Turkey Erythrocyte Plasma Membranes Research Articles

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.

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.

32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor.

A 32P-labelled ATP analog, 3'-O-(4-benzoyl)benzoyl ATP (BzATP) previously shown to be an agonist at P2Y-purinergic receptors (Boyer J. L., and Harden T. K. (1989) Mol. Pharmacol. 36, 831-835), has been used as a probe for the P2Y-purinergic receptor on turkey erythrocyte plasma membranes. In the absence of light, [32P]BzATP bound to membranes with high affinity (KD approximately 5 nM), and in a saturable and reversible manner. The binding of [32P]BzATP was competitively inhibited by ATP and ADP analogs (2-methylthioadenosine 5'-triphosphate greater than adenosine 5'-O-(2-thiodiphosphate) greater than BzATP greater than ATP greater than beta,gamma-methyleneadenosine 5'-triphosphate greater than 5'-adenylylimidodiphosphate) with pharmacological specificity consistent with that of a P2Y-purinergic receptor. Guanine nucleotides (guanosine 5'-O-(3-thiotriphosphate) greater than GTP greater than guanosine 5'-O-(2-thiodiphosphate) greater than GMP) noncompetitively inhibited the binding of radioligand. Photolysis of [32P] BzATP-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 53,000 Da. Photolabeling was inhibited in a concentration-dependent manner by ATP and ADP analogs with a potency order characteristic for a P2Y-purinergic receptor and was modulated by guanine nucleotides. A protein of approximately 53,000 daltons was also labeled by [32P]BzATP in membranes from several other tissues known to express the P2Y-purinergic receptor. These results suggest that [32P]BzATP can be used to label covalently the P2Y-purinergic receptor and that this radioprobe will be a useful reagent for further characterization and purification of the P2Y-purinergic receptor.

Amino acid specific ADP-ribosylation: specific NAD: arginine mono-ADP-ribosyltransferases associated with turkey erythrocyte nuclei and plasma membranes.

Turkey erythrocytes contain NAD:arginine mono-ADP-ribosyltransferases which, like cholera toxin and Escherichia coli heat-labile enterotoxin, catalyze the transfer of ADP-ribose from NAD to proteins, to arginine and other low molecular weight guanidino compounds, and to water. Two such ADP-ribosyltransferases, A and B, have been purified from turkey erythrocyte cytosol. To characterize further the class of NAD:arginine ADP-ribosyltransferases, the particulate fraction was examined; 40% of erythrocyte transferase activity was localized to the nucleus and cell membrane. Transferase activity in a salt extract of a thoroughly washed particulate preparation was purified 36,000-fold by sequential chromatography on phenyl-Sepharose, (carboxymethyl) cellulose, concanavalin A-Sepharose, and NAD-agarose. Subsequent DNA-agarose chromatography separated two activities, termed transferases C and A', which were localized to the membrane and nucleus, respectively. Transferase C, the membrane-associated enzyme, was distinguished from the cytosolic enzymes by a relative insensitivity to salt and histone; transferase C was stimulated 2-fold by 300 mM NaCl in contrast to a 20-fold stimulation of transferase A and a 50% inhibition of transferase B. Similarly, histones, which stimulate transferase A 20-fold, enhanced transferase C activity only 2-fold. Transferase A', the nuclear enzyme, was retained on DNA-agarose. It was similar to transferase A in salt and histone sensitivity. Gel permeation chromatography showed slight molecular mass differences among the group of enzymes: A, 24,300 daltons (Da); B, 32,700 Da; C, and A', 25,500 Da. The affinities of transferase C for NAD and agmatine were similar to those of the cytosolic transferases A and B.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional activation of beta-adrenergic receptors by thiols in the presence or absence of agonists.

Treatment of beta-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained beta-adrenergic receptors, purified to greater than or equal to 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (Gs) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0 degrees C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of Gs by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding, an effect about equivalent to the addition of beta-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTP gamma S binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity, and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTP gamma S binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on Gs alone. Thus, the site of action of DTT appears to be on the beta-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes.

Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor.

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.

Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase.

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characterization of insulin receptors. I. Hydrodynamic properties of receptors from turkey erythrocytes.

Insulin receptors from turkey erythrocyte plasma membranes were solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate). Their hydrodynamic properties were determined by sedimentation analyses in H2O and D2O, and gel filtration on Sepharose 4B. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peaks I and II. In Triton X-100, the sedimentation coefficient (s20,w), partial specific volume (Vc), and Stokes radius (a) for peaks I and II are, respectively, 10.2 +/- 0.5 S and 6.6 +/- 0.5 S, 0.75 +/- 0.02 ml/g, and 0.76 +/- 0.02 ml/g, and 89 +/- 3 A and 76 +/- 3 A, to yield Mr = 410,000 +/- 75,000 and 235,000 +/- 55,000, respectively, for the protein-Triton X-100 complex. The corresponding values in deoxycholate solution are: 10.7 +/- 0.5 S and 6.9 +/- 0.5 S, 0.71 +/- 0.03 ml/g and 0.70 +/- 0.04 ml/g, and 86 +/- 3 A and 69 +/- 3 A for peaks I and II, respectively, to yield 360,000 +/- 65,000 and 180,000 +/- 45,000, respectively, for the molecular weight of the protein-deoxycholate complex. These data are consistent with a model whereby each receptor species binds to one micelle of the appropriate detergent. In agreement with this model, it was also found that, in both Triton X-100 and deoxycholate, concentrations higher than the critical micellar concentration are required in order to maintain discrete receptor species in solution. At concentrations below the critical micellar concentration, the receptors aggregate to a broad band that sediments faster than 11.3 S. This is typical of membrane proteins that are stabilized in solution by insertion into detergent micelles. Based on these results, the protein molecular weights of peaks I and II are estimated to be 355,000 +/- 65,000 and 180,000 +/- 45,000, respectively. When membranes are treated with the reducing agent dithiothreitol, peak I is converted to peak II. This fact, together with the estimates obtained for the protein molecular weights of the two receptor species, suggests that peak I is a disulfide-linked dimer of peak II. The sedimentation characteristics of insulin receptors in many different cell types appear to be similar. As with turkey erythrocytes, detergent extracts of membranes from rat liver contained two native receptor species whose sedimentation coefficients were similar to peaks I and II. However, in all the other cell types examined, including rat adipocytes, rat heart muscle, 3T3-L1 adipocytes, 3T3-C2 fibroblasts, and FAO hepatoma cells, peak I (the native dimer) was the predominant species observed.

Structural characterization of insulin receptors. II. Subunit composition of receptors from turkey erythrocytes.

In the preceding paper (Aiyer, R. A. (1983) J. Biol. Chem. 258, 14992-14999), the hydrodynamic properties of insulin receptors from turkey erythrocyte plasma membranes solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate) were characterized. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peak II whose protein molecular weight (Mp) is 180,000 +/- 45,000 and its disulfide-linked dimer, peak I (Mp, 355,000 +/- 65,000). This paper describes the subunit composition of these species determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Insulin receptors were covalently attached to [125I]iodoinsulin with disuccinimidyl suberate. After solubilization in Triton X-100 or deoxycholate, peaks I and II were separated by sedimentation and subjected to SDS-PAGE; the constituent polypeptides were then identified by autoradiography. Under reducing conditions, both peaks I and II yield a major band of apparent molecular weight (Mapp) of 135,000; this band most likely represents the insulin-binding subunit (alpha). Minor bands of lower molecular weight are also seen whose significance is not entirely obvious. Under nonreducing conditions, peak I yields bands at Mapp = 230,000 and at greater than 240,000, while peak II yields bands at Mapp = 120,000 and 200,000. When these bands were cut out of the gel and subjected to SDS-PAGE following reduction with 10% beta-mercaptoethanol, all of them produced a single band that migrated with Mapp = 135,000. These results indicate that the alpha subunit is linked by disulfide bonds to at least one more subunit (beta). It is also apparent that the alpha subunit travels with higher mobility (Mapp = 120,000) under nonreducing conditions, suggesting the presence of intrachain disulfide bonds. Thus, peak II has a minimum subunit composition of alpha beta, where alpha is the insulin-binding subunit with a minimum Mr = 120,000-135,000 and beta has a minimum Mr = 80,000-90,000. And peak I, the disulfide-linked dimer of peak II, has a minimum subunit composition of alpha 2 beta 2. These results were further confirmed by cross-linking of protein subunits with glutaraldehyde, an (alpha, omega)-dialdehyde that reacts with amino groups. Within the limits of error, these molecular weights are in agreement with those estimated from the hydrodynamic properties of the detergent-solubilized, native receptor species reported in the preceding paper.

Functional reconstitution of beta-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase.

A procedure for the functional reconstitution of beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. beta-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of beta-adrenergic agonists. This stimulation of the activation of G/F was specific for beta-adrenergic agonists and could be specifically blocked by beta-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active beta-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F by receptor was reconstituted.

The regulatory component of adenylate cyclase. Purification and properties of the turkey erythrocyte protein.

The regulatory component (G/F) of adenylate cyclase has been purified from turkey erythrocyte plasma membranes by adaptation of procedures developed for purification of the rabbit liver protein. The major modifications entail inclusion of high concentrations of NaCl to facilitate extraction and reconstitution of the protein. A typical preparation yields 200 micrograms of protein with a reconstitutive specific activity of 3-4 mumol . min-1 mg-1. Turkey erythrocyte G/F contains two putative subunits of 35,000 and 45,000 daltons. The 52,000-dalton polypeptide that appears to be a component of rabbit liver G/F is lacking. In solution, G/F behaves as a particle with Mr = 81,000. This value is reduced to 50,000 in the presence of activating ligands, suggesting dissociation of subunits. Activation of G/F by guanine nucleotide analogs is markedly accelerated in the presence of high concentrations of Mg2+. Reconstitutive and physical properties of the protein are also affected by fluoride. Cyc- S49 lymphoma membranes reconstituted with turkey erythrocyte G/F acquire properties that are characteristic of the turkey adenylate cyclase system; at least certain differing characteristics of adenylate cyclase systems are thus dictated by the nature of their G/F.

Hormonal control of protein phosphorylation in turkey erythrocytes. Phosphorylation by cAMP-dependent and Ca2+-dependent protein kinases of distinct sites in goblin, a high molecular weight protein of the plasma membrane.

In previous studies, a correlation was observed between isoproterenol-responsive Na+-K+ co-transport in turkey erythrocytes and increased phosphorylation of goblin, an Mr = 230,000 protein of the turkey erythrocyte plasma membrane. The phosphorylation of specific sites in goblin has now been analyzed by tryptic fingerprinting. Three major phosphopeptides were detected in goblin prepared from intact, 32P-labeled erythrocytes. One of the peptides 1, was maximally phosphorylated in the absence of hormonal agents. Two additional peptides, 2 and 3, were phosphorylated only following exposure of cells to the beta-adrenergic agonist isoproterenol, to cAMP plus isobutylmethylxanthine, or to cholera toxin. In cells stimulated by isoproterenol, phosphorylation of goblin peptides 2 and 3 could be selectively and completely reversed by subsequent addition of the beta-adrenergic antagonist propranolol. Addition of either cAMP or of Ca2+ plus calmodulin to purified turkey erythrocyte plasma membranes increased incorporation of 32P into goblin. Peptides 2 and 3 of goblin were phosphorylated by addition to the membranes of cAMP or of purified cAMP-dependent protein kinase. Two additional goblin peptides, 4 and 5, were phosphorylated in the plasma membrane preparation by addition of purified calmodulin plus Ca2+, whereas peptides 2 and 3 of goblin were not phosphorylated under these conditions. Peptide 1 did not incorporate 32P in the plasma membranes under any condition tested. Both calmodulin and cAMP-dependent protein kinase were identified directly in turkey erythrocytes. The three major phosphopeptides of goblin phosphorylated in intact cells (peptides 1, 2, and 3) contained phosphothreonine and represented distinct phosphorylation sites. In contrast, the two phosphopeptides of goblin phosphorylated in plasma membranes by addition of Ca+/calmodulin (peptides 4 and 5) contained phosphoserine. It is concluded that goblin, a plasma membrane protein possibly involved in the hormonal regulation of Na+-K+ co-transport, contains at least 3 distinct threonine residues and 1 or more serine residues which serve as specific substrates for three or more distinct protein kinases of the turkey erythrocyte, namely a cAMP-dependent enzyme, a Ca2+/calmodulin-dependent enzyme, and a third enzyme with undetermined regulatory control.

Purification of β-adrenergic receptors: The search for an adequate affinity adsorbent

Several agonist and antagonist adrenergic agents were linked to spacer arms on agarose, and tested for their ability to absorb β-adrenergic receptors from solubilized turkey erythrocyte plasma membranes. By comparing the adsorption characteristics of the ligand-bearing gels with those of the corresponding spacer arm-agarose conjugates, only a newly synthetized alprenolol-agarose conjugate behaved as a biospecific affinity adsorbent.

Ligand specificity and characteristics of the β-adrenergic receptor in turkey erythrocyte plasma membranes

Detailed kinetic studies of adenylate cyclase activation by catecholamines in turkey erythrocyte plasma membranes were carried out in order to characterize the ligand specificity of the β -adrenergic receptor. A general kinetic method was used to evaluate the affinity of the β -receptor towards full-agonists, partial agonists and antagonists. This method allowed us to study ligands that bind with high affinity as well as ligands that bind with low affinity. Direct binding studies of [ 3 H]propranolol to the β -receptor were used to study the ligand specificity and the modulation of that specificity by regulatory ligands known to activate or inhibit adenylate cyclase stimulation by l -catecholamines. A very close correspondence between the binding constants obtained by the kinetic method and the binding method was found. It was found that the allosteric activator of adenylate cyclase, guanylylimidodiphosphate, and the allosteric inhibitor, Ca 2+ , do not affect propranolol binding.

Effects of beta-adrenergic catecholamines on potassium transport in turkey erythrocytes.

Isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a 2- to 4-fold increase in bidirectional potassium fluxes in turkey erythrocytes. Ouabain, which does not alter catecholamine-stimulated cellular cyclic AMP, inhibits potassium influx by 50 to 70%, does not alter potassium outflux or isoproterenol-stimulated potassium influx, but increases isoproterenol-stimulated potassium outflux. Stimulation of potassium transport by isoproterenol can be reproduced by adding cyclic AMP to the medium and is inhibited by propranolol or dichloroisoproterenol but not by phentolamine. Theophylline at concentrations which inhibit cyclic nucleotide phosphodiesterase in isolated turkey erythrocyte plasma membranes by greater than 90%, does not augment isoproterenol stimulation of cellular cyclic AMP or of potassium transport but does potentiate stimulation of potassium influx produced by adding cyclic AMP to the medium. Isoproterenol-stimulated cellular cyclic AMP increases steadily for at least 2 hours. Potassium transport, however, increases rapidly, becomes maximal after 20 to 30 min of incubation, and thereafter decreases progressively so that after 2 hours of incubation potassium fluxes are only slightly greater than for the control. Ouabain prolongs the duration of catecholamine-stimulated potassium influx and potassium outflux, reflecting its ability to relieve the refractoriness developed by turkey erythroyctes to endogenous cyclic AMP.