By John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin
This quantity emphasizes equipment for the assay, purification, and characterization of adenylyl cyclases, guanine nucleotide-dependent regulatory proteins (G proteins), and guanylyl cyclases.
Key Features
* Adenylyl cyclase: decision of activity
* practise of fabrics valuable in purification of parts of hormonally responsive cyclase systems
* Purification guanine nucleotide-dependent regulatory proteins
* Purification and characterization of g proteins: labeling and quantitating of G proteins
* Reconstitution guanylyl cyclase: assay
* training of fabrics used for research of the cyclase
* Purification
* law
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Additional resources for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
Example text
W. Eaton, Science 217, 948 (1982). 5 D. L. Confer, A. S. Slungaard, E. Graf, S. S. Panter, and J. W. Eaton, Adv. Cyclic Nucleotide Res. 17, 183 (1984). 6 G. E. Ward, D. L. Garbers, and V. D. Vacquier, Science 227, 768 (1985). 7 D. Cassel and T. Pfeuffer, Proc. Natl. Acad. Sci. A. 75, 2669 (1978). g S. H. Leppla, Proc. Natl. Acad. Sci. A. 79, 3162 (1982). 9 S. H. Leppla, Ado. Cyclic Nucleotide Res. 17, 189 (1984). [3] PREPARATION OF Ot-32p-LABELED NUCLEOTIDES 37 volume of 1 M perchloric acid and the [32p]cGMP purified as described below.
5 volume of guanylyl cyclase (about 20 to 50 nmol cGMP formed/min) to 1 volume of [o~-32p]GTP from Stage 4. Depending on the preparation of guanylyl cyclase, 50 to 90% conversion to [32p]cGMP should occur within 3 to 4 hr at room temperature. The reaction is terminated by the addition of an equal 4 D. L. Confer and J. W. Eaton, Science 217, 948 (1982). 5 D. L. Confer, A. S. Slungaard, E. Graf, S. S. Panter, and J. W. Eaton, Adv. Cyclic Nucleotide Res. 17, 183 (1984). 6 G. E. Ward, D. L. Garbers, and V.
L'2 Subsequently, following a wash to remove extraceUular [3H]adenine, the cells are further incubated in flesh medium containing the test substance(s) and appropriate controls. During the incubation step, the cells are permitted to respond to the extracellular challenge. The incubation is terminated by the addition of perchloric acid (PCA), which inactivates the metabolism of the cell, denatures the cellular proteins, and permits extraction of all cellular soluble nucleotides, including the [3H]adenine nucleotides.