scholarly journals Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase

1980 ◽  
Vol 186 (3) ◽  
pp. 749-754 ◽  
Author(s):  
C A Doberska ◽  
A J S MacPherson ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Activation Process ◽  
Liver Plasma Membrane ◽  
Adp Ribosylation ◽  
A Subunit

1. Cholera toxin was shown to require the presence of GTP to activate rat liver plasma-membrane adenylate cyclase. ATP did not affect the activation process. 2. Cholera toxin catalysed the incorporation of 32P from NAD labelled in the alpha-phosphate group of the ADP moiety into a rat liver plasma-membrane protein with a subunit mol.wt. of 42 500. This is taken to demonstrate ADP-ribosylation. The ADP-ribosylation of this protein also required GTP and was unaffected by ATP. 3. Nicotinamide inhibited both the activation of adenylate cyclase by cholera toxin and the ADP-ribosylation of the protein of 42 500 subunit mol wt. Neither the activation nor the ADP-ribosylation could be reversed by treatment with nicotinamide in the presence of cholera toxin.

Alterations of adenylyl cyclase-linked G proteins in rat liver during aging

1996 ◽  
Vol 270 (1) ◽  
pp. E126-E132 ◽  
Author(s):  
A. T. Eakes ◽  
T. K. Hymer ◽  
M. J. Rosenthal ◽  
J. Moss ◽  
M. S. Katz
Keyword(s):  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
G Proteins ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Adp Ribosylation ◽  
Old Rats ◽  
Gs Alpha

beta-Adrenergic stimulation of adenylyl cyclase in rat liver increases during aging. We examined whether this increase is related to alterations in the stimulatory and inhibitory G proteins (Gs and Gi) linked to adenylyl cyclase. Levels of immunoreactive alpha- and beta-subunits of Ga and Gi in liver plasma membranes from 6-, 12-, 18-, and 24-mo-old rats were unchanged with age, as was pertussis toxin-catalyzed [32P]ADP ribosylation of Gi alpha. Cholera toxin-catalyzed [32P]ADP ribosylation of Ga alpha and Gs bioactivity, assessed as reconstitution of adenylyl cyclase activity in S49 cyc- cell membranes, increased two- to threefold between 6 and 12-18 mo, and declined by 24 mo. Recombinant ADP ribosylation factor (ARF) enhanced cholera toxin labeling of Gs alpha at all ages, yet abolished the increase in toxin labeling at 12-18 mo. Auto-ADP ribosylation of the cholera toxin A1 peptide also increased transiently with age. Alteration of Gs alpha, as reflected by increased cholera toxin labeling and Gs bioactivity, may be involved in the regulation of beta-adrenergic-responsive adenylyl cyclase in rat liver during aging. Moreover, changes in endogenous ARF levels could contribute to age differences in cholera toxin labeling of Gs alpha.

scholarly journals Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action

1988 ◽  
Vol 253 (3) ◽  
pp. 735-743 ◽  
Author(s):  
M Janicot ◽  
J P Clot ◽  
B Desbuquois
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Acidic Medium ◽  
Isolated Hepatocytes ◽  
Surface Generation ◽  
Lag Phase ◽  
Single Class ◽  
Endosomal Membrane ◽  
A Subunit

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.

scholarly journals Lack of vasopressin receptors in liver, but not in kidney, of ob/ob mice

1983 ◽  
Vol 216 (2) ◽  
pp. 475-480 ◽  
Author(s):  
F Assimacopoulos-Jeannet ◽  
B Cantau ◽  
G van de Werve ◽  
S Jard ◽  
B Jeanrenaud
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Metabolic Response ◽  
Isolated Hepatocytes ◽  
The Other ◽  
Kidney Medulla ◽  
Liver Plasma Membrane ◽  
Liver Plasma Membranes ◽  
Vasopressin Receptors

The activity of phosphorylase a was measured in isolated hepatocytes from fed lean and ob/ob mice after addition of vasopressin, angiotensin, phenylephrine and glucagon. The binding of these hormones to purified liver plasma membranes was also determined. In hepatocytes of ob/ob mice, no increase in phosphorylase a was measured after addition of vasopressin, whereas the other hormones promoted an increase in the activity of the enzyme. No specific vasopressin receptors could be measured on purified liver plasma membrane of ob/ob mice. A decrease in the number of receptors for angiotensin and glucagon, without modification of the affinity, was also observed. No restoration of the number of vasopressin receptors was observed in liver of ob/ob mice starved for 3 days or in younger (5-6 weeks) animals. Vasopressin receptors and vasopressin-stimulated adenylate cyclase, measured on purified kidney medulla membranes, were similar in both lean and ob/ob mice. The data indicate a selective lack of vasopressin receptors and metabolic response in liver of the ob/ob mouse.

scholarly journals The interactions between the activatory guanine nucleotide binding protein and the catalytic subunit of adenylate cyclase in rat liver plasma membranes

1985 ◽  
Vol 231 (1) ◽  
pp. 39-46 ◽  
Author(s):  
S K-F Wong ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Rat Liver ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Lag Time ◽  
Gtp Binding ◽  
Rat Liver Plasma Membranes ◽  
Maximal Extent

Three GTP-binding proteins of 50 kDa, 45 kDa and 28 kDa were identified by photoaffinity labelling with [gamma-32P]GTP-gamma-azidoanilide (A-GTP) in the rat liver plasma membrane. Pertussis toxin catalysed ADP-ribosylation of a single protein of 40 kDa. A-GTP had no effect on the basal labeling by pertussis toxin. After u.v. irradiation of the membrane in the presence of A-GTP, the GTP-dependent ADP-ribosylation by cholera toxin was increased, while the basal labelling was not affected. These results suggest that A-GTP interacts specifically with the activatory GTP-binding protein (Gs) and does not interact with the inhibitory GTP-binding protein (Gi). The effects of partial photoinactivation of Gs of the rat liver plasma membrane adenylate cyclase system by A-GTP were studied. U.v. irradiation in the presence of increasing concentrations of the analogue caused progressive decrease in the maximal extent of activation by guanosine 5′-[γ-thio]triphosphate, but the Ka was not affected. The rate of activation of liver adenylate cyclase by guanosine 5′-[γ-thio]triphosphate is temperature-dependent. The lag time increased from 0.5 min at 30 degrees C to 2.0-2.5 min at 15 degrees C in the presence of 10 microM-guanosine 5′-[γ-thio]triphosphate. However, Ka remains unaffected by lowering the temperature. Photoinactivation by A-GTP or competitive inhibition by guanosine 5′-[β-thio]diphosphate decreases the maximal extent of activation by guanosine 5′-[γ-thio] triphosphate, but the lag time remains unaffected. The present results support the idea that Gs is tightly associated with the catalytic subunit under basal conditions. The present results also indicate that the transition of an inactive Gs to its active form is the rate-limiting step of the activation of adenylate cyclase by guanosine 5′-[γ-thio]triphosphate in the intact rat liver plasma membranes.

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