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: decision of activity
* guidance of fabrics worthy 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
* instruction of fabrics used for examine of the cyclase
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Extra info for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
L] ASSAY OF ADENYLYL CYCLASE CATALYTIC ACTIVITY 21 Example 2. An analogous though simpler calculation is used for activities when unlabeled c A M P is used for sample recovery and is the same whether [a-32p]ATP or [3H]ATP is used as substrate tracer. Velocity = (sample cpm - no e n z y m e cpm) × A T P concentration × reaction volume/fraction of sample counted/(substrate cpm - no enzyme cpm) × cAMP standard Az59/sample A259/time/protein cAMP standard A259 is the optical density at 259 nm that would represent 100% r e c o v e r y of the added unlabeled cAMP.
25 Since comparable separation of ATP and cAMP can be achieved with columns packed with ZRCO325 or A120 3 ,26 it is likely that adsorption to the insoluble inorganic salts, rather than coprecipitation with them, is the basis of the separation of cAMP from the multivalent nucleotides and hence the basis of their usefulness in assays of adenylyl or guanylyl cyclases. It is important to emphasize that since none of these salt combinations will separate cAMP from adenosine, [a-32p]ATP is 23 R. 24 G.
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. <--24 m m - > E E o CO tion FIG. 2. Apparatus for column chromatography. of the racks supporting the alumina columns should allow the eluate from them to drip directly into scintillation vials.
Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase by John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin