Adrenocorticotropin- and opiate-like hormones from pituitaries of the sockeye salmon Oncorhynchus nerka

1987 ◽  
Vol 65 (4) ◽  
pp. 386-391 ◽  
Author(s):  
T. B. Ng ◽  
W. K. Hon ◽  
D. R. Idler
Keyword(s):  
Rat Brain ◽  
Sockeye Salmon ◽  
Oncorhynchus Nerka ◽  
Opiate Receptors ◽  
Opiate Receptor ◽  
Exchange Chromatography ◽  
Acetone Powder ◽  
Rat Brain Membranes ◽  
Acetone Water ◽  
Opiate Peptides

The pituitaries of vitellogenic sockeye salmon (Oncorhynchus nerka) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate which formed upon the addition of a copious volume of acetone to the extract, designated acid acetone powder, was subjected to salt fractionation and desalting, followed by ion-exchange chromatography on CM-cellulose. An unadsorbed fraction (S-1) and four adsorbed fractions (S-2, S-3, S-4, and S-5) were obtained. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal decapsular cells to produce corticosterone and by their immunoreactivities in an adrenocorticotropin-specific radioimmunoassay. The steroidogenic activities of all fractions, except S-4, were blocked by corticotropin inhibiting peptide. Opiate activity was detected in the fractions by their ability to inhibit the binding of either [3H]naloxone or (D-ala2,D-leu5)-[3H]enkephalin to rat brain membranes. There was a discrepancy in the potencies of the five fractions in the two opiate radioreceptor assays, indicating the presence of opiate peptides with different affinities of binding to the μ- and δ-opiate receptors of the rat brain. There was a separation between adrenocorticotropic and opiate receptor binding activities, suggesting that the activities were due to separate molecular entities.

Adrenocorticotropin- and β-endorphin-like substances in brains of the freshwater snake Ptyas mucosa

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1012-1018 ◽  
Author(s):  
T. B. Ng ◽  
Alfred S. L. Ng ◽  
C. C. Wong
Keyword(s):  
Molecular Weight ◽  
Ammonium Acetate ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Exchange Chromatography ◽  
Acetone Powder ◽  
Concentration Gradients ◽  
Adrenal Cells ◽  
Rat Brain Membranes ◽  
Acetone Water

Snake (Ptyas mucosa) brains (400 g) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate (5.6 g) that formed upon addition of five volumes of acetone to the extract, designated acid–acetone powder, was subjected to gel filtration on Sephadex G-25. A large unretarded peak (SB-1) with molecular weight greater than 5000 and a small retarded peak (SB-2) with molecular weight smaller than 5000 were obtained. They were then separately subjected to ion-exchange chromatography on CM-cellulose. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal cells to produce corticosterone. Opiate activity was detected in the fractions by their ability to inhibit the binding of (D-Ala2,D-Leu5)-[tyrosyl-3,5-3H]enkephalin to rat brain membranes and their cross-reactivity in a β-endorphin radioimmunoassay. Adrenocorticotropic and opiate activities were found to be concentrated in fractions strongly adsorbed on CM-cellulose, which were eluted by combined pH and ammonium acetate concentration gradients. There appeared to be a separation between adrenocorticotropic and opiate activities, suggesting that they were due to separate molecular entities.Key words: adrenocorticotropin, β-endorphin, snake brains.

scholarly journals Purification of the G-protein G13 from rat brain membranes

1994 ◽  
Vol 303 (1) ◽  
pp. 135-140 ◽  
Author(s):  
R Harhammer ◽  
B Nürnberg ◽  
K Spicher ◽  
G Schultz
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
G Proteins ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain Membranes ◽  
Rat Brain Membranes

Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.

Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

1987 ◽  
Vol 65 (11) ◽  
pp. 2338-2345 ◽  
Author(s):  
Gordon T. Bolger ◽  
Kendall A. Marcus ◽  
Ronald Thibou ◽  
Phil Skolnick ◽  
Ben Avi Weissman
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Divalent Cations ◽  
Opiate Receptors ◽  
Opiate Receptor ◽  
Affinity Binding ◽  
High Affinity ◽  
Regulatory Effects ◽  
In Vitro Effects

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5′-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity μ-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris–HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+,2.9 mM and Li, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the μ-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris–HCl. In 50 mM Tris–HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris–HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited, [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris–HCl, respectively. These findings indicate that the in vitro effects of Na+ at μ- and possibly other opiate receptors in rat brain are dependent on the concentration of Tris–HCl used in the assay buffer, lower concentrations of Tris-HCl revealing novel regulatory effects for Na+ at μ-opiate receptors.

scholarly journals Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of Gα12 purified from rat brain

1996 ◽  
Vol 319 (1) ◽  
pp. 165-171 ◽  
Author(s):  
Rainer HARHAMMER ◽  
Bernd NÜRNBERG ◽  
Christian HARTENECK ◽  
Daniela LEOPOLDT ◽  
Torsten EXNER ◽  
...  
Keyword(s):  
Rat Brain ◽  
G Proteins ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Subunit Exchange ◽  
Apparent Homogeneity ◽  
Rat Brain Membranes

G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of Gα12 from rat brain membranes. Gα12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified Gα12 bound guanosine 5´-[γ-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active Gα12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of Gα12 and Gα13 for Gβγ. Gα12 required a higher Mg2+ concentration for AlF4--induced dissociation from immobilized Gβγ than did Gα13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G12 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active Gα12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.

Hemodynamic effects of hemorrhage and subsequent naloxone treatment in conscious rabbits

1984 ◽  
Vol 247 (3) ◽  
pp. R497-R505 ◽  
Author(s):  
J. C. Schadt ◽  
M. D. McKown ◽  
D. P. McKown ◽  
D. Franklin
Keyword(s):  
Vascular Resistance ◽  
Opiate Receptors ◽  
Peripheral Resistance ◽  
Opiate Receptor ◽  
Receptor Blockade ◽  
Hemodynamic Effects ◽  
Abrupt Decrease ◽  
Arterial Blood ◽  
Opiate Peptides ◽  
Conscious Rabbits

The central and peripheral hemodynamic effects of rapid hemorrhage and subsequent opiate receptor blockade were studied in conscious rabbits. With hemorrhage of less than 12 ml/kg, mean arterial blood pressure (BP) was maintained by an increase in total peripheral resistance (TPR). Cardiac output (CO) declined in spite of an increase in heart rate (HR). Blood loss greater than 13 ml/kg resulted in an abrupt decrease in BP that was largely due to a decline in TPR. CO continued to decline gradually as it did early in hemorrhage. HR also decreased at the transition to hypotension. Subsequent opiate receptor blockade with naloxone (3 mg/kg) produced a prompt increase in BP and a decrease in HR. An increase in TPR accounted for the rise in BP. CO did not change significantly after naloxone. Therefore the hypotension associated with hemorrhage results from a decline in peripheral vascular resistance that is reversible by opiate receptor blockade with naloxone. These results are consistent with the involvement of opiate receptors and endogenous opiate peptides centrally and/or peripherally in control of vascular resistance during acute hemorrhagic hypotension.

scholarly journals Dimeric Dermorphin Analogues as µ-Receptor Probes on Rat Brain Membranes

1989 ◽  
Vol 264 (1) ◽  
pp. 354-362
Author(s):  
L H Lazarus ◽  
A Guglietta ◽  
W E Wilson ◽  
B J Irons ◽  
R de Castiglione
Keyword(s):  
Rat Brain ◽  
Brain Membranes ◽  
Rat Brain Membranes ◽  
Dermorphin Analogues
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