NDLI: Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.

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Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.

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Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.

Content Provider	PubMed Central
Author	Pillai, R. Kytle, K. Reyes, A. Colicelli, J.
Abstract	Saccharomyces cerevisiae strain PP5 has a phosphodiesterase (PDE) deficiency that results in heat-shock sensitivity due to the intracellular accumulation of cAMP. This strain also carries the cam mutation, which confers permeability to cAMP and, as shown here, to other compounds. Expression of rat type IV PDE in these cells caused them to revert to heat-shock resistance. Treatment of the transformed PP5 cells with rolipram, an antidepressant in humans and a potent inhibitor of type IV PDEs, reinstated sensitivity to heat shock. The biochemical properties of deletion mutants of this PDE were determined, and an active enzyme of minimum length was created. Reversion to heat-shock resistance was then used to select for PDE mutants refractory to the inhibitory effects of rolipram. Four mutants (A1, A2, A3, and A5) were isolated. Each carries a single point mutation; two have mutations in the same codon. Each mutant showed distinct properties, based on analysis of their substrate kinetics and IC50 values for a variety of inhibitors. Mutant A5 had a reduced activity for substrate, mutants A1 and A3 showed no change in substrate kinetics, and mutant A2 displayed an increase in activity. For most mutants, the drug resistance was confined to the class of drug used in the selection. This study shows that it is possible to recreate in yeast cells the susceptibility of mammalian enzymes to pharmacological agents. Our study also demonstrates that such systems can be used to select rare mutants useful in the analysis of drug-protein interactions.
Starting Page	11970
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	24
Volume Number	90
Language	English
Publisher Date	1993-12-15
Access Restriction	Open
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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