Certificate of Analysis and Data Sheet

Source:

Catalog No.
PKA-204

Description :

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. The recombinant PKA regulatory subunit II a is a dimeric 90kDa protein. Recombinant Protein Kinase A is purified by proprietary chromatographic techniques.

Physical Appearance:

Sterile Filtered clear soultion.

Formulation:

Recombinant 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蚓.

Storage:

Recombinant PKA should be stored at 4蚓 if entire vial will be used within 2-4 weeks. For long term storage it is recommended to store at -20蚓.
Please avoid freeze-thaw cycles.

Purity:

Greater than 95.0% as determined by:
(a) Analysis by reducing and non-reducing SDS-PAGE Silver Stained gel.

Dimers and aggregates:

Less than 1% as determined by silver-stained SDS-PAGE gel analysis.

Biological Activity:

Prospec's recombinant 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).

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蚓 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:

This material is offered by ProSpec-TechnoGene for research, laboratory or further evaluation purposes.

Gene:

Name:PRKAR2A
Synonyms:PKR2, PRKAR2

Protein synonyms/aliases:

cAMP-dependent protein kinase type II-alpha regulatory subunit.

Protein Family:

Belongs to the cAMP-dependent kinase regulatory chain

Protein Domains:

Domains:
•IPR003117   cAMP-dependent protein kinase regulator, type II PKA R subunit
•IPR000595   Cyclic nucleotide-binding

Protein links:

Precursor- Protein structure and amino acid sequence:

Latest Publications:

1. Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions.
J Mol Biol 2004 Apr 9;337(5):1183-94
2. Deletion of type IIalpha regulatory subunit delocalizes protein kinase A in mouse sperm without affecting motility or fertilization.
J Biol Chem 1999 Aug 20;274(34):24131-6

採購找生工，預算最輕鬆。

回頁首↑ | 回首頁←

---

中華民國95年06月06日更新