Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase by John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie

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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: choice of activity
* training of fabrics priceless 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
* training of fabrics used for learn of the cyclase
* Purification
* law

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Extra info for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase

Example text

To achieve full membrane solubilization in cases of high membrane content, it is advisable to boil the test tubes for 3 min at this stage. This also facilitates the rate of chromatography. Hence, use heatstable tubes. 3. The mixtures in the reaction tubes are then diluted and decanted onto chromatography columns for purification of sample cAMP. 4. Assay blanks are prepared by omitting enzyme or by adding enzyme after the stopping solution. 5. A disadvantage of this procedure is that all radioactive compounds, including unused substrate [a-32p]ATP, [ct-32P]ADP, [32p]AMP, [32p]p i , as well as degradation products of [3H]cAMP, are passed with the labeled cAMP onto the chromatography column and are typically eluted in a fall through fraction that must be collected and 2s y.

R. Russell, and D. Gospodarowicz, J. Biol. Chem. 251, 2344 (1976). 31 C. Nakai and G. Brooker, Biochim. Biophys. Acta 391, 222 (1975). 32 R. Counis and S. Mongongu, Anal. Biochem. 84, 179 (1978). 16 ADENYLYLCYCLASE [11 then poured into columns ( - 0 . 6 x 4 cm). After each use, Dowex 50 columns are regenerated by washing with 5 ml of 1 N HC1, then stored until reused. Before use the columns are washed 3 times with 10 ml of water. Columns can be reused dozens of times. If flow rates decrease, columns should be regenerated with NaOH, water, and HCI as above.

6 ml of zinc acetate containing [3H]cAMP used to stop the reaction, counted under comparable quench conditions used to count the samples. [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.

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