inhibit or activate Adenylate Cyclase. The
fastest disappearance, after PGI, addition, is seen for bindingsites exposed by agonists, that do not activate Gi.
ANALYSIS OF TEE INWJNOCYTOCBBMIC BEUAVIODR ON FRKE%E-FEACTlJRJi OF erbB-2 PROTEIIYS Lavinia V. Lotti, Antonio Pavan, Claudia Zompetta, Luigi Frati and Maria Rosaria Claudia Di Lazsaro, Dipartimento di Medicina Sperimentale. Torrisi. Universita’ di Rowa “La Sapiensa” .Rome, Italy. We have recently observed, using fracture-label the behaviour on freeze-fracture of technique, epidermal growth factor (EGF) receptors in A431 Binding of the ligand to the receptors as cells. well as receptor cross-linking induced a cbangement of the partition during fracture of the receptors from the outer to the inner leaflet of the freeze-fractured plasma membranes, probably as a consequence of receptor aggregation and next to analyze the activation. We decided partition on fracture of gp185, product of the homologous to the EGF erbB-2 proto-oncogene, which can be activated by point receptor gene, mutations in the transmembrane region. NIB-3T3 expressing the normal or the mutant activated proteins (a gift of Dr. Aaronson) were fixed, freezeembedded in 30% bovine serum albumin, fractured and thawed. The immunollbellng was performed using an anti-gel85 monoclonal antibody, followed by protein A colloidal gold conjugates. In all cells the labeling was associated with the outer leaflets of the freeze-fractured plasma membranes. pur results show that, during freezefracture , the gp185 behave differently from the EGF receptors when activated, suggesting that the mechanism of activation of gp185 may not involve protein aggregation.
P251
P250
1990
141
BETA-ADRENOCEPTOR SUBTYPES MODULATE
DIFFERENTIALLY TRE ADEh’YLATE-CYCLASE
Piantanelli. INRCA , Lucia Claudio Viticchi. Ctr of Biochemistry, Via Gerontol. Res. Dept., Birarelli 8, Ancona, 60100 Italy. aging a progressive impairment of the During to the beta-adrenergic stimulation responsiveness Receptor density is a step deeply was observed. in the trasmission of the stimulation. involved Beta-adrenoceptor density was found decreased in brain cortex of old mice and this decrease is subtype, while completely in charge to the beta-l the beta-2 remains unchanged during aging. The differential effect of the two beta-adrenoceptor subtypes on the adenylate-cyclase activity was examined in order to study the influence of the different age-related behaviour of the beta-l and beta-2 adrenoceptors. Brain cortex membranes derived from 3- and 22-month old mice were used to assay the adenylate-cyclase activity under basal or IPR-stimulated conditions and in presence of nonhydrolyzable GTP anolog Gpp(NB)p. A the consistent decrease in the IPR-stimulated conditions was put in evidence when the adenylatecyclase activity measured in membrane of old to the young one. The animal was compared inactivation of the beta-l subtype selective allows to the disappearence of these age-related suggesting that the capability to difference responde to the stimulation is strictly linked to the beta-l receptors availability.
BIOCHEMICAL, IMMUNOLOGICAL AND FUNCTIONAL STUDIES OF AN a-BUNGAROTOXIN RECEPTOR PURIFIED FROM CHICK OPTIC LOBE C. Gotti, A. Esparis-Ogando, M. Moretti, *W. Hanke and F. Clementi, CNR Ctr. for Cytopharmacol., 20129 Milan, Italy., *Inst. Neurobiology, Univ. of Dusseldorf, Dusseldorf, ERG. a-Bungarotoxin (aBgtx) is a specific marker for the muscle type nicotinic aceylcholine receptor (AChR). This toxin is also used as a probe for studying neuronal AChR. The results of these studies clearly indicate that at least two types of aBgtx binding sites with nicotinic choline@ pharmacology exist in the nervous tissue. One in which the aBgtx binding site and the AChR exist as a single molecular entity; the other, where there is a clear physical dissociation between the aBgtx binding sites and the functional AChR. In order to elucidate further the structure and function of neuronal aBgtx binding sites and to clarify the relationship between the two types of aBgtx binding sites we purified the aBgtx receptor present on the chick optic lobe. Analysis of the purified receptor by SDSPAGE shows that this receptor is composed of three polypeptides of Mr 67000, 58000 and 52000. Ligand binding to blots of purified receptor reveals that only the polypeptide of Mr 58000 is bound by izsI-aBgtx. An immunological approach was also used to characterize the aBgtx receptor purified from chick optic lobe and to compare it with other knwon nicotinic receptors. For this study we used polyclonal antibodies raised against muscular AChRs, neuronal aBgtx receptor and a monoclonal antibody which specifically recognize a funtional neuronal AChR. To verify if the purified receptor represents only an aBgtx binding site or also a functional nicotinic receptor, purified receptor has been reconstituted in a planar lipid bilayer. We have shown that in this system cholinergic agonists activate functional ion channels which are blocked by d-Tubocurarine and the activated channels have properties comparable to those exhibited by other neuronal AChRs.
P252
P249
REGULATION INDEPENDENT
Reports, Vol. 14, Abstracts Supplement
OF GP IIB/IIIA EXPOSURE OF SIGNAL GENERATION.
Jan-Willem N. Akkerman and Gijsbert van Willigen, Dept. of Haematology, Univ. Hosp. Utrecht, The Netherlands. When human platelets are stimulated with Platelet Activating Factor, ADP or epinephrine in the absence of fibrinogen and 1a51-fibrinogen is added at different time fall in exposed intervals thereafter, an exponential fibrinogen bindingsites is seen. Thus, these agonists IIB/IIIA exposure of glycoprotein induce a transient bindingsites for complex. In contrast, thrombin-exposed (coagulation prevented by fibronectin and fibrinogen PPACK) remain stable for as long as 30 min (22°C). even when thrombin is removed from its receptor 15 set later, with an excess of hirudin and signal generation via are terminated and Phospholipase-C and Ca 2+-mobiliration (30uM formation is abolished thromboxane-A, However, exposed sites close rapidly indomethacin). following addition of a Protein Kinase-C (PK-C) inhibitor (JuM staurosporin) or PGI, (IOng/ml), which raises that maintenance of exposed binCAMP, indicating and a low CAMP dingsites requires an active PK-C comparison between PC&-induced content. A different bindingsites exposed by disappearance of agonists reflects the degree of coupling between the different receptors and Gi and G,, the GTP-binding proteins that inhibit or activate Adenylate Cyclase. The
fastest disappearance, after PGI, addition, is seen for bindingsites exposed by agonists, that do not activate Gi.
ANALYSIS OF TEE INWJNOCYTOCBBMIC BEUAVIODR ON FRKE%E-FEACTlJRJi OF erbB-2 PROTEIIYS Lavinia V. Lotti, Antonio Pavan, Claudia Zompetta, Luigi Frati and Maria Rosaria Claudia Di Lazsaro, Dipartimento di Medicina Sperimentale. Torrisi. Universita’ di Rowa “La Sapiensa” .Rome, Italy. We have recently observed, using fracture-label the behaviour on freeze-fracture of technique, epidermal growth factor (EGF) receptors in A431 Binding of the ligand to the receptors as cells. well as receptor cross-linking induced a cbangement of the partition during fracture of the receptors from the outer to the inner leaflet of the freeze-fractured plasma membranes, probably as a consequence of receptor aggregation and next to analyze the activation. We decided partition on fracture of gp185, product of the homologous to the EGF erbB-2 proto-oncogene, which can be activated by point receptor gene, mutations in the transmembrane region. NIB-3T3 expressing the normal or the mutant activated proteins (a gift of Dr. Aaronson) were fixed, freezeembedded in 30% bovine serum albumin, fractured and thawed. The immunollbellng was performed using an anti-gel85 monoclonal antibody, followed by protein A colloidal gold conjugates. In all cells the labeling was associated with the outer leaflets of the freeze-fractured plasma membranes. pur results show that, during freezefracture , the gp185 behave differently from the EGF receptors when activated, suggesting that the mechanism of activation of gp185 may not involve protein aggregation.
P251
P250
1990
141
BETA-ADRENOCEPTOR SUBTYPES MODULATE
DIFFERENTIALLY TRE ADEh’YLATE-CYCLASE
Piantanelli. INRCA , Lucia Claudio Viticchi. Ctr of Biochemistry, Via Gerontol. Res. Dept., Birarelli 8, Ancona, 60100 Italy. aging a progressive impairment of the During to the beta-adrenergic stimulation responsiveness Receptor density is a step deeply was observed. in the trasmission of the stimulation. involved Beta-adrenoceptor density was found decreased in brain cortex of old mice and this decrease is subtype, while completely in charge to the beta-l the beta-2 remains unchanged during aging. The differential effect of the two beta-adrenoceptor subtypes on the adenylate-cyclase activity was examined in order to study the influence of the different age-related behaviour of the beta-l and beta-2 adrenoceptors. Brain cortex membranes derived from 3- and 22-month old mice were used to assay the adenylate-cyclase activity under basal or IPR-stimulated conditions and in presence of nonhydrolyzable GTP anolog Gpp(NB)p. A the consistent decrease in the IPR-stimulated conditions was put in evidence when the adenylatecyclase activity measured in membrane of old to the young one. The animal was compared inactivation of the beta-l subtype selective allows to the disappearence of these age-related suggesting that the capability to difference responde to the stimulation is strictly linked to the beta-l receptors availability.
BIOCHEMICAL, IMMUNOLOGICAL AND FUNCTIONAL STUDIES OF AN a-BUNGAROTOXIN RECEPTOR PURIFIED FROM CHICK OPTIC LOBE C. Gotti, A. Esparis-Ogando, M. Moretti, *W. Hanke and F. Clementi, CNR Ctr. for Cytopharmacol., 20129 Milan, Italy., *Inst. Neurobiology, Univ. of Dusseldorf, Dusseldorf, ERG. a-Bungarotoxin (aBgtx) is a specific marker for the muscle type nicotinic aceylcholine receptor (AChR). This toxin is also used as a probe for studying neuronal AChR. The results of these studies clearly indicate that at least two types of aBgtx binding sites with nicotinic choline@ pharmacology exist in the nervous tissue. One in which the aBgtx binding site and the AChR exist as a single molecular entity; the other, where there is a clear physical dissociation between the aBgtx binding sites and the functional AChR. In order to elucidate further the structure and function of neuronal aBgtx binding sites and to clarify the relationship between the two types of aBgtx binding sites we purified the aBgtx receptor present on the chick optic lobe. Analysis of the purified receptor by SDSPAGE shows that this receptor is composed of three polypeptides of Mr 67000, 58000 and 52000. Ligand binding to blots of purified receptor reveals that only the polypeptide of Mr 58000 is bound by izsI-aBgtx. An immunological approach was also used to characterize the aBgtx receptor purified from chick optic lobe and to compare it with other knwon nicotinic receptors. For this study we used polyclonal antibodies raised against muscular AChRs, neuronal aBgtx receptor and a monoclonal antibody which specifically recognize a funtional neuronal AChR. To verify if the purified receptor represents only an aBgtx binding site or also a functional nicotinic receptor, purified receptor has been reconstituted in a planar lipid bilayer. We have shown that in this system cholinergic agonists activate functional ion channels which are blocked by d-Tubocurarine and the activated channels have properties comparable to those exhibited by other neuronal AChRs.
P252