Recombinant (cAMP free) Protein Kinase A regulatory subunit-I A

• Cat.No.: rcfPKArIa-09
• Product Overview: PKA regulatory subunit I a Recombinant is a dimeric 90 kDa protein. PKAR-I alpha is purified by proprietary chromatographic techniques.

Specification

• Source: E.coli
• Tag: Non
• Description: 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.
• Physical Appearance: Sterile Filtered clear solution (0.185mg/ml).
• Purity: Greater than 95% as determined by SDS-PAGE.
• 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.
• 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.
• Storage: 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.

Gene Information

• Synonyms: 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); PRKAR1A; CAR; CNC; CNC1; DKFZp779L0468; MGC17251; PKR1; PRKAR1; TSE1; PPNAD1; Tissue-specific extinguisher 1; cAMP-dependent protein kinase regulatory subunit RIalpha; cAMP-dependent protein kinase type I-alpha regulatory chain; cAMP-dependent protein kinase, regulatory subunit alpha 1; protein kinase A type 1a regulatory subunit; protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific extinguisher 1); tissue-specific extinguisher 1.

Citation

Crystal structure of diamondback moth ryanodine receptor Repeat34 domain reveals insect-specific phosphorylation sites

Journal: BMC Biology    PubMed ID: 31597572    Data: 2019/10/9

Authors: Tong Xu, Zhiguang Yuchi

Article Snippet:Phosphorylation reactions were performed at 30 °C or 18 °C overnight in 50-μl volumes.Phosphorylation reactions were performed at 30 °C or 18 °C overnight in 50-μl volumes.. The reaction mixture for PKA contained 10 mM HEPES (pH 7.4), 50 mM KCl, 20 mM MgCl 2 , 0.2 mM ATP, 2 mM 2-mercaptoethanol, 0.8 mg/ml DBM RyR Repeat34, and 200 U PKA (Creative BioMart).. The unit of PKA is defined as the amount of enzyme required to incorporate 1 pmol of phosphate into casein in 1 min. DBM PKA and human PKA share a relatively high sequence identity and a conserved activity center, so we chose to use the commercial human PKA for this assay.The unit of PKA is defined as the amount of enzyme required to incorporate 1 pmol of phosphate into casein in 1 min. DBM PKA and human PKA share a relatively high sequence identity and a conserved activity center, so we chose to use the commercial human PKA for this assay.

Comparison of repeat domains. a Superpositions of the DBM RyR Repeat34 crystal structure (gray) with mouse RyR2 Repeat34 crystal structure (blue; PDB ID 4ETV) and rabbit RyR1 Repeat12 crystal structure (red; PDB ID 5C30). The main structural differences are labeled. b Comparison of the surface views of the three structures in panel A. The distance between the two ends of each horseshoe is labeled. c Plot showing per residue root mean square deviation (RMSD) for the mouse RyR2 Repeat34 and rabbit RyR1 Repeat12 crystal structures relative to DBM RyR Repeat34 crystal structure. d Sequence alignment of DBM Repeat34, mouse RyR2 Repeat34, and rabbit RyR1 Repeat12. Secondary structure elements for DBM RyR Repeat34 are indicated above the sequence. Phosphorylation sites in DBM are colored in blue, green, and pink depending on their location within the 3-dimensional structure of the domain (same color scheme as in Fig. ). Phosphorylation sites in RyR2 are colored in turquoise. The classic PKA site RyR2 S2808 and the equivalent PKA site DBM RyR S2946 are highlighted in yellow. Residues contributing to the glycerol binding are highlighted in orange

New Optical Imaging Reporter-labeled Anaplastic Thyroid Cancer-Derived Extracellular Vesicles as a Platform for In Vivo Tumor Targeting in a Mouse Model

Journal: Scientific Reports    PubMed ID: 30201988    Data: 2018/9/10

Authors: Prakash Gangadaran, Xiu Juan Li, Byeong-Cheol Ahn

Article Snippet:Stability of Free Rluc protein (Creative BioMart) was evaluated by measuring Rluc activity at 0, 1, 2 and 3 hours.. The appropriate substrate coelenterazine was added to each well.The appropriate substrate coelenterazine was added to each well.

EV-CAL-62/Rluc showed EV-specific Rluc activity and serum stability in vitro . ( A ) Representative bioluminescent imaging of an in vitro luciferase assay in EVs from CAL-62 and CAL-62/Rluc cells. ( B ) Quantitative in vitro luciferase data in EVs are expressed as the mean ± SD. ( C ) Western blot analysis of the Rluc protein in EVs from CAL-62, CAL-62/Rluc cells, detected by means of Rluc-specific antibodies. TSG served as loading control. ( D ) Stability of Rluc in EV-CAL62/Rluc and recombinant Rluc protein in serum. Time course of stability of Rluc at 37 °C in 20% FBS/PBS buffer. ( E ) Representative confocal images of Rluc (green) and DiD (red) in EV-CAL62/Rluc/DiD or EV-CAL62/DiD or EV-CAL62 in CAL62 cells. Scale bars: 10 μm.

A new bioluminescent reporter system to study the biodistribution of systematically injected tumor-derived bioluminescent extracellular vesicles in mice

Journal: Oncotarget    PubMed ID: 29299117    Data: 2017/12/15

Authors: Prakash Gangadaran, Xiu Juan Li, Byeong-Cheol Ahn

Article Snippet:Stability of Free Rluc protein (Creative BioMart) was evaluated by measuring Rluc activity at 0, 1, 2, 3, 4 and 5 hours.. The appropriate substrate coelenterazine was added to each well.The appropriate substrate coelenterazine was added to each well.

( A , B ) Representative bioluminescent imaging of an in vitro luciferase assay in EVs from CAL-62, CAL-62/Rluc cells, MDA-MB-231 and MDA-MB-231/Rluc. Quantitative in vitro luciferase assay in EV's data are expressed as mean ± SD. ( C ) Western blot analysis of the Rluc protein in cells and EVs from CAL-62, CAL-62/Rluc & MDA-MB-231 and MDA-MB-231/Rluc cells, detected by means of Rluc-specific antibodies. β-Actin served as loading control. ( D ) RT-PCR analysis of Rluc mRNA in cells and EVs from CAL-62, CAL-62/Rluc & MDA-MB-231 and MDA-MB-231/Rluc cells. GAPDH was used as a loading control.

EV-CAL-62/Rluc and EV-MDA-231/Rluc showed high serum stability and Rluc protein incorporated inside the EVs ( A ) Rluc-binding capacity of EVs in serum. Time course of binding of Rluc in EV-CAL-62/Rluc and EV-MDA-231/Rluc at 37°C in 20% FBS/PBS buffer. Rluc activity was measured in serum. ( B ) Stability of Rluc activity of EV-CAL-62/Rluc and EV-MDA-231/Rluc in serum. Time course of stability of Rluc in EV at 37°C in 20% FBS/PBS buffer. ( C ) Stability of Rluc activity of Free Rluc protein in serum. Time course of stability of Rluc at 37°C in 20% FBS/PBS buffer. ( D , E ) EV-CAL-62/Rluc and EV-MDA-231/Rluc treated with proteinase K and without proteinase K and activity of Rluc was measured by IVIS. Data are expressed as mean ± SD. ( F ) Western blot analysis of the Rluc protein in cytosolic and membrane fraction of EV-CAL-62/Rluc & EV-MDA-231/Rluc, detected by means of Rluc-specific antibodies. Alix and CD63 served as loading control for cytosolic and membrane fraction respectively.

( A ) Representative in vivo bioluminescent imaging (BLI) of mice injected with Free Rluc ( n = 3) or PBS ( n = 3). Coelenterazine was injected via the same route at 1, 10 and 30 min and 1, 3 and 24 hours after initial administration to visualize free Rluc protein. ( B ) Quantitation of Free Rluc signal from regions corresponding to the lung, liver, spleen and kidney after EV administration; the values are expressed as mean ± SD. * P < 0.05 (Student's t -test). ( C ) Quantification of bioluminescent signals in lungs, liver, spleen, and kidneys at 3 minutes and 24 hours (Free Rluc or PBS). The values are expressed as mean ± SD, * P < 0.05, (Student's t -test).