Recombinant Human GGT5 Protein, CF

Catalog #: 7209-GT Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
7209-GT-010

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Recombinant Human GGT5 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to hydrolyze glutathione to Glu and Cys-Gly. The specific activity is >265 pmol/min/μg, as measured under the described conditions.

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human GGT5 protein Ser30-Tyr586, with an N-terminal 6-His tag.

Accession #

P36269

N-terminal Sequence
Analysis

His and Thr388

Predicted Molecular Mass

60 kDa

SDS-PAGE

24-27(mature), 41-46 (mature) & 64-66 (pro) kDa, reducing conditions

Product Datasheets

7209-GT

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

7209-GT

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 degreesC as supplied. 3 months, -20 to -70 degreesC under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 50 mM Tris, 0.05% Tween® 20, pH 8.0
Recombinant Human GGT5 (rhGGT5) (Catalog # 7209-GT)
Recombinant Human NQO-1 (rhNQO-1) (Catalog # 7567-DH)
Glutamate dehydrogenase (GIDH) (Sigma, Catalog # G7882), 200 U/mL stock in 50 mM Tris, 0.05% Tween 20, pH 8.0
Nicotinamide adenine dinucleotide ( beta -NAD) (Sigma, Catalog # N6522), 100 mM stock in deionized water
Resazurin (Catalog # AR002)
Glutathione, reduced (GSH) (Amresco, Catalog # 399), 250 mM stock in deionized water
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhGGT5 to 1 µg/mL in Assay Buffer.
Prepare substrate mixture containing 2 mM GSH, 100 U/mL GIDH, 2 mM beta -NAD, 40 µM Resazurin, and 4 µg/mL rhNQO-1 in Assay Buffer.
Load 50 µL rhGGT5 into wells of plate. Include 50 µL Assay Buffer for a Substrate Blank.
Start reaction by adding 50 µL substrate mixture to all wells.
Read the plate at excitation and emission wavelengths of 540 and 585 nm, respectively, for five minutes in kinetic mode.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard Resorufin (Sigma, Catalog # R3257)

Per Well:

rhGGT5: 0.050 µg
GSH: 1 mM
GIDH: 5 U
beta -NAD: 1 mM
rhNQO-1: 0.2 μg
Resazurin: 0.02 mM

Reconstitution Calculator

Background: GGT5

Gamma-glutamyl transpeptidase-5 (GGT5) is expressed as a pro form, which undergoes autocatalytic cleavage in the endoplasmic reticulum to become the two chain mature form (1). The mature form of GGT is a membrane protein embedded into the outer surface of the plasma membrane through its N-terminal transmembrane domain. It cleaves the gamma-glutamyl peptide bond of glutathione (GSH) and glutathione S-conjugates and transfers the g-glutamyl moiety to acceptors (2). By breaking down extracellular GSH into its constitutive amino acids, its activity is critical for maintaining GSH and cysteine homeostasis. GGT1 and GGT5 are the most fully characterized members of the GGT family (2). Elevated activity of GGT1 in blood has been used to measure liver damage such as that found in hepatocellular carcinoma or alcoholic liver (3). GGT5, also known as g-glutamyl leukotrienase (GGL), may play an important role in leukotriene D4 synthesis and in inflammatory processes.

References

Kinlough C.L. et al. (2005) Methods Enzymol. 401:426.
Heisterkamp N. et al. (2008) Hum. Genet. 123:321.
Whitfield J.B. (2001) Crit. Rev. Clin. Lab. Sci. 38:263.
Han B. et al. (2002) Am. J. Path. 161:481.

Long Name

gamma-Glutamyltransferase 5

Entrez Gene IDs

2687 (Human); 23887 (Mouse)

Alternate Names

DKFZp566O011; EC 2.3.2.2; gamma-glutamyl cleaving enzyme; Gamma-glutamyl transpeptidase-related enzyme; gamma-glutamyl transpeptidase-related protein; gamma-glutamyltransferase 5; Gamma-glutamyltransferase-like activity 1FLJ92733; Gamma-glutamyltranspeptidase 5; GGT 5; GGT5; GGTLA1; GGT-rel