• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <0.01 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to hydrolyze p-nitrophenylacetate. The specific activity is >330 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived His19-Leu565, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His19

• Predicted Molecular Mass

  62 kDa

• SDS-PAGE

  55-60 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 7.5

• Reading Buffer: 50 mM Tris, pH 8.0

• Recombinant Mouse Carboxylesterase 1/CES1 (rmCES1) (Catalog # 7929-CE)

• Substrate: 4-Nitrophenyl acetate (4-NPA) (Sigma, Catalog # N-8130), 100 mM stock in Acetone

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rmCES1 to 12 ng/μL in Assay Buffer.

2. Dilute Substrate to 2 mM in Assay Buffer.

3. Load 50 μL of 12 ng/μL rmCES1 into a clear microplate in triplicate and start the reaction by adding 50 μL of 2 mM 4-NPA. Include a Substrate Blank consisting of 50 μL of Assay Buffer and 50 μL of 2 mM 4?NPA.

4. Incubate for 10 minutes at room temperature.

5. Add 100 μL Reading Buffer to each well (this will not stop the reaction).

6. Read immediately at a wavelength of 410 nm (bottom read) in endpoint mode.

7. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Nitrophenol (Sigma, Catalog # 241326).

Per Well:

• rmCES1:  0.6 μg

• Substrate:  0.5 mM

Background: Carboxylesterase 1/CES1

Carboxylesterase 1 is a member of a large family of carboxylesterases that are responsible for the hydrolysis of ester and amide bonds (1, 2). They have broad substrate specificity ranging from small molecule esters such as phenylester to long chain fatty acid esters and thioesters. They play a major role as determinants of pharmacokinetic behavior for most therapeutic agents containing an ester. By de?esterification, they can activate or inactivate the agents. They also participate in detoxification of drugs such as cocaine and heroin in serum and liver. The resulting de?esterified metabolites are secreted out in urine. They can also detoxify organophosphate and carbamate analogues used in agrochemicals or chemical nerve agents, such as malathion, sarin, tabun, and VX. In addition to the hydrolytic activity, they can perform transesterification, a reaction important for cholesterol homeostasis. Carboxylesterase deficiency may be associated with non?Hodgkin lymphoma or B?cell lymphocytic leukemia. CES?1 shares the serine hydrolase fold observed in other esterases (3). Ces1G is a rat and mouse specific protein that is expressed predominantly in liver, but also in kidney and lung (4).

• References:

1. Redinbo, M.R. and Potter, P.M. (2005) Drug Discovery Today         10:313.

2. Satoh, T. and Hosokawa, M. (2006) Chem. Biol. Interactions         162:195.

3. Fleming, C.D.         et al. (2007) Biochemistry         46:5603.

4. Ellinghaus P.         et al. (1998) Biochim. Biophys. Acta.         1397:175.

• Entrez Gene IDs:

  1066 (Human); 12623 (Mouse); 29225 (Rat)

• Alternate Names:

  ACAT; Carboxylesterase 1; CES1; Ces-1; Egasyn; HMSE1; REH; SES1; Ses-1; TGH