Preparation and application of GTP-agarose matrices

Pfeuffer, Elke; Pfeuffer, Thomas

doi:10.1016/0076-6879(91)95163-e

Cited by 3 publications

(1 citation statement)

References 13 publications

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“…For the partially purified preparation, the homogenate was produced in accordance with the method of Katz et al 11 and plasma membranes were prepared from this by the method of Pilkis and Johnson, 12 with the exception that 1 mM Tris-HCl buffer (pH 7.4) was used as the buffer throughout the procedure. For the production of the highly purified adenylyl cyclase preparation, the method of Pfeuffer et al 13 was employed.…”

Section: Enzyme Preparationmentioning

confidence: 99%

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast‐atom bombardment mass spectrometry

Newton

Bayliss

Geyschem

et al. 1997

Rapid Commun. Mass Spectrom.

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The enzyme adenylyl cyclase catalyses the conversion of adenosine 5'-triphosphate (ATP) to adenosine-3',5'-cyclic monophosphate (cyclic AMP), and is an important pharmaceutical target. Quantitation of this enzyme's activity has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The kinetic data obtained are in good agreement with those obtained by the conventional radiometric assay, and this mass spectrometry-based assay offers the facility to monitor the turnover of several components of the incubation simultaneously. This is utilized to study the relative efficiencies of two ATP-regenerating systems, three phosphodiesterase inhibitors and two modified substrates, and to monitor the uptake and conversion of two competing substrates, adenosine 5' triphosphate and 2'-deoxyadenosine-5-triphosphate, to cyclic AMP and to cyclic deoxyAMP, respectively.

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Section: Enzyme Preparationmentioning

confidence: 99%

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast‐atom bombardment mass spectrometry

Newton

Bayliss

Geyschem

et al. 1997

Rapid Commun. Mass Spectrom.

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Antibody immobilization to high-performance liquid chromatography supports

Clarke

Beckwith

Jackson

et al. 2000

Journal of Chromatography A

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Wise

Thomas

Carr³

et al. 1997

Plant Molecular Biology

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The Arabidopsis G alpha subunit, GP alpha1, was expressed within Escherichia coli by co-transformation with the expression vector and the dnaY gene which encodes tRNA(Arg)(AGA/AGG) Isolation of the recombinant GP alpha1 in a highly pure form could be achieved by a combination of anion exchange and dye affinity chromatography or by a single step affinity procedure via chromatography on 4-amino-anilido-GTP agarose. The recombinant protein yielded by both procedures was highly active and bound GTPgammaS with an apparent Kd in the nM range. GTPgammaS binding was stimulated two-fold in the presence of Zn2+ compared with that in the presence of Mg2+, Mn2+ or Ca2+.

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Preparation and application of GTP-agarose matrices

Cited by 3 publications

References 13 publications

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast‐atom bombardment mass spectrometry

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast‐atom bombardment mass spectrometry

Antibody immobilization to high-performance liquid chromatography supports

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