scholarly journals Quantification of the α and β subunits of the transducing elements (Gs and Gi) of adenylate cyclase in adipocyte membranes from lean and obese (ob/ob) mice

1990 ◽  
Vol 268 (1) ◽  
pp. 83-89 ◽  
Author(s):  
N Bégin-Heick
Keyword(s):  
Adenylate Cyclase ◽  
Bacterial Toxin ◽  
Beta Subunits ◽  
Obese Mice ◽  
Adp Ribosylation ◽  
Beta Peptide ◽  
Adenylate Cyclase Activation ◽  
Dissociation Model ◽  
Gs Alpha ◽  
Kinetics Of

The abundance of the alpha and beta subunits of the GTP-binding proteins (G-proteins) that transduce hormonal messages to adenylate cyclase was assessed in adipocyte membranes from lean (+/+) and obese (ob/ob) mice, using ADP-ribosylation with bacterial toxin and immunodetection. Both methods revealed two Gs alpha species (48 and 42 kDa) in the membranes. Compared with those of lean mice, the membranes from obese mice contained substantially less of the 48 kDa species of Gs alpha, as assessed by both methods. ADP-ribosylation by pertussis toxin showed that only half as much ADP-ribose was incorporated into Gi alpha in the membranes from obese as compared with lean mice. Immunodetection revealed two separate Gi alpha peptides (39 and 40 kDa) and showed that the 40 kDa species was less abundant in the membranes from obese mice, whereas the amount of the 39 kDa species was similar in membranes from both lean and obese animals. Based on ADP-ribosylation assays, in membranes from lean mice the ratio Gs alpha/Gi alpha was 1:16, whereas in the membranes from obese mice it was 1:10. Similar amounts of immunodetectable beta peptide were found in both types of membranes. On the basis of the currently accepted dissociation model of adenylate cyclase activation, the decrease in the abundance of the Gi alpha subunit in adipocyte membranes from obese mice could account for the abnormal kinetics of the enzyme in these membranes.

Effect of acidosis on PTH-dependent renal adenylate cyclase in phosphorus deprivation: role of G proteins

1990 ◽  
Vol 258 (6) ◽  
pp. F1640-F1649
Author(s):  
E. Bellorin-Font ◽  
R. Starosta ◽  
C. L. Milanes ◽  
C. Lopez ◽  
N. Pernalete ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Adenylate Cyclase ◽  
Maximal Activity ◽  
Phosphate Deprivation ◽  
Adp Ribosylation ◽  
Phosphate Depletion ◽  
Cortical Membranes ◽  
And Function ◽  
Gs Alpha

These studies examine the regulation of adenylate cyclase in renal cortical membranes from phosphate-deprived and phosphate-deprived acidotic dogs. Enzyme stimulation by parathyroid hormone (PTH) was decreased in phosphate deprivation [Vmax 1,578 +/- 169 vs. 2,581 +/- 219 pmol adenosine 3',5'-cyclic monophosphate (cAMP).mg protein-1 x 30 min-1 in controls, P less than 0.01]. Metabolic acidosis further decreased PTH-stimulated activity. Membranes from phosphate-deprived dogs showed a decrease in Gs alpha-content by cholera toxin-dependent ADP-ribosylation (174 +/- 18 arbitrary units vs. 266.4 +/- 13.6 in controls, P less than 0.01). Metabolic acidosis further decreased Gs alpha-content, P less than 0.01. Gi content by pertussis-dependent ADP-ribosylation was also lower in phosphate-deprived and phosphate-deprived acidotic animals. Gs function was examined by its property to protect the catalytic unit from inactivation by N-ethylmaleimide when preincubated with GTP gamma S. In controls, protection of inactivation was 80% of the maximal activity, whereas in phosphate deprivation protection was less than 50%. In conclusion, metabolic acidosis enhances adenylate cyclase resistance to PTH in phosphate deprivation. These alterations are associated with a decrease in the content and function of Gs alpha, suggesting a role of Gs in the renal adaptation to phosphate depletion and acidosis.

scholarly journals The effect of iloprost on the ADP-ribosylation of Gsα (the α-subunit of Gs)

1989 ◽  
Vol 261 (3) ◽  
pp. 841-845 ◽  
Author(s):  
L Molina y Vedia ◽  
R D Nolan ◽  
E G Lapetina
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Present Evidence ◽  
Adp Ribosylation ◽  
Specific Antisera ◽  
Membrane Bound ◽  
Gs Alpha ◽  
Adp Ribosyltransferase

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.

Evidence for vasopressin activation of adenylate cyclase by subunit dissociation

1986 ◽  
Vol 250 (1) ◽  
pp. C115-C123 ◽  
Author(s):  
K. L. Skorecki ◽  
A. S. Verkman ◽  
C. Y. Jung ◽  
D. A. Ausiello
Keyword(s):  
Molecular Weight ◽  
Adenylate Cyclase ◽  
Regulatory Protein ◽  
Beta Subunits ◽  
Radiation Inactivation ◽  
Subunit Dissociation ◽  
Molecular Weights ◽  
Guanosine Diphosphate ◽  
Dissociation Model ◽  
Hormonal Activation

Radiation inactivation is used to probe the sequence of subunit interactions involved in the activation of adenylate cyclase by vasopressin in cultured renal epithelial cells (LLC-PK1) based on our previous analysis of the radiation inactivation of multimeric enzymes [Verkman et al., Am. J. Physiol. 250 (Cell Physiol. 19): C103-C114, 1986]. For basal adenylate cyclase activity, a concave downward ln(activity) vs. dose relation was observed with limiting slope corresponding to a molecular weight of (169-196) X 10(3). Similar results were obtained with NaF. In contrast, addition of vasopressin, guanylyl imidodiphosphate, or forskolin resulted in transition to a linear ln(activity) vs. dose relation with a slope corresponding to a molecular weight similar to that observed for basal activity. These findings were incorporated into a cyclic dissociation model for the hormonal activation of adenylate cyclase (graph see text) where H is hormone, R is receptor, C is catalytic unit, alpha and beta are subunits of guanyl nucleotide-regulatory protein (G), GTP is guanosine triphosphate, and GDP is guanosine diphosphate. The addition of H favors the dissociation of G into alpha and beta subunits by providing a rapid pathway for addition of GTP to dissociated alpha subunits. The observed target size of the active enzyme species formed corresponds to the composite molecular weights of alpha GTP with C. This model consolidates the radiation inactivation findings as well as the known biochemical characteristics for adenylate cyclase.

Decrease in Gs protein expression may impair adenylate cyclase activation in old kidneys

1993 ◽  
Vol 264 (5) ◽  
pp. F770-F773
Author(s):  
C. T. Liang ◽  
J. Barnes ◽  
H. Hanai ◽  
M. A. Levine
Keyword(s):  
Adenylate Cyclase ◽  
Mrna Level ◽  
Cdna Probe ◽  
Transcription Rate ◽  
Aged Rats ◽  
Total Rna ◽  
Cyclase Activation ◽  
General Decline ◽  
Adenylate Cyclase Activation ◽  
Gs Alpha

The possibility that alteration in stimulatory guanine nucleotide-binding protein (Gs) expression may contribute to the blunting of renal parathyroid hormone (PTH)-stimulated adenylate cyclase in aged rats was examined. Using rat cDNA probe, we identified a Gs alpha-subunit (Gs alpha) of 1.9 kb. Age did not alter the size of Gs alpha mRNA. The level of Gs alpha mRNA [normalized to poly(A)+ RNA] was decreased 23%, which was consistent with our previous report that Gs alpha protein decreased in senescence. In contrast, mRNA level of Gi alpha 2 increased with age. Level of beta-actin mRNA did not change with age. Nuclear runoff assay was performed to determine the transcription rate of Gs mRNA. Synthesis of poly(A)+ RNA and total RNA was reduced 39% and 37%, respectively, in nuclei prepared from old kidney, which suggested a general decline in RNA synthesis capacity in old rats. Our results also showed the transcription rate of Gs alpha mRNA in aged rats was reduced 89%, a decrease far exceeding that observed for total RNA or poly(A)+ RNA. We concluded that the decrease in steady-state level of Gs alpha mRNA was specific and probably was due to a reduction in the transcription activity. Thus alteration in Gs transcription may contribute, at least in part, to the impaired renal adenylate cyclase activation in aged rats.

scholarly journals Gsα is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gsα activity and abundance

1992 ◽  
Vol 288 (1) ◽  
pp. 331-336 ◽  
Author(s):  
L E Donnelly ◽  
R S Boyd ◽  
J MacDermot
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Major Protein ◽  
Adp Ribosylation ◽  
Short Term Exposure ◽  
Significant Difference ◽  
Gs Alpha

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.

Ticlopidine and Clopidogrel (SR 25990C) Selectively Neutralize ADP Inhibition of PGE1-Activated Platelet Adenylate Cyclase in Rats and Rabbits

1991 ◽  
Vol 65 (02) ◽  
pp. 186-190 ◽  
Author(s):  
G Defreyn ◽  
C Gachet ◽  
P Savi ◽  
F Driot ◽  
J P Cazenave ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Direct Effect ◽  
Cyclic Nucleotide ◽  
Camp Accumulation ◽  
Rabbit Platelets ◽  
Camp Levels ◽  
Kinetics Of ◽  
Camp Elevation ◽  
Rat Platelets

SummaryTiclopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGEj-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGEr stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGEX-induced cAMP elevation but this effect seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGEr activated platelet adenylate cyclase in rats and rabbits.

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