By John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin
This quantity emphasizes tools 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 precious 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 resources for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
Note: If cell culture media contain adenine, this method cannot be applied. Care must be taken as per international and local regulations concerning handling and waste disposal of radioactive materials. Acknowledgments I wish to thank Rachel Benjaminfor devoted secretarial assistance, Josepha SchmidtSole for technicalhelp, and Nira Garty for enthusiasmand help. The authoris the Charles W. and Tillie K. Lubin Professor for Hormone Research at The WeizmannInstitute of Science.  PREPARATION OF O~-32p-LABELED NUCLEOTIDES 29  P r e p a r a t i o n of a - a s P - L a b e l e d N u c l e o s i d e T r i p h o s p h a t e s , N i c o t i n a m i d e A d e n i n e D i n u c l e o t i d e , a n d Cyclic N u c l e o t i d e s for U s e in D e t e r m i n i n g A d e n y l y l a n d G u a n y l y l C y c l a s e s a n d Cyclic N u c l e o t i d e P h o s p h o d i e s t e r a s e B y TIMOTHY F.
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.
Incubate (37°) for 90-120 min* in a COz incubator. * The conditions cited are typical for M2R mouse melanoma cells, grown in Dulbecco's modified Eagle's medium: Ham's F12 medium (DMEM : F I2) containing 10% horse serum, 24 ADENYLYL CYCLASE  Incubation. At this stage, prelabeled cells are washed and stimulated to form [3H]cAMP. Place the dish on the table on insulating paper and aspirate the medium. (Caution: Medium is radioactive! 5 ml of test substance (37°), dissolved in incubation medium.