scholarly journals G-proteins in skeletal muscle. Evidence for a 40 kDa pertussis-toxin substrate in purified transverse tubules

1988 ◽  
Vol 254 (2) ◽  
pp. 405-409 ◽  
Author(s):  
M Toutant ◽  
J Barhanin ◽  
J Bockaert ◽  
B Rouot
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Low Salt ◽  
T Tubules

In muscle, it has been established that guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.

scholarly journals In vivoexpression of G-protein β1γ2dimer in adult mouse skeletal muscle alters L-type calcium current and excitation-contraction coupling

2010 ◽  
Vol 588 (15) ◽  
pp. 2945-2960 ◽  
Author(s):  
Norbert Weiss ◽  
Claude Legrand ◽  
Sandrine Pouvreau ◽  
Hicham Bichraoui ◽  
Bruno Allard ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
Calcium Current ◽  
Adult Mouse ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Mouse Skeletal Muscle

scholarly journals Light-dependent GTP-binding proteins in squid photoreceptors

1990 ◽  
Vol 272 (1) ◽  
pp. 79-85 ◽  
Author(s):  
P R Robinson ◽  
S F Wood ◽  
E Z Szuts ◽  
A Fein ◽  
H E Hamm ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Protein G ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Gtp Binding ◽  
Molecular Masses ◽  
Lines Of Evidence

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5′-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.

scholarly journals Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses

1988 ◽  
Vol 252 (1) ◽  
pp. 297-300 ◽  
Author(s):  
H S Banga ◽  
R K Walker ◽  
L K Winberry ◽  
S E Rittenhouse
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Marked Decrease ◽  
Human Platelets ◽  
Adp Ribosylation

Thrombin stimulates phospholipase C and inhibits adenylate cyclase in human platelets. We have studied the effect of purified S1 monomer, the ADP-ribosylating subunit of pertussis toxin, on these receptor-coupled G-protein-dependent activities. ADP-ribosylation of a 41 kDa protein is associated with a marked decrease in the ability of thrombin to inhibit cyclic AMP formation, but has little effect on phospholipase C. Therefore adenylate cyclase and phospholipase C appear to be modulated by different G-proteins.

Regulation of renal cortical Na-HCO3 cotransporter. II. Role of G proteins

1995 ◽  
Vol 268 (3) ◽  
pp. F461-F467 ◽  
Author(s):  
O. S. Ruiz ◽  
Y. Y. Qiu ◽  
L. J. Wang ◽  
J. A. Arruda
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Inhibitory Effect ◽  
Adp Ribosylation ◽  
Protein Activator ◽  
The Difference ◽  
22Na Uptake ◽  
Gamma S

We examined the regulation of the renal cortical basolateral Na-HCO3 cotransporter by G proteins. Na-HCO3 cotransporter activity was measured in highly purified rabbit renal cortical basolateral membranes (BLMV) as the difference in 22Na uptake in presence of HCO3- and gluconate. HCO(3-)-dependent 22Na uptake was significantly inhibited by 10 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a G protein activator. In contrast, addition of 50 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), an inhibitor of G protein, prevented the inhibition of the Na-HCO3 cotransporter activity by GTP gamma S. AlF4-, another G protein activator, also inhibited the activity of the Na-HCO3 cotransporter. This inhibitory effect of G protein on the Na-HCO3 cotransporter activity was not prevented by dideoxyadenosine, an adenylate cyclase inhibitor, or by the protein kinase A inhibitor, suggesting a direct effect of G protein on the cotransporter. To identify the G proteins that mediate the regulation of the Na-HCO3 cotransporter, purified BLMV were ADP ribosylated in presence of cholera toxin or pertussis toxin. Autoradiograms of BLMV incubated with [32P]NAD showed that cholera and pertussis toxins caused ADP ribosylation of 42- and 41-kDa G proteins, respectively. To determine whether the ADP ribosylation by cholera or pertussis toxin was associated with alterations of the Na-HCO3 cotransporter activity, we measured HCO(3-)-dependent 22Na uptake in BLMV treated with 20 micrograms/ml cholera toxin or with 100 ng/ml pertussis toxin. Na-HCO3 cotransporter activity was significantly decreased by both cholera and pertussis toxins.(ABSTRACT TRUNCATED AT 250 WORDS)

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