A Sensitive Cyclic Nucleotide Phosphodiesterase Assay for Transient Enzyme Kinetics

A new assay for cyclic nucleotide phosphodiesterase has been developed by using reverse-phase column chromatography for the separation of product and substrate of the enzymatic reaction. The polar 5'-nucleotides are not retarded by the column, while the more lipophilic cyclic nucleotides bind to the column. Properties such as pH and ionic strength of the incubation mixture or the elution buffer have only minor effects on the elution pattern. The assay by reverse-phase chromatography has several advantages above other assay methods currently employed; it is fast and simple, has a very low blank (0.2%), and is very sensitive (1 fmol). The assay can be used for different substrates (cyclic AMP, cyclic GMP, cyclic IMP) without modification of the conditions. The usefulness of the assay is demonstrated by transient kinetic measurements on a time scale in seconds of a cGMP-dependent cGMP-specific phosphodiesterase from the cellular slime mold Dictyostelium discoideum.

University of Groningen Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in MULTIPLE DEGRADATION PATHWAYS OF CHEMOATrRACTANT MEDIATED CYCLIC GMP ACCUMULATION IN DICTYOSTELIUM

Citation for published version (APA): Haastert, P. J. M. V., Lookeren Campagne, M. M. V., & Kesbeke, F. (1983). Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in Dictyostelium. Biochimica et Biophysica Acta %28BBA%29 -General Subjects, 756(1). https://doi.org/10.1016/0304-4165(83)90025-9 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 28-06-2019 Biochirnica et Biophysica Acta, 756 (1983) (Received September 10th, 1982) Key words: cyclic GMP," Chemotaxis; (Dictyostelium) Chemoattractants induce a transient accumulation of eGMP levels in Dictyostelium. lntraceilular cGMP levels reach a peak at 10 s and prestimulated cGMP levels are recovered at about 30 s. Intracellular and extraceilular cGMP levels were detected simultaneously after stimulation of D. lacteum cells with monapterin and of D. discoideum cells with cAMP. In both species about 20% of the intracellularly accumulated cGMP was secreted. All slime mold species investigated so far contain an intracellular phosphodiesterase specific for cGMP. A mutant of D. discoideum which does not contain this cGMP-specific enzyme shows a strongly retarded decline of intracellular cGMP levels. Secretion of cGMP is in this mutant not sufficient to explain the decline of cGMP levels which indicates the involvement of nonspecific phosphodiesterase in intracellular cGMP regulation. These results show multiple degradation pathways of intraceUularly accumulated cGMP. In wild-type cells about 20% is secreted, 10-20% is hydrolyzed intracellularly by non-specific phosphodiesterase, while the majority (60-70%) is hydrolyzed intracellularly by a cGMP-specific phosphodiesterase. The relationships of intraeeilular regulation of cGMP and cAMP levels are discussed