Advances in cyclic nucleotide research, 10, current methodology

Johnson, George S.

doi:10.1016/0092-8674(79)90371-4

Cited by 37 publications

(39 citation statements)

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“…Unlabelled nucleotides and creatine kinase were from Boehringer Mannheim (Mannheim, FRG). f M e t -L e~-[~ H]Phe (40 -60 Ci/mmol), carrier-free [32P]phosphoric acid for enzymatic preparation of [ Y -~~P I G T P [15], and [35S]GTP[S] (1200-1400 Ci/mmol) were from New England Nuclear (Dreieich, FRG). [8-3H]GDP (10 Ci/mmol) was from Amersham Buchler (Braunschweig, FRG).…”

Section: Methodsmentioning

confidence: 99%

Activation of signal‐transducing guanine‐nucleotide‐binding regulatory proteins by guanosine 5′‐[γ‐thio]triphosphate

Wieland

Ulibarri

Gierschik

et al. 1991

European Journal of Biochemistry

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Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) Enzymes. Creatine kinase (E.C. 2.7.3.2); GTPase (E.C. 3.6.1 .-); nucleoside diphosphokinase (E.C. 2.7.4.6).hormones and neurotransmitters, are composed of three distinct subunits: a guanine-nucleotide-binding CI subunit, a P subunit and a y subunit, the latter two forming a fly dimer under non-denaturing conditions [l -31. Both components, i. e. the cl subunit and the fly dimer, are required for receptorcontrolled activation of G proteins [4-61. However, the exact role of B y subunits, apparently acting in a substoichiometric manner in activation of CI subunits, is not known. The fly dimers of the G protein transducin, found in vertebrate rod outer segment (ROS) membranes, are different from other G protein B y dimers in that apparently only one form of each B and y subunit is present in ROS membranes, while other membranes contain at least two distinct fl subunits, one (P36)

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Section: Methodsmentioning

confidence: 99%

Activation of signal‐transducing guanine‐nucleotide‐binding regulatory proteins by guanosine 5′‐[γ‐thio]triphosphate

Wieland

Ulibarri

Gierschik

et al. 1991

European Journal of Biochemistry

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“…Bound cAMP was removed from the enzyme by gel filtration at 22°C. [Y-'~P]ATP was prepared as described [14]. Protein was determined by the Lowry procedure.…”

Section: Miscellaneous Methodsmentioning

confidence: 99%

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

et al. 1986

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Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 pM. Autophosphorylation of the enzyme decreases the apparent K, value to 7 pM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP.Tritiated cAMP apparently binds at 4°C to one site with a Kd value of 3 pM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (& 0.46 pM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-l to 1.2 min-' by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of CAMP to the high-affinity site from 1.9 pM to 0.17 pM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 pM to 40 yM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1.

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“…[y-32p]GTP and [32p]NAD were synthesized as described [8,9]. SDS-polyacrylamide gel electrophoresis and autoradiography were performed as described in [2].…”

Section: Introductionmentioning

confidence: 99%

Receptor‐mediated ADP‐ribosylation of a phospholipase C‐stimulating G protein

1987

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In membranes of myeloid differentiated HL 60 cells, the chemotactic peptide FMLP stimulates phospholipase C via a pertussis toxin-sensitive G protein. FMLP markedly stimulates the cholera toxin-dependent ADP-ribosylation of a 40 kDa protein in these membranes. This effect of FMLP is inhibited by GTP and GTP [S], and is almost completely abolished in membranes of pertussis toxin-pretreated HL 60 cells. Treatment of HL 60 membranes with cholera toxin and NAD markedly inhibits FMLP-stimulated high affinity GTPase. These results suggest that a 40 kDa G protein sensitive to both pertussis and cholera toxin functionally interacts with the formyl peptide receptor of HL 60 cells and, thus, very likely is the G protein that stimulates phospholipase C in this system.

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Advances in cyclic nucleotide research, 10, current methodology

Cited by 37 publications

References 0 publications

Activation of signal‐transducing guanine‐nucleotide‐binding regulatory proteins by guanosine 5′‐[γ‐thio]triphosphate

Activation of signal‐transducing guanine‐nucleotide‐binding regulatory proteins by guanosine 5′‐[γ‐thio]triphosphate

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

Receptor‐mediated ADP‐ribosylation of a phospholipase C‐stimulating G protein

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