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Biomedical subjects

E M Ross

Publications and source records attributed to E M Ross.

At least 91 records · Page 5Linked to original sources
PubMed1985

Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins.

beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate cyclase was estimated to be about 1:10:1. Adenylate cyclase activity in the vesicles was stimulated up to 2.6-fold by beta-adrenergic agonists. Stimulation was dependent on the presence of guanine nucleotide, displayed appropriate beta-adrenergic selectivity and stereoselectivity for agonists, and was blocked appropriately by beta-adrenergic antagonists. Therefore, while additional proteins may modulate adenylate cyclase activity in native membranes, these results show that these three proteins are sufficient for the expression of hormone-stimulated adenylate cyclase.

Adenylyl Cyclases↗
PubMed1985

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.

Adenylyl Cyclases↗
PubMed1985

GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates.

The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [alpha-32P]- or [gamma-32P] GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S) was used to quantitate the total concentration of Gs. 1) GTPase activity was a saturable function of the concentration of GTP, with Km = 0.3 microM. MgCl2 monotonically increased the activity. The maximum observed turnover number was about 1.5 min-1. 2) During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t 1/2 less than or equal to 5 s at 30 degrees C and t 1/2 approximately 1 min at 0 degrees C. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. 3) The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concentrations of either GTP or MgCl2 (above 0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was constant at 0.3-0.4. 4) The rate of dissociation of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t 1/2 of about 1 min. 5) Lubrol 12A9 potently inhibited the GTPase reaction and the dissociation of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. 6) The beta and gamma subunits of Gs markedly inhibited the dissociation of GDP from Gs in contrast to their ability to stimulate the dissociation of GTP gamma S. 7) GDP, GTP gamma S, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively inhibited the accumulation of Gs-GDP. GTP gamma S and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and Pi did not inhibit. These data are interpretable in terms of the coexistence of two specific mechanistic pathways for the overall GTPase reaction.

Adenylyl Cyclases↗
PubMed1985

The carcinoid syndrome: comparison of 21 necropsy subjects with carcinoid heart disease to 15 necropsy subjects without carcinoid heart disease.

Carcinoid heart disease is a morphologically specific type of cardiac disorder that involves the mural and valvular endocardium on the right side of the heart. Twenty-one subjects (57 percent) (Group I) with carcinoid heart disease and 15 subjects (43 percent) (Group II) without carcinoid heart disease were studied at necropsy. The two groups were similar in mean age (54 years versus 55 years), duration of clinical illness (4.7 years versus 6.3 years), body weight (50 kg versus 52 kg), systemic blood pressure (117/77 mm Hg versus 128/77 mm Hg), blood hematocrit levels (37 percent versus 36 percent), total serum protein levels (6.0 g/dl), and serum albumin levels (2.2 g/dl versus 2.6 g/dl). The two groups were different in the frequency of the presence of precordial murmurs consistent with tricuspid regurgitation and/or pulmonic stenosis (95 percent versus 13 percent), cardiomegaly by chest radiography (38 percent versus 0), low voltage on electrocardiography (47 percent versus 0), and location of the primary site of the carcinoid tumor. Total electrocardiographic 12-lead QRS voltage was similar in each group (105 mm versus 132 mm) (10 mm = 1 mV). Of Group I subjects, 43 percent died of cardiac causes; none of the Group II subjects died of cardiac causes. Of the 21 subjects with carcinoid heart disease, seven had left-sided cardiac involvement, but in none was it of functional significance. Thus, although carcinoid heart disease frequently is the cause of death in patients with the carcinoid syndrome, the development of carcinoid heart disease is not related to the duration of symptoms of the carcinoid syndrome.

Adult↗
PubMed1984

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)

Animals↗
PubMed1984

Catecholamine-stimulated guanosine 5'-O-(3-thiotriphosphate) binding to the stimulatory GTP-binding protein of adenylate cyclase: kinetic analysis in reconstituted phospholipid vesicles.

The stimulatory GTP-binding protein of adenylate cyclase, Gs, and beta-adrenergic receptors were reconstituted into unilamellar phospholipid vesicles. The kinetics of the quasiirreversible binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gs, equivalent to Gs activation by nucleotide, was studied with respect to the stimulation of this process by beta-adrenergic agonists and Mg2+. The rate of GTP gamma S binding displayed apparent first-order kinetics over a wide range of nucleotide, agonist, and Mg2+ concentrations. In the absence of agonist, the apparent first-order rate constant, kapp, was 0.17-0.34 min-1 and did not vary significantly with the concentration of nucleotide. At 50 mM MgCl2, kapp increased somewhat, to 0.26-0.41 min-1, and remained invariant with the nucleotide concentration. In the presence of agonist, kapp was dependent on nucleotide concentration. At 10(-9) M GTP gamma S, the addition of (-)-isoproterenol caused at most a 2-fold stimulation of kapp. However, kapp measured in the presence of isoproterenol increased as an apparently saturable function of the GTP gamma S concentration, such that isoproterenol caused a 17-fold increase in kapp at 1 microM GTP gamma S. The effect of isoproterenol on kapp also appeared to saturate at high isoproterenol concentration, yielding a kapp approximately 6 min-1 at high concentrations of both nucleotide and agonist. These data suggest that the receptor-agonist complex acts by increasing the rate of conversion of a lower affinity Gs-GTP gamma S complex to the stable activated state.

Animals↗
PubMed1984

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles.

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.

Adenylate Cyclase Toxin↗
PubMed1983

Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity.

beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver. These proteins were reconstituted into phospholipid vesicles by addition of phospholipids and removal of detergent by gel filtration. This preparation hydrolyzes GTP to guanosine 5'-diphosphate (GDP) plus inorganic phosphate (Pi) in response to beta-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed min-1 X mol-1 of Gs. This low rate may be limited by the hormone-stimulated binding of substrate, since it is roughly equal to the rate of binding of the GTP analogue guanosine 5'-O-(3-[35S] thiotriphosphate) [( 35S]GTP gamma S) to Gs in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a beta-adrenergic antagonist, is 8-25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 microM GTP, and the response to GTP is formally consistent either with the existence of multiple Km's or of a separate stimulatory site for GTP. The GTPase activity of Gs in vesicles is also stimulated by 50 mM MgCl2 in the presence or absence of receptor. Significant GTPase activity is not observed with Lubrol-solubilized Gs, although [35S]-GTP gamma S binding is increased by Lubrol solubilization.

Adenylyl Cyclases↗
PubMed1983

Infantile spasms and pertussis immunisation.

The possible roles of pertussis immunisation and of other factors in the aetiology of infantile spasms were investigated by analysis of 269 cases reported to the National Childhood Encephalopathy Study. In 34% of the cases an antecedent factor which may have caused infantile spasms was identified; the commonest of these were perinatal hypoxia (38 cases) and tuberous sclerosis (16 cases). Case-control analyses showed no significant association between infantile spasms and pertussis immunisation in the 28 days before onset. There was, however, some clustering of cases immunised with either diphtheria-tetanuspertussis or diphtheria-tetanus vaccines in the 7 days before onset. The excess compared with controls was compensated for by a corresponding deficit over the remaining period up to 28 days. It is suggested that these vaccines do not cause infantile spasms but may trigger their onset in those children in whom the disorder is destined to develop.

Acute Disease↗
PubMed1983

Regulation of the catalytic component of adenylate cyclase. Potentiative interaction of stimulatory ligands and 2',5'-dideoxyadenosine.

Both adenosine and the P site agent 2',5'-dideoxyadenosine (DDA) reversibly inhibit adenylate cyclase activity in two different preparations of the enzyme that lack the stimulatory guanine nucleotide-binding protein, G/F: plasma membranes from the cyc- variant of S49 lymphoma cells and the resolved catalytic component (C) from rabbit liver. P site agents do not compete with any of the activators of C (Mn2+, G/F, forskolin), nor do they diminish the potency of any activator of C. Rather, activation of C increases the effectiveness of P site agents and causes up to a 10,000-fold increase in the potency of DDA. The ability of G/F or forskolin to potentiate P site inhibition is also noted at concentrations much lower than those required for the stimulation of adenylate cyclase activity. These data are inconsistent with a simple two-state allosteric model for the regulation of the activity of C and demand the postulation of either a distinct, inhibited conformation of the enzyme or the existence of a dead-end complex with adenosine bound to the catalytic site.

Adenylyl Cyclase Inhibitors↗