Recombinant c-AMP dependant Protein Kinase A regulatory subunit 2 alpha

Synonyms  cAMP-Dependent Protein Kinase A regulatory subunit-II A, PKA-RII alpha. 

Introduction  cAMP-dependent PKA is a ubiquitous serine/theonine protein kinase present in a variety of tissues (e.g. brain, skeletal muscle, heart). The intracellular cAMP level regulates cellular responses by altering the interaction between the catatytic C and regulatory R subunits of PKA. The inactive tetrameric PKA holoenzyme R2C2 is activated when cAMP binds to R2, which dissociates the tetramer to R2 cAMP 4 and two active catalytic subunits. Free Catalytic subunits of PKA can phosphorylate a wide variety of intracellular target proteins. In response to hormone- induced high cAMP levels, PKA phosphorylates glycogen synthetase (inhibition of the enzyme activity) and phosphorylase kinase to block glycogen synthesis. Different isoforms of catalytic and regulatory subunits suggest specific functions. 

Recombinant c-AMP dependant Protein Kinase A regulatory subunit 2 alphaDescription  The recombinant PKA regulatory subunit II-a is a dimeric 90 kDa protein.

Protein Kinase A is purified by proprietary chromatographic techniques. 

Source  Escherichia Coli. 

Physical Appearance  Sterile Filtered clear solution. 

Formulation  PKA regulatory subunit-II alpha is supplied in 50% glycerol. 

Unit Definition  One unit is defined as the amount of recombinant PKA catalytic subunit alpha, required to incorporate 1nmol of phosphate into the specific substrate peptide kemptide (LRRASlG) in one minute at 30°C. 

Recombinant c-AMP dependant Protein Kinase A regulatory subunit 2 alphaStability  PKA should be stored at 4°C if entire vial will be used within 2-4 weeks. For long term storage it is recommended to store at -20°C.

Avoid multiple freeze-thaw cycles. 

Purity  Greater than 95% as determined by SDS-PAGE. 

Biological Activity  PKA regulatory subunit alpha specifically inhibits PKA catalytic subunit (Ki about 0.7nM). The binding is not dependent on the presence of ATPMg, however, the regulatory subunit will be phosphorylated at the binding site by PKA C alpha upon binding. Activity can be restored by adding the second messenger cAMP (Kact about 100nM). Subcellular localisation is regulated via interaction of PKA RII with so-called A-kinase anchoring proteins (AKAPs). 

Recombinant c-AMP dependant Protein Kinase A regulatory subunit 2 alphaAssay Conditions  Roskoski-AssayProtein kinase activity can be measured using a modified radioactive assay according to Roskoski et al. 

 : The assay will be performed in a mixture containing 50mM MOPS (pH7.0), 10mM MgCI2, 0.25 mg/ml bovine serum albumin, 100 IJM Kemptide (peptide substrate), 100 IJM unlabeled ATP mixed with [y_32p] ATP (500-1000 cpm/pmol) and Ca subunit in a final volume of 50 IJI. Reaction is started by addition of the Ca subunit and can be stopped after a 5 minute incubation at 30°C by spotting the reaction mix onto Whatman P-81 filters and soaking the filters four times in 75mM phosphoric acid (10 ml per sample) for at least 5 minutes. After four washing steps rinse filters with ethanol, dry and count.

Roskoski, R., Jr. (1983) Methods Enzymol. 99, 3-6

For the detection of phosphorylation in substrate proteins the phosphotransferase reaction can alternatively be stopped by taking aliquots of the mixture and adding SDS sample buffer. The phosphorylation status of the substrate proteins can subsequently be analysed using SDS PAGE and autoradiography.

Zimmermann, B. (1999) Journal of Biological Chemistry.274, 9, 5370-78.