scholarly journals Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes

1975 ◽  
Vol 146 (1) ◽  
pp. 269-271 ◽  
Author(s):  
S Van Heyningen ◽  
C A King
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Erythrocyte Membranes ◽  
Subunit A ◽  
Subunit B

Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes.

scholarly journals Activation by cholera toxin of adenylate cyclase solubilized from rat liver

1976 ◽  
Vol 157 (3) ◽  
pp. 785-787 ◽  
Author(s):  
S Van Heyningen
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Thiol Group ◽  
Subunit B

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

1981 ◽  
Vol 209 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Robert W. Downs ◽  
Sharon A. Reen ◽  
Michael A. Levine ◽  
Gerald D. Aurbach ◽  
Allen M. Spiegel
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Erythrocyte Membranes ◽  
Adp Ribosylation

scholarly journals Persistent activation of Gsα through limited proteolysis by calpain

2000 ◽  
Vol 347 (3) ◽  
pp. 733-740 ◽  
Author(s):  
Kaori SATO-KUSUBATA ◽  
Yukiko YAJIMA ◽  
Seiichi KAWASHIMA
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Active Form ◽  
Limited Proteolysis ◽  
Specific Inhibitor ◽  
Activation Mechanism ◽  
Calpain Inhibitor ◽  
Pituitary Cells ◽  
Rat Pituitary

Treatment of rat pituitary GH4C1 cell membranes with calpain, a calcium-activated cysteine protease, increased adenylate cyclase activity, and this activity was inhibited by a calpain inhibitor, leupeptin. Calpain treatment potentiated the activity of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]), but did not attenuate MnCl2 action on adenylate cyclase, suggesting that calpain acted at the G-protein level, rather than directly on adenylate cyclase. This calpain stimulation of adenylate cyclase was inhibited by an antibody raised against the C-terminal portion of Gsα, but not by anti-Gi2α or anti-Gβ antibodies. Furthermore, it was shown that Gsα is more susceptible to calpain-mediated proteolysis than Gi2α or Gβ. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of Gsα in GH4C1 cell membranes. Proteolysis of Gsα by μ-calpain involved sequential cleavages at two sites, resulting in the generation of a 39 kDa fragment first, and then a 20 kDa fragment, from the C-terminus. Treatment of GH4C1 cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH4C1 cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH4C1 cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of Gsα, which were produced by cholera toxin or pituitary adenylate cyclase-activating polypeptide. These results suggest that calpain sustains adenylate cyclase in an active form through the cleavage of Gsα to an active Gsα fragment. This is a novel calpain-dependent activation mechanism of Gsα and, thus, of adenylate cyclase in rat pituitary cells.

scholarly journals Activity of covalently cross-linked cholera toxin with the adenylate cyclase of intact and lysed pigeon erythrocytes

1977 ◽  
Vol 168 (3) ◽  
pp. 457-463 ◽  
Author(s):  
S van Heyningen
Keyword(s):  
Cell Surface ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Dimethyl Ester ◽  
Lag Phase ◽  
Ganglioside Gm1 ◽  
Whole Cells ◽  
Subunit B

Reaction of cholera toxin with NN'-bis(carboximidomethyl)tartaramide dimethyl ester produced several cross-linked species that had subunit B (which binds to the cell surface) and peptides A1 (which activates adenylate cyclase) and A2 all covalently joined together. This cross-linded material had activity with pigeon erythrocytes that was comparable in all respects with that of native toxin. It activated the adenylate cyclase of whole cells, showing a characteristic lag phase, and this activation was increased if the cells had been preincubated with ganglioside GM1, but abolished if the protein had been preincubated with the ganglioside. It activated the enzyme in lysed cells more strongly and without the lag phase. These results show that the toxin is active even when peptide A1 cannot be released from the rest of the molecule.

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.

Sign in / Sign up

Export Citation Format

Share Document