Inhibition of pertussis toxin catalyzed ADP-ribosylation of G-proteins by membrane depolarization in rat brain synaptoneurosomes

1991 ◽  
Vol 126 (1) ◽  
pp. 87-90 ◽  
Author(s):  
Malca Cohen-Armon ◽  
Mordechai Sokolovsky
Keyword(s):  
Rat Brain ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Membrane Depolarization ◽  
Adp Ribosylation

scholarly journals Erratum

1991 ◽  
Vol 11 (4) ◽  
pp. 706-706
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Equilibrium State ◽  
Rat Brain ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Adp Ribosylation ◽  
Gtp Binding ◽  
Reconstituted System ◽  
Α Subunits

Ischemia of Rat Brain Decreases Pertussis Toxin-Catalyzed [32P] ADP Ribosylation of GTP-Binding Proteins (Gi1 and G0) in Membranes Katsunobu Takenaka, Yasunori Kanaho, Koh-ichi Nagata, Noboru Sakai, Hiromu Yamada, Yoshinori Nozawa [ Originally published in Journal of Cerebral Blood Flow and Metabolism 1991;11:155–160] On page 158 of the above, arrows were erroneously deleted from the equation in the following passage: Heterotrimers of G proteins that bind GDP to α subunits seem to be the preferred substrates for PTcatalyzed ADP ribosylation since guanine nucleotides (GDP and GTP) and 13'Y subunits stimulate ADP ribosylation in the reconstituted system and in membranes (Tsai et aI., 1984). These results indicate that the G proteins may exist at the equilibrium state as shown below: This omission was the result of a typesetting error, which the publisher regrets.

Ischemia of Rat Brain Decreases Pertussis Toxin-Catalyzed [32P]ADP Ribosylation of GTP-Binding Proteins (Gi1 and G0) in Membranes

1991 ◽  
Vol 11 (1) ◽  
pp. 155-160 ◽  
Author(s):  
Katsunobu Takenaka ◽  
Yasunori Kanaho ◽  
Koh-Ichi Nagata ◽  
Noboru Sakai ◽  
Hiromu Yamada ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Molecular Basis ◽  
Guanine Nucleotides ◽  
Qualitative And Quantitative ◽  
Adp Ribosylation ◽  
Quantitative Changes ◽  
Control Stimulation ◽  
Stimulation Of

As an approach to understanding the molecular basis of the pathophysiology of cerebral ischemia, we examined qualitative and quantitative changes in pertussis toxin substrates, Gi1 and G0, in the membrane of rat cerebral cortex after decapitation. Within 1 min after decapitation, the extent of pertussis toxin-catalyzed [32P]ADP ribosylation of the G proteins in the cerebral cortex membrane was significantly decreased and the magnitude of the decrease became slightly larger upon further incubation of the decapitated brain. Addition of guanine nucleotides, GTP and GDP, or the purified βγ subunits of transducin to the membranes of control and ischemic cerebral cortex stimulated [32P]ADP ribosylation of the G proteins. The stimulation of [32P]ADP ribosylation in the control situation by guanine nucleotides was almost to the same extent as that in ischemia. However, the stimulation by transducin βγ subunits was different; the control stimulation was greater than that in ischemia. In immunoblots probed with antibodies against Gi1α G0α and Tβ, the immunoreactivity of the corresponding proteins in ischemia was similar to that in control, suggesting that the amounts of G proteins were not changed in ischemia. These results suggest that ischemia accelerates the dissociation of α–GDP–βγ to α–GDP and free βγ and causes the denaturation of the dissociated α–GDP, thereby decreasing [32P]ADP ribosylation.

scholarly journals Failure of [32P]ADP-ribosylation by pertussis toxin to determine Giα content in membranes from various human tissues. Improved radioimmunological quantification using the 125I-labelled C-terminal decapeptide of retinal transducin

1991 ◽  
Vol 277 (1) ◽  
pp. 223-229 ◽  
Author(s):  
M Böhm ◽  
K Larisch ◽  
E Erdmann ◽  
M Camps ◽  
K Jakobs ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Polyclonal Antiserum ◽  
Covalent Modification ◽  
Striking Difference ◽  
Left Ventricular Myocardium ◽  
Western Blots ◽  
Adp Ribosylation

The quantitative determination of pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G-proteins) in cell membranes is still a problem. Pertussis-toxin-catalysed [32P]ADP-ribosylation strongly relies on the substrate quality of the alpha-subunits and is influenced by the concentration of nucleotides, beta gamma-subunits, the physicochemical properties of the membranes influencing the availability of Gi alpha for pertussis toxin, and covalent modification of Gi alpha. Quantification of immunoreactive material on Western blots can be only imprecisely performed by two-dimensional densitometry. In order to generate a method for quantification of pertussis-toxin-sensitive G-proteins in membranes we have developed a fast and sensitive radioimmunoassay. The C-terminal decapeptide of retinal transducin alpha (KENLKDCGLF) was 125I-labelled and used as tracer. Polyclonal antiserum (DS 4) was raised against this peptide. Gi alpha proteins were determined by competition of solubilized membranes for 125I-KENLKDCGLF binding to DS 4 using dilutions of retinal transducin alpha as standard. The interassay variation was less than 10%, with a sensitivity of 2.5 micrograms/ml. The density of Gi alpha was highest in human adipose tissue, followed by HL60 cells, lung, mononuclear leucocytes, thrombocytes and left ventricular myocardium. A striking difference was observed between the density of Gi alpha and the amount of incorporation of [32P]ADP-ribose into the 40 kDa membrane proteins by pertussis toxin in the same samples. This is also demonstrated by comparison of the weak [32P]ATP-ribosylation of pertussis toxin substrates with the density of immunoreactive Gi alpha on Western blots in tissues such as lung. This study shows that the Gi alpha content can be exactly determined by a sensitive and fast radioimmunoassay using iodinated synthetic peptide homologues of Gi alpha proteins. Radioimmunological quantification of Gi alpha might be able to detect the ‘true’ Gi alpha content of membranes without being hampered by influences on the [32P]ADP-ribosylation reaction. It is concluded that this newly developed method may become an important tool for studying expression of Gi alpha proteins in a variety of tissues or cell types, and for precisely quantifying the changes caused by pathological conditions.

scholarly journals Differential cholera-toxin- and pertussis-toxin-catalysed ADP-ribosylation of G-proteins coupled to formyl-peptide and leukotriene B4 receptors

1993 ◽  
Vol 289 (2) ◽  
pp. 469-473 ◽  
Author(s):  
T M Schepers ◽  
K R McLeish
Keyword(s):  
Cholera Toxin ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Leukotriene B4 ◽  
Guanine Nucleotides ◽  
Dependent Manner ◽  
Functional Responses ◽  
Concentration Dependent Manner ◽  
Adp Ribosylation ◽  
Formyl Peptide

N-Formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B4 (LTB4) induce disparate second-messenger generation and functional responses in neutrophils and HL-60 granulocytes. Receptors for these chemoattractants couple to a common pool of G-proteins which are substrates for both pertussis-toxin- and cholera-toxin-catalysed ADP-ribosylation. The hypothesis that formyl-peptide and LTB4 receptors induce different receptor-specific conformations of activated G-proteins was tested. The ability of pertussis toxin and cholera toxin to ADP-ribosylate G(i) proteins coupled to formyl-peptide or LTB4 receptors in membranes isolated from HL-60 granulocytes was used to assess the conformational state of the alpha subunits. Cholera-toxin-catalysed ADP-ribosylation of alpha 40 (40 kDa alpha subunit) was inhibited by guanosine 5′-[beta gamma-imido]triphosphate and GDP in a concentration-dependent manner. Addition of fMet-Leu-Phe, but not LTB4, re-established cholera-toxin labelling of alpha 40 in the presence of either guanine nucleotide. In the absence of guanine nucleotides, fMet-Leu-Phe and C5a enhanced cholera-toxin-catalysed labelling of alpha 40, whereas LTB4 and platelet-activating factor had no effect. Preincubation with fMet-Leu-Phe, but not LTB4, inhibited pertussis-toxin labelling of alpha 40 in the presence of guanosine 5′-[gamma-thio]triphosphate and in the absence of guanine nucleotides. Preincubation with fMet-Leu-Phe or LTB4 enhanced pertussis-toxin labelling of alpha 40 in the presence of GDP. These data suggest that activated G(i) proteins coupled to formyl-peptide and LTB4 receptors exist in different conformations determined by the receptor with which they interact.

scholarly journals Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3710-3718 ◽  
Author(s):  
Ikuo Uchida ◽  
Ryoko Ishihara ◽  
Kiyoshi Tanaka ◽  
Eiji Hata ◽  
Sou-ichi Makino ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Salmonella Enterica ◽  
Phage Type ◽  
Chinese Hamster ◽  
Adp Ribosylation ◽  
Dependent Modification

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB – encoded by a prophage in S. Typhimurium DT104 – are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [32P]NAD and ArtA, and the label was released by HgCl2, which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA6Arg-Ala and ArtA115Glu-Ala, in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

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