cfPKAR-1a

PKAR-I alpha
(cAMP free) Protein Kinase A regulatory subunit-I A Recombinant
Catalog No. PKA-202

Synonyms:
cAMP free protein kinase type I-alpha regulatory subunit, Tissue-specific extinguisher 1, TSE1, CAR, CNC, CNC1, PKR1, PPNAD1, PRKAR1, PRKAR1A, MGC17251, DKFZp779L0468, Protein Kinase A regulatory subunit-I A, cAMP free Protein Kinase A regulatory subunit-I A, PKAR-I alpha, Regulatory (R) subunit of Protein Kinase A (PKA).

Introduction:
The Regulatory (R) subunit of Protein Kinase A (PKA) inhibits its kinase activity by shielding the Catalytic (C) subunit from physiological substrates. This inhibition is reversed in response to extra-cellular signals that increase cAMP levels in the cytoplasm. Upon cAMP binding to R, C is allosterically released from R, activating a spectrum of downstream signaling cascades. Crystallographic data indicated that a series of distinct conformational changes within CBD-A must occur to relay the cAMP signal from the cAMP binding site to the R:C interaction interface. One critical cAMP relay site within the CBD-A of R has been identified as Asp170 because the D170A mutation selectively reduces the negative cooperativity between the cAMP- and C-recognition sites (i.e. the KD for the R:C complex in the presence of cAMP is reduced by more than 12-fold), without significantly compromising the high affinity of R for both binding partners.

Description:
PKA regulatory subunit I a Recombinant is a dimeric 90 kDa protein.
PKAR-I alpha is purified by proprietary chromatographic techniques.

Source:
Escherichia Coli

Physical Appearance:
Sterile Filtered clear solution.

Formulation:
PKA regulatory subunit-I 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.

Storage Conditions:
PKAR-Ia 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 0.1nM). Activity can be restored by adding cAMP (Kact about 100nM). The binding of the catalytic subunit is dependent on the presence of ATP and Mg.

Assay Conditions:
Roskoski-Assay:
Protein 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 minutes 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

Usage:
Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.