Recombinant Human 6-Phosphogluconate Dehydrogenase/PGD, CF Summary
Details of Functionality |
Measured by its ability to dehydrogenate 6-phosphogluconic acid. The specific activity is >6000 pmol/min/μg, as measured under the described conditions. |
Source |
E. coli-derived human 6-Phosphogluconate Dehydrogenase/PGD protein Ala2-Ala483, with a N-terminal Met and 6-His tag |
Accession # |
|
N-terminal Sequence |
Met |
Protein/Peptide Type |
Recombinant Enzymes |
Gene |
PGD |
Purity |
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Endotoxin Note |
<1.0 EU per 1 μg of the protein by the LAL method. |
Applications/Dilutions
Dilutions |
|
Theoretical MW |
54 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. |
SDS-PAGE |
48 kDa, reducing conditions |
Packaging, Storage & Formulations
Storage |
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.- 6 months from date of receipt, -20 to -70 °C as supplied.
- 3 months, -20 to -70 °C under sterile conditions after opening.
|
Buffer |
Supplied as a 0.2 μm filtered solution in Tris, NaCl, TCEP and Glycerol. |
Purity |
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. |
Assay Procedure |
- Assay Buffer: 25 mM MES, 150 mM NaCl, 10 mM MgCl2, pH 7.0
- Recombinant Human 6-Phosphogluconate Dehydrogenase/PGD (rhPGD) (Catalog # 8964-DH)
- Donor: 6-phosphogluconic acid trisodium salt, 20 mM stock in 50 mM HCl
- Acceptor: beta -Nicotinamide adenine dinucleotide phosphate (NADP+), 50 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader
- Dilute rhPGD to 1 µg/mL in Assay Buffer.
- Prepare Reaction Mixture containing 1 mM NADP+ and 0.4 mM 6-phosphogluconic acid trisodium salt in Assay Buffer.
- Load 50 µL of 1 µg/mL rhPGD in a plate, and start the reaction by adding 50 µL of Reaction Mixture. Include a Control containing 50 µL of Assay Buffer and 50 µL of Reaction Mixture.
- Read at 340 nm (absorbance) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol |
ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) | *Adjusted for Control **Using the extinction coefficient 6220 M -1cm -1 ***Using the path correction 0.32 cm Note: the output of many spectrophotometers is in mOD Per Well:
- rhPGD: 0.05 µg
- NADP+: 0.5 mM
- 6-phosphogluconic acid trisodium salt: 0.2 mM
|
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human 6-Phosphogluconate Dehydrogenase/PGD, CF
Background
6-phosphogluconate dehydrogenase (PGD) is the second dehydrogenase in the pentose phosphate pathway (1). It catalyzes the oxidative decarboxylation of
6-phosphogluconate to ribulose 5-phosphate and CO
2, with concomitant reduction of NADP to NADPH (2). Together with glucose-6-phosphate dehydrogenase (G6PD), these enzymes ensure the reducing environment of the cell cytoplasm (3). However, the activities of both enzymes are strongly inhibited by NADPH. It is not quite clear how the enzymes are regulated to maintain the level of reducing power inside cells (4). Deficiency of PGD is generally asymptomatic, and the inheritance of this disorder is autosomal dominant. Hemolysis results from combined deficiency of PGD and 6-phosphogluconolactonase suggest a synergism of the two enzymopathies (5).
-
Barcia-Vieitez, R. and Ramos-Martinez, J.I. (2114) IUBMB Life 66:775.
- Wamelink, M.M. et al. (2008) J. Inherit. Metab. Dis. 31:703.
- Tsui, S.K.W. et al. (1996) Biochem. Genet. 34:367.
- Zimmer, H.G. (1996) Mol. Cell Biochem. 160-161:101.
- Caprari, P. et al. (2001) Ann. Hematol. 80:41.
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