PRKACA Human

cAMP-Dependent Protein Kinase A catalytic subunit α Human Recombinant
Cat. No.
BT8328
Source
Escherichia Coli.
Synonyms
cAMP-dependent protein kinase alpha-catalytic subunit, EC 2.7.11.11, PKA C-alpha, PKACA, PRKACA, MGC48865, MGC102831.
Appearance
Purity
Greater than 95% as determined by SDS-PAGE.
Usage

THE BioTek'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.

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Description

cAMP-dependent PKA is an ubiquitous serine/threonine 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 catalytic 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 catalytic subunit a is a 41kDa protein. The a-isoform is the predominant form with a broad tissue distribution and can be used for in vitro enzymological studies of neural and hormonal signal transduction or to phosphorylate target proteins in vivo including Ion channels, transcriptional activator proteins and regulatory enzymes of glycogen metabolism.

Product Specs

Description
cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase found in various tissues, including the brain, skeletal muscle, and heart. PKA regulates cellular responses based on intracellular cAMP levels. When cAMP levels rise, such as in response to hormone signaling, cAMP binds to the regulatory (R) subunits of the inactive PKA holoenzyme (R2C2). This binding causes the tetramer to dissociate into R2 cAMP 4 and two active catalytic (C) subunits. These free catalytic subunits can then phosphorylate a wide range of intracellular target proteins, leading to various downstream effects. For example, PKA phosphorylates glycogen synthase and phosphorylase kinase, inhibiting glycogen synthesis. Different isoforms of catalytic and regulatory subunits suggest specialized functions. This product contains the recombinant PKA catalytic subunit alpha, a 41 kDa protein, known for its broad tissue distribution. This recombinant subunit is suitable for in vitro studies of neural and hormonal signal transduction and in vivo phosphorylation of target proteins like ion channels, transcriptional activators, and enzymes involved in glycogen metabolism.
Formulation
PKA catalytic subunit alpha is supplied in a storage buffer consisting of 20mM MOPS (pH 7), 150mM NaCl, 1mM DTT, 1mM EDTA, and 50% Glycerin.
Stability
For short-term storage (2-4 weeks), store the vial at 4°C. For long-term storage, freeze the product at -20°C. Avoid repeated freeze-thaw cycles to maintain enzyme activity.
Unit Definition
One unit (U) of activity is defined as the quantity of recombinant cAMP-Dependent Protein Kinase catalytic subunit alpha needed to catalyze the incorporation of 1 picomole (pmol) of phosphate into the kemptide peptide substrate (LRRASLG) per minute at a temperature of 30°C.
Specific Activity
The specific activity of this recombinant PKA catalytic subunit alpha exceeds 10,000,000 units per milligram of protein (U/mg).
Purity
The purity of this recombinant PKA catalytic subunit alpha is greater than 95% as determined by SDS-PAGE analysis.
Assay Conditions
Protein kinase activity can be assessed using a modified radioactive assay based on the Roskoski method. The assay is conducted in a 50 μl reaction mixture containing 50mM MOPS (pH 7.0), 10mM MgCl2, 0.25 mg/ml bovine serum albumin, 100 μM Kemptide (peptide substrate), 100 μM unlabeled ATP mixed with [γ-32P] ATP (500-1000 cpm/pmol), and the PKA catalytic subunit. The reaction is initiated by the addition of the catalytic subunit and allowed to proceed for 5 minutes at 30°C. The reaction is then terminated by spotting aliquots onto Whatman P-81 filters. The filters are washed four times with 75mM phosphoric acid (10 ml per sample) for at least 5 minutes each to remove unincorporated [γ-32P] ATP. Following the washes, filters are rinsed with ethanol, dried, and counted using a scintillation counter. For analysis of substrate protein phosphorylation, the phosphotransferase reaction can be stopped by adding SDS sample buffer to aliquots of the reaction mixture. The phosphorylation status of the substrate proteins can then be evaluated by SDS-PAGE followed by autoradiography. References: 1. Roskoski, R., Jr. (1983) Methods Enzymol. 99, 3-6. 2. Zimmermann, B. (1999) Journal of Biological Chemistry. 274, 9, 5370-78.
Synonyms
cAMP-dependent protein kinase alpha-catalytic subunit, EC 2.7.11.11, PKA C-alpha, PKACA, PRKACA, MGC48865, MGC102831.
Source
Escherichia Coli.
Amino Acid Sequence
MGNAAAAKKG SEQESVKEFL AKAKEDFLKK WESPAQNTAH LDQFERIKTL GTGSFGRVML VKHKETGNHY AMKILDKQKV VKLKQIEHTL NEKRILQAVN FPFLVKLEFS FKDNSNLYMV MEYVPGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS LDLIYRDLKP ENLLIDQQGY IQVTDFGFAK RVKGRTWTLC GTPEYLAPEI ILSKGYNKAV DWWALGVLIY EMAAGYPPFF ADQPIQIYEK IVSGKVRFPS HFSSDLKDLL RNLLQVDLTK RFGNLKNGVN DIKNHKWFAT TDWIAIYQRK VEAPFIPKFK GPGDTSNFDD YEEEEIRVSI NEKCGKEFSE F.

Product Science Overview

Introduction

cAMP-Dependent Protein Kinase A (PKA) is a crucial enzyme in cellular signaling pathways. It is a serine/threonine protein kinase that plays a significant role in regulating various cellular processes, including metabolism, gene expression, and cell cycle progression. The catalytic subunit α (Cα) of PKA is one of the primary isoforms responsible for its enzymatic activity.

Structure and Function

PKA exists as a tetrameric holoenzyme composed of two regulatory subunits and two catalytic subunits. In its inactive form, the catalytic subunits are bound to the regulatory subunits. Upon binding of cyclic AMP (cAMP) to the regulatory subunits, the holoenzyme dissociates, releasing the active catalytic subunits .

The catalytic subunit α (Cα) contains the active site responsible for phosphorylating target proteins. The recognition motif for phosphorylation by PKA is RRXS/TY, where Y is typically a hydrophobic residue . This phosphorylation event is critical for modulating the activity of various proteins involved in cellular signaling pathways.

Recombinant Expression

The human recombinant form of the PKA catalytic subunit α is produced using recombinant DNA technology. It is typically expressed in Escherichia coli (E. coli) systems, which allows for high-yield production and easy purification . The recombinant enzyme is often used in research to study PKA’s role in cellular processes and to develop potential therapeutic interventions.

Biological Significance

PKA is involved in numerous cellular functions, including:

  • Regulation of Metabolism: PKA phosphorylates enzymes involved in glycogen, sugar, and lipid metabolism, thereby regulating their activity .
  • Gene Expression: PKA can phosphorylate transcription factors, influencing gene expression and cellular responses to external signals .
  • Cell Cycle Progression: PKA activity is essential for proper cell cycle progression and division .
Historical Context

The discovery of PKA dates back to 1968 when chemists Edmond H. Fischer and Edwin G. Krebs identified its role in phosphorylation and dephosphorylation processes. Their groundbreaking work earned them the Nobel Prize in Physiology or Medicine in 1992 .

Applications in Research

The recombinant form of the PKA catalytic subunit α is widely used in biochemical and pharmacological research. It serves as a valuable tool for studying the enzyme’s structure, function, and regulatory mechanisms. Additionally, it is used in drug discovery efforts to identify compounds that can modulate PKA activity for therapeutic purposes .

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