Guanine nucleotide binding properties of rap1 purified from human neutrophils

G. M. Bokoch, Lawrence Quilliam

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[γ-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein β/γ-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.

Original languageEnglish (US)
Pages (from-to)407-411
Number of pages5
JournalBiochemical Journal
Volume267
Issue number2
StatePublished - 1990
Externally publishedYes

Fingerprint

Guanine Nucleotides
Guanosine Triphosphate
Phosphorylation
Neutrophils
Cyclic AMP-Dependent Protein Kinases
Hydrolysis
Nucleotides
GTPase-Activating Proteins
Proteins
Guanosine
Protein Subunits
GTP-Binding Proteins
Cations
Chemical activation
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Guanine nucleotide binding properties of rap1 purified from human neutrophils. / Bokoch, G. M.; Quilliam, Lawrence.

In: Biochemical Journal, Vol. 267, No. 2, 1990, p. 407-411.

Research output: Contribution to journalArticle

@article{f4a8f3dd473344078aee98746c53c365,
title = "Guanine nucleotide binding properties of rap1 purified from human neutrophils",
abstract = "The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[γ-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein β/γ-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.",
author = "Bokoch, {G. M.} and Lawrence Quilliam",
year = "1990",
language = "English (US)",
volume = "267",
pages = "407--411",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "2",

}

TY - JOUR

T1 - Guanine nucleotide binding properties of rap1 purified from human neutrophils

AU - Bokoch, G. M.

AU - Quilliam, Lawrence

PY - 1990

Y1 - 1990

N2 - The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[γ-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein β/γ-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.

AB - The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[γ-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein β/γ-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.

UR - http://www.scopus.com/inward/record.url?scp=0025240662&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025240662&partnerID=8YFLogxK

M3 - Article

C2 - 2110451

AN - SCOPUS:0025240662

VL - 267

SP - 407

EP - 411

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -