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

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.

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

Biochemical Journal ◽

10.1042/bj2890469 ◽

1993 ◽

Vol 289 (2) ◽

pp. 469-473 ◽

Cited By ~ 8

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.

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Fluorescent labeling of signal-transducing G-proteins. Pertussis toxin-catalyzed etheno-ADP ribosylation of transducin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)77706-0 ◽

1988 ◽

Vol 263 (36) ◽

pp. 19804-19808

Author(s):

V N Hingorani ◽

Y K Ho

Keyword(s):

G Proteins ◽

Pertussis Toxin ◽

Fluorescent Labeling ◽

Adp Ribosylation

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Erratum

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1991.126 ◽

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.

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Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2Dopamine Receptors in the A1Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

Journal of Neurochemistry ◽

10.1111/j.1471-4159.1990.tb04949.x ◽

1990 ◽

Vol 55 (5) ◽

pp. 1631-1638 ◽

Cited By ~ 7

Author(s):

Toshihiko Murayama ◽

Yuriko Itahashi ◽

Yasuyuki Nomura

Keyword(s):

Cerebral Cortex ◽

Adenylate Cyclase ◽

G Proteins ◽

Pertussis Toxin ◽

Rat Cerebral Cortex ◽

Adenylate Cyclase System

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

Neuroscience Letters ◽

10.1016/0304-3940(91)90378-7 ◽

1991 ◽

Vol 126 (1) ◽

pp. 87-90 ◽

Cited By ~ 6

Author(s):

Malca Cohen-Armon ◽

Mordechai Sokolovsky

Keyword(s):

Rat Brain ◽

G Proteins ◽

Pertussis Toxin ◽

Membrane Depolarization ◽

Adp Ribosylation

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Inhibition of interleukin 3 and colony-stimulating factor 1-stimulated marrow cell proliferation by pertussis toxin

Blood ◽

10.1182/blood.v71.5.1187.1187 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1187-1195 ◽

Cited By ~ 30

Author(s):

YX He ◽

E Hewlett ◽

D Temeles ◽

P Quesenberry

Keyword(s):

Bone Marrow ◽

G Proteins ◽

Pertussis Toxin ◽

Cell Surface Receptor ◽

Target Cells ◽

Colony Stimulating Factor ◽

Murine Bone Marrow ◽

Interleukin 3 ◽

Stimulating Factor ◽

Stimulation Of

Abstract Pertussis toxin (PT) catalyzes the ADP-ribosylation of several guanine nucleotide-binding (G) proteins that are involved in the transduction of cell surface receptor-mediated signals. Involvement of such G- proteins in regulation of hematopoiesis by two growth factors, colony- stimulating factor-1 (CSF-1) and interleukin 3 (IL 3), was investigated using pertussis toxin. Continuous or pulse exposure of murine bone marrow cells to pertussis toxin inhibited CSF-1 or IL 3-induced colony formation by approximately 50%. Pertussis toxin inhibition was also demonstrated against partially separated marrow from 5-fluorouracil- treated mice. The toxin effect was blocked by heating (95 degrees C for 30 minutes), by antitoxin antibody and was not associated with increased cAMP levels in target cells. In experiments with murine marrow, toxin-mediated inhibition appeared to involve predominantly the macrophage lineage. IL 3 stimulation of proliferation of the murine marrow-derived factor-dependent cell line FDC-P1, as measured by 3H-TdR incorporation, and CSF-1 stimulation of pure populations of murine bone marrow derived macrophages, as measured by DNA content and cell number, was also inhibited. Analysis of the effects of pertussis toxin on the growth of single cells stimulated by IL 3 demonstrated that this inhibition involved a decreased growth rate rather than a toxic ablation of cells. Phorbol myristate acetate (PMA) stimulated FDC-P1 cells and was able to abrogate the PT inhibition of IL 3 stimulation of these cells, suggesting but not establishing that IL 3 may mediate its proliferative effects through activating protein kinase C.

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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

Biochemical Journal ◽

10.1042/bj2770223 ◽

1991 ◽

Vol 277 (1) ◽

pp. 223-229 ◽

Cited By ~ 26

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.

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Pertussis toxin substrate is a guanosine 5′-[β-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein

Biochemical Journal ◽

10.1042/bj2320191 ◽

1985 ◽

Vol 232 (1) ◽

pp. 191-197 ◽

Cited By ~ 23

Author(s):

S K Wong ◽

B R Martin ◽

A M Tolkovsky

Keyword(s):

Pertussis Toxin ◽

Binding Protein ◽

Alkylating Agents ◽

Guanine Nucleotides ◽

Temperature Sensitive ◽

Gtp Binding Protein ◽

Adp Ribosylation ◽

Gtp Binding ◽

Rat Brain And Liver ◽

Liver Membranes

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5′-[γ-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5′-[β-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5′-[β, γ-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.

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