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.
* Adenylyl cyclase: choice of activity
* practise of fabrics helpful in purification of elements 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
* practise of fabrics used for examine of the cyclase
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Additional resources for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
Exp. Ther. 163, 379 (1968). S. Chan and M. C. Lin, this series, Vol. 38, p. 38. A. White and T. V. Zenser, Anal. Biochem. 41, 372 (1971). [l] ASSAY OF ADENYLYL CYCLASE CATALYTIC ACTIVITY 13 preferred to [3H]ATP as substrate, to avoid the possibility that the [3H]cAMP formed through adenylyl cyclase may be contaminated with [3H]adenosine, [3H]inosine, or other labeled compounds deriving from adenine nucleotides. The characteristics of the insoluble inorganic salts are taken advantage of in the following procedure adapted from Jakobs et al.
A p p a r a t u s . Rapid flow rates for the alumina columns, and consequently short chromatography times, are achieved with glass columns with a large cross-sectional area and a coarse sintered glass plug to retain the alumina (Fig. 2). d. ). , - 0 . , plastic) with spacing identical to that of the racks of scintillation vials to be used. The design of the racks supporting the Dowex 50 columns should be such that the columns can be conveniently mounted aboved the alumina columns so that the eluate of the Dowex 50 columns can drip directly onto the alumina columns.
Following counting, the absorbance at 259 nm is determined on an aliquot of the sample to quantitate recovery of unlabeled cAMP. If recovery is monitored with [3H]cAMP, both [3H]cAMP and [32p]cAMP are eluted with 4 ml of 100 mM imidazole into 10-ml vials to which 5 ml scintillation cocktail is then added. The smaller vials spare expensive cocktail. Sample recovery is determined from dual channel counting. 6. Scintillation counting of 32p by Cerenkov radiation is achieved with a single channel with wide open windows.