Certificate of Analysis and Data Sheet

Source:
E.Coli

Catalog No.
PKA-201

Description :

cAMP-dependent PKA is an 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 I 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-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 0.1nM). 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:PRKAR1A
Synonyms:PKR1, PRKAR1

Protein synonyms/aliases:

cAMP-dependent protein kinase type I-alpha regulatory subunit (Tissue-specific extinguisher 1)
    (TSE1).

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. CpG immunomer DNA enhances antisense protein kinase A RIalpha inhibition of multidrug-resistant colon carcinoma growth in nude mice: molecular basis for combinatorial therapy.
Clin Cancer Res 2005 Aug 15;11(16):5950-5
2. The conformationally dynamic C helix of the RIalpha subunit of protein kinase A mediates isoform-specific domain reorganization upon C subunit binding.
J Biol Chem 2005 Oct 21;280(42):35521-7
3. The second subunit of the replication factor C complex (RFC40) and the regulatory subunit (RIalpha) of protein kinase A form a protein complex promoting cell survival.
Cell Cycle 2005 Feb;4(2):323-9
4. Antisense protein kinase A RIalpha inhibits 7,12-dimethylbenz(a)anthracene-induction of mammary cancer: blockade at the initial phase of carcinogenesis.
Clin Cancer Res 2004 Jul 1;10(13):4568-77
5. Mapping intersubunit interactions of the regulatory subunit (RIalpha) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS).
J Mol Biol 2004 Jul 23;340(5):11 85-96
6. Identification of the protein kinase A regulatory RIalpha-catalytic subunit interface by amide H/2H exchange and protein docking.
Proc Natl Acad Sci U S A 2003 Nov 11;100(23):13264-9

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中華民國95年06月06日更新