Characterization of Mamalian GS-α Proteins Expressed in Yeast
| Autor: | David J. Ecker, M. Gross, M. D. Minnich, Stanley T. Crooke, Julian B. Marsh, David Powers, Tauseef R. Butt, K. Hoyle, Jeffrey M. Stadel |
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| Rok vydání: | 1994 |
| Předmět: |
Gs alpha subunit
Guanine Genes Fungal Saccharomyces cerevisiae Biology Adenylyl cyclase chemistry.chemical_compound GTP-Binding Proteins Cell surface receptor Animals Humans Nucleotide RNA Messenger Transducin Promoter Regions Genetic Pharmacology Regulation of gene expression chemistry.chemical_classification Expression vector Cell Membrane Membrane Proteins RNA Fungal Molecular biology Recombinant Proteins Yeast chemistry Biochemistry Metallothionein Rabbits Copper Adenylyl Cyclases |
| Zdroj: | Journal of Receptor Research. 14:357-379 |
| ISSN: | 0197-5110 |
| Popis: | The guanine nucleotide regulatory protein, GS, mediates transmembrane signaling by coupling membrane receptors to the stimulation of adenylyl cyclase activity. The full length coding sequences for the M(r) = 42-45,000, short form (S), and M(r) = 46-52,000, long form (L), of the alpha-subunits of rat GS were placed in yeast expression vectors under the regulatory control of the copper-inducible CUP1 promoter and transformed into Saccharomyces cerevisiae. In the presence of 100 microM CuSO4, the transformed yeast expressed GS-alpha mRNAs and proteins. In reconstitution experiments, rat GS-alpha(S and L), solubilized from yeast membranes with 1% cholate, conferred NaF-, (-)isoproterenol-, and guanine nucleotide-dependent sensitivity to adenylyl cyclase catalytic units in S49 lymphoma cyc- cell membranes, which are devoid of endogenous GS-alpha. GS-alpha (S) demonstrated twice the activity of GS-alpha(L) in reconstitution assays of fluoride-stimulated adenylyl cyclase activity. Comparison of GS-alpha (S) expressed in yeast with GS purified from rabbit liver or human erythrocytes showed that the crude recombinant protein was fully competent in reconstituting NaF-stimulated adenylyl cyclase activity, but was only 2-5% as potent as purified GS. Addition of bovine brain beta gamma subunits during reconstitution enhanced all parameters of adenylyl cyclase activity for GS-alpha(S and L) obtained from yeast. In contrast, transducin beta gamma only enhanced agonist-stimulated adenylyl cyclase activity for GS-alpha (S and L) following reconstitution. These results demonstrate that the expression of functional mammalian GS-alpha subunits in yeast may be useful for their biochemical characterization. |
| Databáze: | OpenAIRE |
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