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Biomedical subjects

E M Ross

Publications and source records attributed to E M Ross.

At least 73 records · Page 4Linked to original sources
PubMed1990

Chimeric muscarinic cholinergic: beta-adrenergic receptors that activate Gs in response to muscarinic agonists.

The M1-muscarinic cholinergic receptor (M1AChR) stimulates the release of inositol phosphates (IPs) but does not activate adenylyl cyclase. The beta-adrenergic receptor (beta-AR) stimulates adenylyl cyclase but has no effect on IP release. Amino acid sequences corresponding to the second (I2) and third (I3) intracellular loops of the turkey erythrocyte beta-AR and a 12-amino acid segment near the N-terminal end of the I3 region were substituted into the corresponding regions of the human M1AChR. Chimeric receptors that contained either the entire I3 loop or the N-terminal dodecapeptide of that loop both mediated the 2-4-fold stimulation of adenylyl cyclase activity in membrane fractions of COS, A293, or Sf9 cells in response to carbachol. These chimeric receptors also retained the ability to stimulate IP release to the same extent as did the M1AChR. In COS cells transfected with the I3 chimeric receptor, the EC50 for carbachol was approximately 7 microM for the stimulation of adenylyl cyclase and approximately 2 microM for the release of IP; M1AChR-mediated IP release displayed an EC50 of approximately 0.2 microM. Substitution of the I2 region of the beta-AR into the M1AChR did not by itself alter selectivity for signaling. However, the I2+I3 and I2+dodecapeptide combined replacements stimulated adenylyl cyclase fully and caused at most 25% of the maximal stimulation of IP release observed with the M1AChR. Thus, a small region in the third cytoplasmic loop can alter the G proteins to which a receptor is coupled, but interaction among loops is evidently involved in fully determining G protein selectivity.

Adenylyl Cyclases↗
PubMed1989

Pertussis vaccine and severe acute neurological illnesses. Response to a recent review by members of the NCES team.

Dr. A. H. Griffith's article on this subject raises some important issues which require comment. We, like him, regret the controversy over the safety and efficacy of whole cell pertussis vaccines over the last 15 years. It does indeed represent a sorry saga whose principal victims are children, many of whom have not been vaccinated against this unpleasant and sometimes dangerous illness because of fears over safety of the vaccine. The National Childhood Encephalopathy Study (NCES), was set up in 1976 as an independent scientific enquiry into severe acute neurological illnesses associated with pertussis vaccine in an attempt to help resolve the matter. The report on the results concluded that these suggested, but did not prove, that the vaccine may very rarely cause the development of potentially damaging severe acute neurological illnesses in children who were previously apparently neurologically normal. Unfortunately the number of cases in the NCES was too small to allow any firm conclusions on whether or not the vaccine can cause permanent damage. The NCES has since been subject to intense scrutiny and criticism both by those who consider the vaccine can cause permanent neurological damage and by those, such as Dr Griffith, who consider it does not. Regrettably, the controversy continues.

Acute Disease↗
PubMed1988

Rapid binding of guanosine 5'-O-(3-thiotriphosphate) to an apparent complex of beta-adrenergic receptor and the GTP-binding regulatory protein Gs.

When reconstituted phospholipid vesicles that contain purified beta-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the typical receptor-stimulated GTP gamma S binding reaction was preceded by an even more rapid burst of GTP gamma S binding. This burst was studied in detail at 0 degree C. The rate of the burst was second order in nucleotide and Gs [k assoc approximately 2 X 10(7) (M.min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 approximately 15 min at 0 degree C in the presence of agonist and decayed by approximately 3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at at 30 degrees C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.

Animals↗
PubMed1988

The catecholamine binding site of the beta-adrenergic receptor is formed by juxtaposed membrane-spanning domains.

The catecholamine binding domain of the turkey erythrocyte beta-adrenergic receptor was mapped by determining the sites of covalent labeling of the purified receptor by two beta-adrenergic photoaffinity reagents, [125I]iodocyanopindolol-diazirine (ICYP-da) and [125I] iodoazidobenzylpindolol (IABP). Both labels were incorporated at two separate sites. By sequencing a labeled peptide, one site of labeling was found to lie at Trp330 in the extracellular half of the seventh membrane span. This position is homologous to the retinal attachment site in rhodopsin. The second labeled site was isolated on an 8000-Da peptide and immunoprecipitated using sequence-directed antibodies. This site lies in membrane spans 3-5. Labeling of the two sites was equal using ICYP-da and 3-10-fold greater in the span 7 site using IABP. These data indicate that the catecholamine binding site is formed from the juxtaposition of span 7 and spans 3-5 in a tertiary structure probably similar to that of rhodopsin.

Affinity Labels↗
PubMed1988

Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins).

Mastoparan, a peptide toxin from wasp venom, is a nonspecific secretagogue. We show here that mastoparan increases the GTPase activity and the rate of nucleotide binding of several purified GTP-binding regulatory proteins (G proteins) whose function is to couple cell-surface receptors to intracellular mediators. Mastoparan accelerated guanosine-5'-(3-O-thiotriphosphate binding and consequent G protein activation in part by promoting the dissociation of bound GDP, the mechanism by which receptors regulate G proteins. ADP-ribosylation by pertussis toxin, which uncouples receptors from G proteins, selectively inhibited mastoparan-stimulated activation. Like receptors, mastoparan was more potent if the G protein was reconstituted in phospholipid vesicles and was active at micromolar concentrations of Mg2+. The structure of mastoparan in a lipid bilayer is similar to that predicted for a cationic intracellular loop of G protein-coupled receptors. Mastoparan thus displays a novel mode of toxicity by acting directly on G proteins to mimic the role normally played by agonist-liganded receptors.

Adenosine Diphosphate Ribose↗
PubMed1988

Beta-adrenergic receptors display intramolecular disulfide bridges in situ: analysis by immunoblotting and functional reconstitution.

The molecular nature of mammalian beta-adrenergic receptors in situ was probed using immunoblotting and functional reconstitution techniques. Membrane proteins of cells replete with beta-adrenergic receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose and then probed with anti-receptor antibodies. When cell membranes were first treated with agents that cleave disulfides of proteins, immunoblots of these membranes revealed intense immunoreactive bands with electrophoretic mobility similar to that of protein standards of Mr 65,000-67,000, comigrating with purified, reduced, and alkylated beta-adrenergic receptors. However, when cell membranes were prepared under anaerobic conditions, solubilized in the presence of agents that alkylate thiols, and denatured in the absence of added thiols, immunoblotting revealed receptor with Mr 55,000, rather than 65,000. This faster electrophoretic mobility is associated with the presence of intramolecular disulfides in the purified receptor and demonstrates that beta-adrenergic receptors possess intramolecular disulfide bridges in situ. Purified receptors that demonstrate this faster mobility (Mr 55,000 under nonreducing conditions) were co-reconstituted into phospholipid vesicles with the stimulatory GTP-binding protein GS and their ability to catalyze the binding of [35S]guanosine-5'-O-(3-thio)triphosphate to GS was measured. Agonist (isoproterenol) as well as thiol increased the receptor-promoted activation of GS. Taken together, these data demonstrate that native beta-adrenergic receptors possess one or more intramolecular disulfide bridges in situ, reduction of which causes functional activation of the receptor.

Animals↗
PubMed1987

The hydrophobic tryptic core of the beta-adrenergic receptor retains Gs regulatory activity in response to agonists and thiols.

The function of structural domains of the beta-adrenergic receptor were probed by studying the ability of tryptic fragments of the receptor to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) to the GTP-binding regulatory protein, Gs. beta-Adrenergic receptor purified from turkey erythrocytes was treated with trypsin under nondenaturing conditions. Such treatment decreased beta-adrenergic ligand binding activity by only 15-25%. Active components of the limit digest were repurified by affinity chromatography on alprenolol-agarose and then reconstituted with purified Gs into unilamellar phospholipid vesicles. After reconstitution, the proteolyzed receptor was able to catalyze agonist-stimulated binding of GTP gamma S to Gs at a rate and extent equivalent to that of the nonproteolyzed receptor. The proteolyzed receptor was also partially activated upon reduction by dithiothreitol, as previously reported for the intact receptor (Pedersen, S.E., and Ross, E.M. (1985) J. Biol. Chem. 260, 14150-14157). The repurified, active tryptic digest contained two detectable peptides. One, of approximately 2 X 10(4) Da, contained either four or five of the amino-terminal membrane-spanning domains plus the intervening hydrophilic loops but not the amino-terminal extracellular, glycosylated peptide. The second, of 9,000-10,000 Da, was composed essentially of the two carboxyl-terminal membrane-spanning domains and the intervening extracellular, hydrophilic loop. These data indicate that most of the large intracellular hydrophilic loop and the hydrophilic, carboxyl-terminal region of the receptor are not necessary for the agonist-stimulated regulation of Gs.

Adrenergic beta-Agonists↗
PubMed1987

The effect of activating ligands on the intrinsic fluorescence of guanine nucleotide-binding regulatory proteins.

The intensity of the tryptophan fluorescence of the alpha subunits of guanine nucleotide-binding regulatory proteins increases when they bind guanosine 5'-O-(3-thio)triphosphate (GTY gamma S). The kinetics of the fluorescence enhancement and of the measured binding of [35S]GTP gamma S are well correlated. The addition of Mg2+ to the nucleotide-bound proteins causes a further, rapid increase in the fluorescence intensity. Similar effects result from exposure of the proteins to F- and Mg2+, and the required concentration of F- is reduced by the inclusion of Al3+. It is presumed that the more highly fluorescent state of the G protein alpha subunits represents their active conformation.

Aluminum↗
PubMed1986

Effect of Al3+ plus F- on the catecholamine-stimulated GTPase activity of purified and reconstituted Gs.

The effects of Al3+ and F- on the catecholamine-stimulated GTPase cycle were studied by using reconstituted phospholipid vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. Al3+/F- activated reconstituted Gs to levels previously reported for detergent-solubilized, purified Gs, although both activation and deactivation were faster in the reconstituted preparation. Under these conditions, Al3+/F- did not inhibit by more than 15% the beta-adrenergic agonist-stimulated GTPase activity of the vesicles nor did it significantly inhibit the rates of GTP binding, GTP hydrolysis, or GDP release. When Mg2+ (50 mM) was used instead of agonist to promote GTP hydrolysis in the receptor-Gs vesicles, Al3+/F- was found to inhibit GTP gamma S binding, GDP release, and steady-state GTPase activity to unstimulated levels. These data can be interpreted as indicating that the receptor catalyzes nucleotide exchange by Gs faster or more efficiently than does Mg2+.

Aluminum↗
PubMed1986

Catecholamine-stimulated GTPase cycle. Multiple sites of regulation by beta-adrenergic receptor and Mg2+ studied in reconstituted receptor-Gs vesicles.

The regulation of the intermediary steps of the catecholamine-stimulated GTPase cycle by beta-adrenergic agonists and Mg2+ was investigated using unilamellar phosphatidylethanolamine-phosphatidylserine vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. The steady-state turnover number of the agonist-stimulated GTPase, normalized according to the receptor-responsive pool of Gs, was 0.8 min-1 for untreated vesicles and 1.7 min-1 for vesicles that had been treated with dithiothreitol to activate the receptors. The binding and release of [alpha-32P]GTP, [3H] GTP, and [gamma-32P]GTP were used to measure the binding and hydrolysis of GTP and the release of GDP. Agonist-liganded receptor stimulated both the binding of GTP and the release of the GDP product, and GDP release per se did not appear to be the mechanism by which receptor stimulated the binding of GTP. Both processes displayed apparent first order rate constants of about 0.5 min-1 for untreated vesicles and both rates increased about 5-fold after dithiothreitol treatment. Both processes were formally catalytic with respect to receptor, in that several (up to 8) molecules of Gs were stimulated per molecule of receptor. The hydrolysis of Gs X GTP to Gs X GDP was unaltered by agonist and occurred with a rate constant of about 4 min-1. The rates of these partial reactions were consistent with the overall rate of steady-state hydrolysis and with the ability of the agonist-liganded receptor to promote the formation of sufficient Gs X GTP to fully stimulate adenylate cyclase in a native membrane. The Mg2+ dependence of agonist-stimulated, steady-state GTPase activity appeared to consist of at least two, distinct Mg2+-requiring processes. Very low concentrations of Mg2+ (approximately 20 nM) were required for hydrolysis of Gs X GTP, and 10 microM Mg2+ was required to maximize the initial rate of agonist-stimulated [alpha-32P] GTP binding.

Adrenergic beta-Agonists↗
PubMed1986

Severe atherosclerotic coronary artery disease, healed myocardial infarction and chronic congestive heart failure: analysis of 81 patients studied at necropsy.

Observations are described in 81 necropsy patients (aged 29 to 91 years [mean 62]; 77 [95%] men) with severe congestive heart failure (CHF) more than 3 months in duration, left ventricular (LV) transmural scar and greater than 75% cross-sectional area narrowing by atherosclerotic plaque of 1 or more of the 4 major epicardial coronary arteries. The duration of symptoms from initial onset of acute myocardial infarction (59 patients) or CHF (18 patients) or angina pectoris (2 patients) to death ranged from 0.5 to 18 years (mean 7.1) (2 unknown). Angina pectoris occurred at some time, however, in 31 patients (38%). Cause of death was CHF in 48 patients (59%), sudden (arrhythmia) in 16 (20%), acute myocardial infarction in 11 (14%), and emboli in 6 (7%). The heart weight ranged from 410 to 800 g (mean 585). Left or right ventricular thrombi or both occurred in 37 patients (46%), only 4 (10%) of whom had systemic emboli; of the 44 patients without intracardiac thrombi, none had any form of emboli. The severity of coronary narrowing was variable. In 24 patients (30%) only 1 artery was narrowed greater than 75% in cross-sectional area; in 22 patients (27%), 2 arteries were so narrowed; in 32 patients (39%), 3 arteries; and in 3 patients (4%), 4 arteries. The size of the LV scar also varied. Of the 81 patients, 58 (72%) had large scars (involving greater than 40% of the LV wall); 10 (12%) had moderate-sized scars (6 to 40% of the LV wall); and 13 (16%) had small scars (less than or equal to 5% of the LV wall).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1986

Severe atherosclerotic coronary arterial narrowing and chronic congestive heart failure without myocardial infarction: analysis of 18 patients studied at necropsy.

Observations are reported on 18 patients (aged 38 to 73 years ([mean 58]; 16 [89%] men) studied at necropsy who had had chronic congestive heart failure (CHF) more than 3 months in duration, greater than 75% cross-sectional area (XSA) narrowing of 1 or more of the 4 major epicardial coronary arteries, and no left ventricular fibrosis or necrosis. Duration of symptoms from onset of CHF to death ranged from 0.3 to 13 years (mean 5.7). Angina pectoris occurred in 2 patients (11%). The mode of death was CHF in 12 (67%), sudden (arrhythmia) in 5 (28%), and emboli in 1 (5%). Heart weight ranged from 410 to 890 g (mean 632). Of 72 major epicardial coronary arteries (right, left main, left anterior descending, left circumflex) in the 18 patients, 30 (42%) were narrowed 76 to 100% in XSA by atherosclerotic plaque (right = 10, left main = 0, left anterior descending = 9 and left circumflex = 11). A mean of 1.7 of 4 major epicardial coronary arteries per patient were narrowed 76 to 100% in XSA by atherosclerotic plaque. In 10 patients, each 5-mm segment of the 4 major coronary arteries was examined histologically (mean 53 per patient): 23 segments (3%) were narrowed 96 to 100% in XSA by atherosclerotic plaque; 58 (11%), 76 to 95; 93 (18%), 51 to 75%; 209 (40%), 26 to 50%, and 146 (28%), 0 to 25%. Left and right ventricular thrombi were found in 9 patients (50%); of the 9 patients, 1 had a systemic embolus; of the 9 patients without intraventricular thrombi, none had systemic emboli.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗