Forskolin stabilizes a functionally coupled state between activated guanine nucleotide-binding stimulatory regulatory protein, Ns, and catalytic protein of adenylate cyclase system in rat erythrocytes

Guanine nucleotide-binding stimulatory regulatory protein of adenylate cyclase system, Ns, in rat erythrocytes was activated by the treatment with guanylyl 5'-imidodiphosphate or NaF-AlCl3 in the presence of Mg2+. The activation was counterbalanced to the basal state either by the removal of Mg2+ or by the addition of beta(gamma)-subunit of N protein of this system. The depression from the activated state was markedly protected by the coexistence of forskolin at the time of the deactivation depending on the dose of forskolin. EC50 of forskolin for the stabilizing effect was much lower than that for the stimulation of adenylate cyclase activity. These data indicate that forskolin has an effect on the interaction between Ns and catalytic unit of adenylate cyclase system in addition to the direct effect on the catalytic unit.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.     

The selective effects of charged local anaesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma membranes

The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal. These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit. The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme. The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.     

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.     

Interaction of the stimulatory and inhibitory regulatory proteins of the adenylyl cyclase system with the catalytic component of cyc-S49 cell membranes

The mechanism by which Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases, regulate the activity of the catalytic component (C) of adenylyl cyclase was investigated using cyc-S49 cell membranes which contain a functional inhibitory regulatory protein (Ni) but not the active subunit of the stimulatory regulatory protein (Ns). To this end, purified Ns protein was preactivated (Ns) in solution with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and Mg2+, and then added to cyc- membranes under conditions where Ni was either unactivated or activated (Ni) by GTP gamma S and Mg2+. Activation of Ni in cyc- membranes resulted in a lowered expression of Ns activity under all conditions tested. Upon dilution of the reactants (Ns and cyc- membranes) the reconstituted activity declined in proportion to the dilution with an approximate t 1/2 of 30-45 min, being unaffected by activation of Ni. Postactivation of Ni after reconstitution of cyc- membranes with Ns resulted in a time-dependent decline in Ns activity to a level that was the same as that obtained when Ns was added to cyc- membranes with preactivated Ni. These data indicated that the effects of Ns on C are of a reversible type. The following indicated that Ns and Ni affect C activity in a noncompetitive manner: (a) the per cent reduction in Ns activity due to activation of Ni was constant and independent of the concentration of Ns, (b) double reciprocal plots of activities reconstituted in control and Ni-containing cyc- membranes versus Ns concentration were linear with an unaltered apparent Km for Ns, and (c) the onset of inhibition of C prereconstituted with Ns was much faster (approximate t 1/2 = 2-5 min) than expected if it were due to occupancy of a common site on C left vacant by Ns.(ABSTRACT TRUNCATED AT 400 WORDS)     

3H]GDP release from rat and hamster adipocyte membranes independently linked to receptors involved in activation or inhibition of adenylate cyclase. Differential susceptibility to two bacterial toxins

When rat adipocyte membranes had been labeled with [3H]GTP in the presence of a beta-adrenergic agonist, the subsequent [3H]GDP release was stimulated by beta-agonists or agonists (e.g. glucagon and secretin) of other "activatory" receptors involved in activation of adenylate cyclase, but was not stimulated by agonists (e.g. prostaglandin E1 and adenosine) of "inhibitory" receptors involved in cyclase inhibition. On the contrary, agonists of inhibitory receptors were effective in stimulating GDP release from hamster adipocyte membranes that had been labeled via inhibitory alpha 2-adrenergic receptors, but an activatory receptor agonist such as isoproterenol was not. Thus, the guanine nucleotide regulatory protein (Ni) involved in adenylate cyclase inhibition is an entity distinct from the regulatory protein (Ns) involved in cyclase activation, and multiple activatory or inhibitory receptors are coupled to a respective common pool of Ns or Ni. Preactivated cholera toxin added together with NAD enhanced GDP release from rat adipocyte membranes prelabeled with isoproterenol but was without effect on the release from hamster adipocyte membranes that had been labeled with an alpha-agonist. In sharp contrast, the active subunit of islet-activating protein, pertussis toxin, failed to alter GDP release from the former membrane but completely abolished inhibitory agonist-induced stimulation of GDP release from the latter membrane preparation in the presence of NAD. Thus, the site of action of cholera toxin is Ns, while that of islet-activating protein is Ni. The function of Ni to communicate between inhibitory receptors and adenylate cyclase was lost when it was ADP-ribosylated by islet-activating protein.     

Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, Ns and Ni.

Platelet-activating factor (PAF, 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine) and the stable thromboxane-receptor agonist U44069 (9 alpha, 11 beta-epoxymethanoprostaglandin H2) stimulated GTPase activity in platelet membranes in a dose-dependent fashion, yielding Ka values of 12 nM and 27 nM respectively. The degree of GTPase activation elicited by these agents was found to be additive with the GTPase activation due to either the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins when activated by prostaglandin E1 and adrenaline (+propranolol) respectively. Treatment of membranes with either cholera or pertussis toxins, which inhibited markedly the receptor-mediated stimulation of the GTPase activities of Ns and Ni respectively, had no or only a small effect, respectively, on the GTPase activity stimulated by PAF and U44069. It is suggested that PAF and U44069, which stimulate inositol phospholipid metabolism in platelets, exert actions through a guanine nucleotide regulatory protein which is distinct from Ns and Ni.     

Inhibitory regulation of adenylyl cyclase in the absence of stimulatory regulation. Requirements and kinetics of guanine nucleotide-induced inhibition of the cyc- S49 adenylyl cyclase

cyc- S49 cell membranes contain an adenylyl cyclase activity which is stimulated by forskolin and inhibited by guanine nucleotides and NaF. These inhibitory effects are mediated by an inhibitory guanine nucleotide-binding regulatory component (Ni) affecting the adenylyl cyclase catalytic unit (Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Manclark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709). Since cyc- S49 cells do not contain a stimulatory guanine nucleotide-binding regulatory component (Ns), these membranes were used to study the requirements and kinetics of activation of Ni in the absence of Ns. Activation of Ni by guanyl-5'-yl imidodiphosphate was time-dependent (i.e. hysteretic) and pseudo-irreversible. Although GTP and guanosine 5'-(beta-thio)diphosphate could prevent the inhibition caused by guanyl-5'-yl imidodiphosphate if added simultaneously with it, they could not reverse the inhibited state induced by previous exposure to guanyl-5'-yl imidodiphosphate. Activation of Ni had an absolute requirement for Mg2+. Unlike the activation of Ns, however, which requires millimolar concentrations of Mg2+ in the absence of hormonal stimulation, activation of Ni requires only micromolar concentrations of the divalent cation. These results support the contention that hormones which activate Ni or Ns do so by altering different parameters of a similar activation mechanism.     

A novel form of gastric inhibitory polypeptide (GIP) isolated from bovine intestine using a radioreceptor assay. Fragmentation with staphylococcal protease results in GIP1-3 and GIP4-42, fragmentation with enterokinase in GIP1-16 and GIP17-42

Sodium cholate and digitonin were used to solubilize alpha2-adrenergic receptors from rat and calf brain. Sodium cholate extracted 40-50% of the membrane protein and 25-30% of the binding capacity. Digitonin extracted only 20-30% of the membrane protein and only 10-15% of the binding capacity of the native membranes. Both detergents were removed by dialysis in the presence of phospholipids, and the solubilized protein was precipitated upon addition of poly(ethyleneglycol) and magnesium. In the solubilization/reconstitution process no purification of the alpha2-adrenergic receptor was obtained, most probably due to its inactivation by the solubilization conditions. The reconstituted protein(s) tested for binding properties, using p-[3H]aminoclonidine and/or [3H]clonidine, maintained the pharmacological profile of the native alpha2-adrenergic receptor. The potency order of various alpha2-agonists and alpha2-antagonists as well as their stereoselectivity were identical to those of the native alpha2-receptor. Specific receptor binding decreases in the presence of the guanyl nucleotides GTP or guanosine 5'-[beta, gamma-imido]-triphosphate but not ATP, thus indicating a co-solubilization of GTP regulatory components (stimulatory protein Ns or inhibitory protein Ni or both). Adenylate cyclase activity of the reconstituted preparation is stimulated threefold by sodium fluoride, suggesting the presence of both Ns-protein and the catalytic unit (C) in the reconstituted protein(s).     

Carbamylcholine inhibits beta-adrenergic receptor-coupled Gs protein function proximal to adenylate cyclase

The specific mechanism by which the inhibitory guanine nucleotide binding protein (Gi) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the beta gamma subunit, which is dissociated with activation of Gi, inhibits the function of the stimulatory guanine nucleotide binding protein (Gs) by reducing the concentration of the free alpha s subunit. In the present study, Gs protein function is determined by measuring cholera toxin-blockable, isoproterenol-induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this beta-adrenergic receptor-coupled Gs protein function. Pretreatment of the cardiac ventricle membrane with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist-receptor coupled effect through Gi on Gs protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.     

Ethanol alters the adenosine receptor-Ni-mediated adenylate cyclase inhibitory response in rat brain cortex in vitro

It has been suggested that ethanol stimulates adenylate cyclase in vitro through an increased function of Ns, the activatory component of adenylate cyclase. Because of the interaction of Ns with Ni, the adenylate cyclase inhibitory component, we have studied the effect of ethanol (0.05-0.2 M) on Ni-mediated adenylate cyclase inhibition caused by the adenosine analog N6-phenylisopropyladenosine (N6-PIA) in brain cortical membranes. Ethanol did not alter N6-PIA binding to the adenosine Ri-receptors, stimulated slightly basal adenylate cyclase activity but abolished adenylate cyclase inhibition due to N6-PIA, suggesting an effect of ethanol on the inhibitory coupling pathway. This was further supported by loss of the adenylate cyclase inhibitory response to GTP (greater than 10(-5) M). It thus seems that, besides its effect on the Ns system, ethanol may also impair Ni-mediated adenylate cyclase responses in rat cerebral cortex.     

Isolation and immunocytochemical location of the nitrite-oxidizing system in Nitrospira moscoviensis

A membrane-associated nitrite-oxidizing system of Nitrospira moscoviensis was isolated from heat-treated membranes. The four major proteins of the enzyme fraction had apparent molecular masses of 130, 62, 46, and 29 kDa, respectively. The nitrite-oxidizing activity was dependent on the presence of molybdenum. In contrast to the nitrite oxidoreductase of Nitrobacter hamburgensis X14, the activity of the nitrite-oxidizing system of Ns. moscoviensis increased when solubilized by heat treatment. Electron microscopy of the purified enzyme revealed uniform particles with a size of approximately 7 × 9 nm. SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153–3, which recognize the β-subunit of the nitrite oxidoreductase from Nitrobacter, reacted with a protein of 50 kDa in Ns. moscoviensis. This protein corresponded to the protein of 46 kDa of the purified enzyme and contained a b-type cytochrome. Using electron microscopic immunocytochemistry and the monoclonal antibodies Hyb 153–3, the nitrite-oxidizing system of Ns. moscoviensis was shown to be located in the periplasmic space. Here a periodic arrangement of membrane-associated particles was found on the outside of the cytoplasmic membrane in the form of a hexagonal pattern. It is supposed that these particles represent the nitrite-oxidizing system in Nitrospira. Received: 22 August 1997 / Accepted: 1 November 1997     

Protein kinase C activators and bradykinin selectively inhibit vasopressin-stimulated cAMP synthesis in MDCK cells

To evaluate a possible modulation by protein kinase C of hormonal, cAMP-mediated effects on renal epithelial cells, we studied the effect of protein kinase C activators and of bradykinin on intracellular cAMP accumulation in MDCK cells. A 15-min pretreatment of cells with phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetylglycerol induced a dose-dependent inhibition of vasopressin-stimulated cAMP synthesis, but not of basal or glucagon-, prostaglandin E2-, and forskolin-stimulated cAMP generation. 4 alpha-Phorbol 12,13-didecanoate, inactive on protein kinase C, did not affect cAMP accumulation. Bradykinin (0.1-10 microM) also inhibited the stimulatory effect of vasopressin on cAMP synthesis in a concentration-dependent manner, but affected neither basal cAMP content, nor its stimulation by glucagon, prostaglandin E2 and forskolin. The effect of activators of protein kinase C and of bradykinin occurred while renal prostaglandin synthesis was blocked with indomethacin. The inhibitory effect of protein kinase C activators and bradykinin on cAMP generation was reversed by the protein kinase C inhibitor H7, was enhanced by monensin, one effect of which is to block the recycling of membrane receptors, and persisted when the GTP-binding protein N1 was blocked with 1 mM Mn2+. Our data suggest that: protein kinase C can modulate the tubular effects of vasopressin by inhibiting cAMP generation; this effect is not mediated by renal prostaglandins, and might result from a direct action on the vasopressin receptor, or on its coupling with Ns; the modulation by bradykinin of vasopressin effects are likely to be exerted, at least partly, through activation of protein kinase C.     

Functional changes in the properties of beta-adrenoreceptors of pigeon erythrocytes after treatment with a catalytic subunit of cAMP-dependent protein kinase

beta-Adrenoreceptors were solubilized by deoxycholate from pigeon erythrocyte plasma membranes treated with N-ethylmaleimide. Removal of the detergent resulted in the incorporation of receptors into phospholipid vesicles as well as in the reconstitution of their biological activity. After fusion of vesicles containing reconstituted receptors to vesicles containing the Ns protein and a catalytic component, the hormonal activation of the enzyme was restored. When prior to fusion the beta-adrenoreceptor-containing vesicles were preincubated with the catalytic subunit of cAMP dependent protein kinase, the hormone-induced activation of the enzyme diminished by 45-50%. The decrease of activation is due to the increase in the lag phase of the enzyme activation in the presence of isoproterenol and Gpp(NH)p as well as to the loss of activity in the steady-state phase of activation. Phosphorylation of beta-adrenoreceptors decreased the concentration of the ternary isoproterenol-receptor-Ns protein complex involved in the activation of adenylate cyclase. Thus, the phosphorylation of receptors is responsible for the disturbances in the mechanism of hormonal signal transmission that are similar to those observed in adenylate cyclase desensitization.     

Generalized approach to the regulation and integration of gene expression

The volume of electron flow through the cbb3 branch of the electron transport chain and the redox state of the quinone pool generate signals that regulate photosynthesis gene expression in Rhodobacter sphaeroides. An inhibitory signal is generated at the level of the catalytic subunit of the cbb3 cytochrome c oxidase and is transduced through the membrane-localized PrrC polypeptide to the PrrBA two-component activation system, which controls the expression of most of the photosynthesis genes in response to O2. The redox state of the quinone pool is monitored by the redox-active AppA antirepressor protein, which determines the functional state of the PpsR repressor protein. The antirepressor/repressor system as well as a modulator of AppA function, TspO, together with FnrL and PrrA stringently control photopigment gene expression. These regulatory elements, together with spectral complex-specific assembly factors, control the ultimate cellular levels and composition of the photosynthetic membrane.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

Heterologous desensitization by 1,25-dihydroxyvitamin D-3 of cyclic AMP response to parathyroid hormone in osteoblast-like cells and the role of the stimulatory guanine nucleotide regulatory protein

The influence of 1,25-dihydroxyvitamin D-3 on the cAMP response to parathyroid hormone was studied in the osteoblast-like rat osteosarcoma cells ROS 17/2.8. The stimulation by parathyroid hormone of cAMP production in intact cells and of adenylate cyclase activity in isolated plasma membranes was attenuated by 1,25-dihydroxyvitamin D-3 treatment. This was associated with a reduction of the stimulatory guanine nucleotide regulatory protein, as demonstrated by a lower response to NaF and guanosine 5'-[beta, gamma-imido]triphosphate, and by a lower activity of solubilized plasma membrane extracts in the reconstitution assay. 1,25-dihydroxyvitamin D-3 blunted also the cAMP response to parathyroid hormone in cells incubated with the glucocorticoid dexamethasone, where a higher activity of the adenylate cyclase catalytic unit was observed. Thus, the two steroids appear to affect distinct levels of the adenylate cyclase system. Furthermore, the two hormones also showed an antagonistic effect upon the production of osteocalcin, an osteoblast-specific extracellular matrix protein. The release of this non-collagenous matrix protein by ROS 17/2.8 cells was increased by 1,25-dihydroxyvitamin D-3 and decreased by dexamethasone.     

Molecular characteristics of glutamate receptors in the mammalian brain

Summary The strong excitatory activity of L-glutamic acid on central nervous system neurons is thought to be produced by interaction of this amino acid with specific neuronal plasma membrane receptors. The binding of L-glutamate to these surface receptors brings about an increase in membrane permeability to Na⁺ and Ca²⁺ ions presumably through direct activation of ion channels linked to the membrane receptors. The studies described in this paper represent attempts to define the subcellular distribution and pharmacological properties of the recognition site for L-glutamic acid in brain neuronal preparations, to isolate and explore the molecular characteristics of the receptor recognition site, and, finally, to demonstrate the activation of Na⁺ channels in synaptic membranes following the interaction of glutamate with its receptors.Radioligand binding assays with L-[³H] glutamic acid have been used to demonstrate a relative enrichment of these glutamate recognition sites in isolated synaptic plasma membranes. The specific binding of L-[³H] glutamate to these membrane sites exhibits rapid association and dissociation kinetics and rather complex equilibrium binding kinetics. The glutamate binding macromolecule from synaptic membranes has been solubilized and purified and was shown to be a small molecular weight glycoprotein (M_T 13 000). This protein tends to form aggregates which have higher specific activity at low concentrations of glutamate than the M_T 13 000 protein has. The overall affinity of the purified protein is lower than that of the high affinity sites in the membrane. Nevertheless, the purified protein exhibits pharmacological characteristics very similar to those of the membrane binding sites. On the basis of its pharmacological properties this protein belongs in the category of the physiologic glutamate preferring receptors.By means of differential solubilization of membrane proteins with Na-cholate, it was shown that this recognition site is an intrinsic synaptic membrane protein whose binding activity is enhanced rather than diminished by cholate extraction of the synaptic membranes. The role of membrane constituents in regulating the binding activity of this protein has been explored and a possible modulation of glutamate binding by membrane gangliosides has been demonstrated. Finally, this glutamate binding glycoprotein is a metalloprotein whose activity is dependent on the integrity of its metallic (Fe) center. This is a clear distinguishing characteristic of this protein vis-à-vis the glutamate transport carriers.The presence of functional glutamate receptors in synaptosomes and resealed synaptic plasma membranes has also been documented by the demonstration of glutamate-activated Na⁺ flux across the membrane of these preparations. The bidirectionality, temperature independence, and apparent desensitization of this stimulated flux following exposure to high concentrations of glutamate are properties indicative of a receptor-initiated ion channel activation. It would appear, then, that the synaptic membrane preparations provide a very useful system for the study of both recognition and effector function of the glutamate receptor complex.     

Solubilization and reconstitution of the sodium-and-chloride-coupled glycine transporter from rat spinal cord

Synaptic membranes from rat spinal cord were solubilized in the presence of 2% sodium cholate, phospholipids and 15% ammonium sulphate. The soluble extract was incorporated into liposomes consisting of asolectin and crude rat brain lipids. Reconstitution of the functional transporter protein was achieved by removal of detergent by gel filtration. Several parameters proved to be important for optimal reconstitution efficiency: (a) the lipid composition of the liposomes, (b) the type of detergent, and (c) the phospholipid/protein and detergent/protein ratio during reconstitution. In the reconstituted system, the transport of glycine showed a specific activity about twice that of native vesicles. The ionic dependence of the transport, the inhibitory effect of nigericin in the presence of external sodium and the stimulatory effect of valinomycin in the presence of internal potassium on glycine transport were preserved and more clearly observed in the reconstituted system. These results indicate that, in this preparation, the glycine transporter protein retains the same features displayed in the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity and inhibitor sensitivity.     

Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N

beta-Adrenergic receptors from turkey erythrocyte membranes have been purified 1000-4000-fold using alprenolol-Sepharose affinity chromatography. Addition of deoxycholate solubilized egg phosphatidylcholine to the beta-adrenergic receptor, that is 5-10% pure and in 0.1% digitonin, followed by Sephadex G-50 gel filtration in buffers containing 30 mM MgCl2 results in 65-70% of the receptor being incorporated into phospholipid vesicles. The beta-adrenergic receptor as detected by photoaffinity labeling using [125I]azidobenzylpindolol in membranes and after alprenolol-Sepharose chromatography is a Mr = 40,000 peptide. Addition of deoxycholate extracts of human erythrocyte membranes, which contain the guanine nucleotide stimulatory regulatory protein of adenylate cyclase (Ns) but not beta-adrenergic receptor, were used to reconstitute a guanine nucleotide-mediated change in agonist affinity for the receptor. These results demonstrate that the alprenolol-Sepharose affinity purified beta-adrenergic receptor is functional in both ligand binding and coupling to Ns. The procedure is rapid, efficient and should be generally applicable to beta-adrenergic receptor and Ns from several different membrane systems.