Turkey Erythrocyte Membranes Research Articles

Potent agonist action of 2‐thioether derivatives of adenine nucleotides at adenylyl cyclase‐linked P2y‐purinoceptors

1. Analogues of adenine nucleotides inhibited beta-adrenoceptor-stimulated cyclic AMP accumulation in C6 rat glioma cells with a pharmacological selectivity consistent with that for involvement of a P2Y-purinoceptor. 2. The inhibitory effect of adenine nucleotides was completely prevented by pretreatment of cells with pertussis toxin. 3. The capacity of a series of recently synthesized 2-thioether analogues of adenine nucleotides to inhibit cyclic AMP accumulation was examined. Several ATP analogues, e.g. 2-cyclohexylthio and 2-hexylthio ATP, inhibited cyclic AMP accumulation with EC50 values of approximately 30 pM. These values represent 100,000 fold increases in potency over ATP. 4. Analogues of ADP exhibited the same remarkable increase in potency relative to their natural congener and diphosphates were at least as potent as the corresponding triphosphates at the C6 cell P2Y-purinoceptor. 5. The relative potencies of a broad series of agonists at the C6 cell receptor did not correspond to the relative potencies of the same compounds for activation of P2Y-purinoceptors on turkey erythrocyte membranes. Some agonists, particularly 2-thioether derivatives were more potent for stimulation of the C6 cell receptor, whereas other agonists were more potent in the turkey erythrocyte system. 6. These results add further support to the view that the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat glioma cells is a different subtype from the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes and several mammalian tissues.

M-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that the GTP analogue is resistant to hydrolysis. The apparent Ka for activation of turkey erythrocyte adenylate cyclase by m-AcAGTP was found to be 0.21 microM, a value similar to that for guanosine 5'-[beta,gamma-imido]triphosphate. m-AcAGTP also effectively inhibited the light-dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [alpha-32P]m-AcAGTP, followed by immunoprecipitation of G-protein Gq, identified a labelled protein band with the mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this protein was enhanced 9-fold in blue over red illuminated membranes, containing metarhodopsin and rhodopsin respectively. Labelling of alpha-subunits of heterotrimeric G-proteins was also demonstrated in turkey erythrocyte membranes. The ease of preparation of m-AcAGTP and the chemical properties of the photoreactive acetophenone make this affinity label an important new tool in studies of cellular phenomena mediated by guanine nucleotide-binding proteins.

Ca inhibits adenylyl cyclase in turkey erythrocyte membranes in a calmodulin independent manner

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 16S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration ([Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 μM, whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.

Unoccupied β-adrenoceptor-induced adenylyl cyclase stimulation in turkey erythrocyte membranes

The influence of β-adrenoceptor ligands and a β-adrenoceptor-derived peptide was studied on stimulation of adenylyl cyclase in membrane preparations of turkey erythrocytes. In the absence of receptor ligands, the hydrolysis-resistant GTP analogs, guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β,γ-imino]triphosphate, caused a slow, time-dependent increase in adenylyl cyclase activity, which was accelerated and potentiated by the additional presence of the agonist, isoproterenol. In contrast, the β-adrenoceptor antagonists, propranolol and pindolol, almost completely prevented stimulation of adenylyl cyclase by the GTP analogs alone, i.e. in the absence of an agonist, in a concentration-dependent and stereo-selective manner. This antagonist action was mimicked by a peptide corresponding to the second cytoplasmic loop of the turkey erythrocyte β-adrenoceptor. On the other hand, GTP analog-preactivated cyclase activity and enzyme stimulation by fluoride and forskolin were not or only slightly β-adrenoceptors can cause significant G s protein and subsequent adenylyl cyclase activation, and that this unoccupied receptor action can be blocked by β-adrenoceptor antagonists, thus exhibiting by themselves a negative intrinsic activity.

Novel competitive antagonists for P 2 purinoceptors

Binding of the radioligand [ 35S]adenosine 5′- O-(2-thiodiphosphate) (ADPβ 35S) to P 2y purinoceptors on turkey erythrocyte membranes was used to determine the affinity of suramin and various suramin congeners belonging to different structure classes (large urea, small urea, dibenzamides and benzamides) for these receptors. Suramin was shown to be a competitive antagonist with a K i value of 7.3±2.2 μM. The simple benzamide compound XAMR0721 (8-(3,5-dinitrophenylene carbonylimino)-1,3,5- naphthalene trisulfonate, trisodium salt) displays a high affinity for the P 2y purinoceptor ( K i value of 19±6 μM). Similar to suramin, compound XAMR0721 is a competitive antagonist at P 2y purinoceptors. In contrast to suramin, which is a potent inhibitor of the ecto-nucleotidase activity in human blood cells (44±2% residual activity at 100 μM), compound XAMR0721 is hardly active in this assay (93±1% residual activity at 100 μM). So XAMR0721, the first competitive antagonist for P 2 purinoceptors that is able to discriminate between P 2 purinoceptor affinity and ecto-nucleotidase activity, is an interesting pharmacological tool for the characterization of P 2 purinoceptor mediated effects.

Structure Activity Relationships for Derivatives of Adenosine-5'-Triphosphate as Agonists at P(2) Purinoceptors: Heterogeneity Within P(2X) and P(2Y) Subtypes.

The structure-activity relationships for a variety of adenine nucleotide analogues at P(2x)- and P(2Y)-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N(6)-substituents, and a uridine base instead of adenine), the ribose moiety (2' and 3'-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P(2Y)-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P(2X)-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P(2X)- or the P(2Y)-purinoceptors. Certain analogues displayed selectivity or specificity within the P(2X)- or P(2Y)-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2',3'-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P(2Y)-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P(2X)-purinoceptors. The potent agonist N(6)-methyl ATP and the somewhat less potent agonist 2'-deoxy-ATP were selective for P(2Y)-purinoceptors in the guinea pig taenia coli, but were inactive at P(2X)-purinoceptors and the vascular P(2Y)-purinoceptors. 3'-Benzylamino-3'-deoxyATP was very potent at the P(2X)-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P(2Y) receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P(2X)- and P(2Y)-purinoceptor classes.

Characterization of a membrane-associated, receptor and G-protein responsive phosphoinositide-specific phospholipase C from avian erythrocytes

We describe the reconstitution and purification of a membrane-associated phosphoinositide-specific phospholipase C (PIC) from turkey erythrocyte ghosts. This PIC is responsive to a G-protein coupled to P 2y purinergic receptors which are expressed in turkey erythrocytes. Reconstitution is achieved by adding partially purified PIC to [ 3H]inositol-prelabelled turkey erythrocyte membranes depleted of their endogenous PIC (acceptor membranes). PIC activity is associated with a 52 kDa polypeptide on SDS-polyacrylamide gel electrophoresis. Addition of a 307-fold purified enzyme to the acceptor membranes has no effect on basal PIC activity, but markedly increases the response to GTPγS and P 2y-purinergic receptor activation.

Halothane regulates G-protein-dependent phospholipase C activity in turkey erythrocyte membranes

The ability of halothane to stimulate phospholipase C (PLC) was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes by measuring [3H]inositol phosphate formation ([3H]InsP) in the presence and absence of G-protein activation. In the presence of guanosine 5'-3-O-(thio)triphosphate) (GTP gamma S), halothane (0.5-10 mM) caused a dose-dependent activation of PLC. The EC50 value for halothane-induced PLC activation was 2.8 +/- 0.3 mM. Halothane (0.1-30 mM) had no effect on PLC activity in the absence of G-protein activation and did not affect Ca(2+)-dependent PLC activity. The activation of PLC by GTP gamma S occurred after an initial lag period of 60 s which was followed by a linear increase in [3H]InsP. Halothane dose-dependently decreased the lag period for GTP gamma S-induced PLC activation (minimal value 15 s) and increased the rate of [3H]InsP formation at all time points following this lag. As a result, halothane shifted the EC50 value for GTP gamma S-induced PLC activation to the left (4-fold) and increased its maximal response. Halothane also caused a dose-dependent activation of PLC in the presence of AlF4-. Half-maximal stimulation of AlF4(-)-activated PLC occurred with an EC50 value of 2.9 +/- 0.4 mM halothane, which is similar to the halothane dose giving half-maximal stimulation of PLC in the presence of GTP gamma S. At low doses (0.1-0.3 mM) halothane inhibited both isoproterenol- and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S)-induced [3H]InsP formation, whereas at higher concentrations it stimulated PLC independent of the presence of these agonists. At concentrations chosen to reflect their different membrane/buffer partition coefficients, both hexanol (5 mM) and benzyl alcohol (20 mM) fluidized turkey erythrocyte membranes to the same degree as halothane (5 mM). However, these agents had no effect on GTP gamma S- or AlF(4-)-induced PLC activity, indicating that halothane-induced PLC activation was not secondary to changes in bulk lipid fluidity properties. Halothane also stimulated [3H]inositol bisphosphate and [3H]inositol trisphosphate formation in intact erythrocytes. These data demonstrate that the anesthetic halothane can stimulate G-protein-dependent PLC activity and modify the responsiveness of this signaling system to activation by receptor-linked agonists.

Binding of the radioligand [ 3Sadenosine 5'- O-(2-thiodiphosphate) and intracellular calcium response in rat liver parenchymal cells

The use of the radioligand [ 35S]adenosine 5'- O-(2-thiodiphosphate) (ADP β 35S) for the determination of P 2y-purinoceptors on turkey erythrocyte membranes has recently been described. In the present study, we were able to demonstrate specific binding of this radioligand in intact rat liver parenchymal cells. Within 10 min a thermodynamic equilibrium was obtained which lasted for 25 min with a subsequent decline. Displacement studies with several nucleotides were performed yielding K i values of 1.5 ± 0.47 μM for UTP, 1.8 ± 0.35 μM for adenosine 5'- O-(2-thiodiphosphate) (ADPβS), 31 ± 6.2 μM for ATP and 35 ± 6.1 μM for GTP. In addition, we showed that ADP β 35S is highly resistant to degradation by ecto-nucleotidases, with only 14.5 ± 1.4% of total ADP β 35S present being degraded after 1 hr, and that the binding of ADP β 35S to its binding sites was modulated by EDTA. The K i value of ATP shifted to 8.1 ± 1.2 μM upon the addition of 1 mM EDTA to the incubation medium. In these rat liver parenchymal cells all nucleotides promoted calcium entry in a dose-dependent manner with ec 50 values of 3.5 ± 0.22 μM for UTP, 20.7 ± 3.1 μM for ATP, 38.3 ± 6.4 jμM for ADPβS and 73.6 ± 13.7 μM for GTP, with GTP being a partial agonist. Based on the data derived from the present study we discuss the possible correlation between binding and functional experiments and conclude that the described receptor resembles most closely the P 2u-purinoceptor and/or “nucleotide receptor”, in that UTP is at least as active as ATP.

The GppNHp-activated adenylyl cyclase complex from turkey erythrocyte membranes can be isolated with its beta gamma subunits.

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5'-[beta, gamma-imido]triphosphate (Gpp[NH]p) and separated under low-detergent and low-salt conditions using conventional molecular-sieve chromatography followed by high-pressure ion-exchange and molecular-sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the beta gamma subunits copurify with the cyclase. The stoichiometry of the cyclase to the alpha subunit of the stimulatory guanosine-nucleotide-binding regulatory protein (alpha s) to the beta subunit is close to unity, demonstrating that the beta gamma subunits do not dissociate from the Gs.cyclase complex (Gs, guanosine-nucleotide-binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high-salt concentrations, the beta gamma subunits could be separated from the alpha s.cyclase complex. Previously reported results on bovine brain cyclase also showed that the Gs.cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar-Sinai, A., Minich, M. and Levitzki, A. (1990) J. Biol. Chem. 265, 9999-10,004]. These results, using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that beta gamma subunits are not released from Gs upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosine-nucleotide-binding regulatory protein cannot be via shifting the alpha s to beta gamma equilibrium as is commonly believed, and an alternate hypothesis is proposed.

G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.

Guanosine nucleotides regulate hormone binding of insulin receptors.

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5'-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.

Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes

The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.

Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.

Multisite contacts involved in coupling of the β‐adrenergic receptor with the stimulatory guanine‐nucleotide‐binding regulatory protein

Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

Phosphatidylinositol synthase and phosphatidylinositol/inositol exchange reactions in turkey erythrocyte membranes

Unlike human erythrocytes, those from avian species, such as turkeys and chicks, rapidly incorporate myo-[3H]inositol into membrane phospholipids. The mechanisms regulating [3H]Ins labelling of phosphatidylinositol have been investigated using turkey erythrocyte membranes. In the absence of added nucleotides, [3H]inositol incorporation appears to proceed via phosphatidylinositol/inositol exchange, with a Km for inositol of 0.01 mM. The reaction was dependent upon divalent cations, either Mg2+ or Mn2+, with the latter metal ion being the more effective. [3H]Inositol incorporation was accelerated by CMP, especially when the concentration of Ins was greater than the Km for the exchange reaction. CMP-dependent labelling of PtdIns had a Km for inositol of 0.3 mM and for CMP of 0.015 mM. Divalent cations were also required for this reaction: activity peaked at 0.5 mM-Mn2+ and declined at higher concentrations. At relatively high concentrations, Mg2+ was more effective than Mn2+, with peak activity being achieved above 10 mM. CMP-dependent incorporation of [3H]inositol appears to reflect an exchange reaction catalysed by PtdIns synthase. Definitive evidence for the occurrence of PtdIns synthase in turkey erythrocyte membranes was obtained by demonstrating the formation of [14C]CMP-phosphatidate from [14C]CMP. The radioactivity could be efficiently chased from [14C]CMP-phosphatidate in the presence of unlabelled inositol. The detection of PtdIns synthase activity in morphologically simple turkey erythrocytes should help to clarify the subcellular distribution of this important component of the phosphatidylinositol cycle.

Binding of α‐ and βγ‐subunits of G0 to β1‐adrenoceptor in sealed unilamellar lipid vesicles

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid-rich vesicles with average diameters ranging over 150-200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30-70 nm (fraction II). When the lipid mixture containing dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with alpha- and beta gamma-subunits of Go-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a beta 1-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside-out. On co-reconstitution of either alpha o or beta gamma alone with beta 1-adrenoceptors, some of these subunits appear together with beta 1-adrenoceptors in the small vesicle fraction II, but much more alpha o was bound to the receptor in the presence of beta gamma-subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that beta gamma-subunits can bind to the non-activated beta 1-receptor where they may serve as an anchor for alpha-subunits. Secondly, the binding of alpha o- and beta gamma-subunits to the beta 1-adrenoceptors enhances the basal GTPase activity of alpha o. Thirdly, since the binding domains of the beta 1-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the beta-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213-215 (1987)] for the transducin-rhodopsin interactions. Finally, the binding of Go-subunits from bovine brain to a beta 1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.

The αβ monomer of the insulin receptor has hormone-responsive tyrosine kinase activity

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.

Irreversible Activation and Photoaffinity Labeling of a P2Y‐Purinergic Receptor Coupled to Phospholipase C Activation in Turkey Erythrocyte Membranes

Irreversible Activation and Photoaffinity Labeling of a P_2Y‐Purinergic Receptor Coupled to Phospholipase C Activation in Turkey Erythrocyte Membranes

Solubilization of a Guanine Nucleotide‐Sensitive Form of the P2Y‐Purinergic Receptor from Turkey Erythrocyte Membranes

Solubilization of a Guanine Nucleotide‐Sensitive Form of the P_2Y‐Purinergic Receptor from Turkey Erythrocyte Membranes