Identification and purification of a novel G protein from neutrophils

Dickey, Burton F.; Pyun, Hae Yung; Navarro, Javier

doi:10.1016/0014-5793(87)80237-5

Cited by 24 publications

(10 citation statements)

References 18 publications

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“…C6 glioma cells, rat fat-cells, rabbit heart, neutrophils, chromaffin cells, astrocytes, neurons and adenohypophysis [28][29][30][31]4,5]. A 40 kDa species has also recently been purified from pig brain [32] and from neutrophils [33,34]. The presence of a G-protein in Ttubule membranes was further ascertained by the immunoreactivity detected with an anti-fl-subunit antiserum.…”

Section: Discussionmentioning

confidence: 99%

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

Toutant

Barhanin

Bockaert

et al. 1988

Biochemical Journal

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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.

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Section: Discussionmentioning

confidence: 99%

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

Toutant

Barhanin

Bockaert

et al. 1988

Biochemical Journal

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“…However, this G-protein appeared to be partially inactivated during purification, resulting in a lower specific activity compared with Gil or Go; to achieve the same maximal extent of GTP[35S] binding or to yield a comparable extent of [32P]ADP-ribosylation by pertussis toxin, 3-4-fold higher concentrations of GL than of G1l and Go were required. Previous reports (Gierschik et al, 1987;Dickey et al, 1987) indicated that Gi2 of neutrophils was highly unstable after solubilization in cholate solution unless stabilizers such as Al3+-For ethylene glycol were added. In the present study, we employed the detergent Lubrol without the use of stabilizers throughout the purification.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain

Kanaho¹,

Crooke²,

Stadel³

1989

Biochemical Journal

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The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.

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“…amine4,Sdione and [3H]tryptamine-4,5-dione were prepared through the electrochemical oxidation of 5-HT and ['HIS HT, respectively (Chen et al, 1989). The major brain G proteins Gi and Go were prepared as previously described by Dickey et al (1987) based on a modification of the technique of Sternweis and Robishaw (Sternweis and Robishaw, 1984). Guanosine-5'-O-(3-thotriphosphate) (GTP-7-S) binding was determined as previously described by Northrup et al (1982).…”

Section: General Methodsmentioning

confidence: 99%

Modification of Brain Guanine Nucleotide‐Binding Regulatory Proteins by Tryptamine‐4,5‐Dione, a Neurotoxic Derivative of Serotonin

Fishman

Rubins

Chen

et al. 1991

Journal of Neurochemistry

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We have recently characterized a novel oxidation product of serotonin (5-hydroxytryptamine, 5-HT), tryptamine-4,5-dione, which increases 5-HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide-binding regulatory proteins Gi and Go to [3H]tryptamine-4,5-dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the alpha subunits of Gi and Go (approximately 40,000). The binding of [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to Gi and Go and pertussis toxin-catalyzed [32P]ADP-ribosylation of the G protein alpha subunits were both inhibited in a dose-dependent manner by tryptamine-4,5-dione. Thus, neurotoxins such as tryptamine-4,5-dione may exert their effects through specific interactions with G proteins.

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Identification and purification of a novel G protein from neutrophils

Cited by 24 publications

References 18 publications

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

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

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain

Modification of Brain Guanine Nucleotide‐Binding Regulatory Proteins by Tryptamine‐4,5‐Dione, a Neurotoxic Derivative of Serotonin

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