Aminopeptidase A Protein, Human (sf9, His)

Aminopeptidase A protein has calcium-regulating activity and plays a key role in central hypertension by preferentially cleaving N-terminal acidic residues, including angiotensin II. The specificity of this enzyme highlights its involvement in the regulation of blood pressure-related peptides, suggesting potential implications for the physiological mechanisms of hypertension. Aminopeptidase A Protein, Human (sf9, His) is the recombinant human-derived Aminopeptidase A protein, expressed by Sf9 insect cells , with N-His labeled tag.

The Aminopeptidase A protein serves a pivotal role in the regulation of central hypertension, demonstrating a calcium-modulated preference for cleaving N-terminal acidic residues from peptides, including angiotensin II. This enzymatic activity highlights its involvement in the modulation of peptides associated with blood pressure regulation, suggesting a potential impact on the physiological mechanisms underlying hypertension. The calcium-dependent specificity of Aminopeptidase A underscores its intricate regulatory role in the processing of bioactive peptides, emphasizing its significance in cardiovascular function and potential relevance in the context of hypertensive conditions.

Measured by its ability to cleave the fluorogenic peptide substrate, Glu-7-amido-4-methyl coumarin (Glu-AMC) and the specific activity is >2000 pmol/min/μg.

Materials
Assay buffer: 25 mM Tris, 50 mM CaCl?, 0.2 M NaCl, pH 8.0
Aminopeptidase A Protein, Human (sf9, His) (HY-P72824)
Substrate: Glu-7-amido-4-methylcoumarin
Standard: 7-amido-4-methylcoumarin
Black 96-well Plate
Fluorescence Microplate Reader

Procedure
1. Dilute 7-amido-4-methylcoumarin in assay buffer to concentrations of 0, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 μmol/L. Add 100 μL of each concentration to the wells to initiate the reaction, with three replicates per concentration. Read the fluorescence in kinetic mode for 5 minutes with excitation and emission wavelengths of 380 nm and 460 nm (top read) . Plot the concentration of the standard on the x-axis and the measured RFU on the y-axis to create the standard curve and obtain the standard curve equation.
2. Dilute Human Aminopeptidase A to 0.4 μg/mL in assay buffer.
3. Dilute the substrate to 200 μM in assay buffer.
4. Add 100 μL of 0.4 μg/mL Human Aminopeptidase A and 200 μM substrate to the wells of the black 96-well plate. Include a control with assay buffer and substrate only. Perform three replicates for each condition.
5. Read the RFU values in kinetic mode for 5 minutes with excitation and emission wavelengths of 380 nm and 460 nm, respectively.
6. Calculate enzyme activity:

Human

Sf9 insect cells

N-His

Q07075/NP_001968.3 (R41-G957)

2028  [NCBI]

Extracellular Domain

RSCDSSGDGGPGTAPAPSHLPSSTASPSGPPAQDQDICPASEDESGQWKNFRLPDFVNPVHYDLHVKPLLEEDTYTGTVSISINLSAPTRYLWLHLRETRITRLPELKRPSGDQVQVRRCFEYKKQEYVVVEAEEELTPSSGDGLYLLTMEFAGWLNGSLVGFYRTTYTENGQVKSIVATDHEPTDARKSFPCFDEPNKKATYTISITHPKEYGALSNMPVAKEESVDDKWTRTTFEKSVPMSTYLVCFAVHQFDSVKRISNSGKPLTIYVQPEQKHTAEYAANITKSVFDYFEEYFAMNYSLPKLDKIAIPDFGTGAMENWGLITYRETNLLYDPKESASSNQQRVATVVAHELVHQWFGNIVTMDWWEDLWLNEGFASFFEFLGVNHAETDWQMRDQMLLEDVLPVQEDDSLMSSHPIIVTVTTPDEITSVFDGISYSKGSSILRMLEDWIKPENFQKGCQMYLEKYQFKNAKTSDFWAALEEASRLPVKEVMDTWTRQMGYPVLNVNGVKNITQKRFLLDPRANPSQPPSDLGYTWNIPVKWTEDNITSSVLFNRSEKEGITLNSSNPSGNAFLKINPDHIGFYRVNYEVATWDSIATALSLNHKTFSSADRASLIDDAFALARAQLLDYKVALNLTKYLKREENFLPWQRVISAVTYIISMFEDDKELYPMIEEYFQGQVKPIADSLGWNDAGDHVTKLLRSSVLGFACKMGDREALNNASSLFEQWLNGTVSLPVNLRLLVYRYGMQNSGNEISWNYTLEQYQKTSLAQEKEKLLYGLASVKNVTLLSRYLDLLKDTNLIKTQDVFTVIRYISYNSYGKNMAWNWIQLNWDYLVNRYTLNNRNLGRIVTIAEPFNTELQLWQMESFFAKYPQAGAGEKPREQVLETVKNNIEWLKQHRNTIREWFFNLLESG

Approximately 115.5 kDa

• 
  ≥ 90%, as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM, Tris 500 mM NaCl, 10 % glycerol, pH 7.4.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.