Expression of functional human glutaminase in baculovirus system: affinity purification, kinetic and molecular characterization.
Article Details
- CitationCopy to clipboard
Campos-Sandoval JA, Lopez de la Oliva AR, Lobo C, Segura JA, Mates JM, Alonso FJ, Marquez J
Expression of functional human glutaminase in baculovirus system: affinity purification, kinetic and molecular characterization.
Int J Biochem Cell Biol. 2007;39(4):765-73. Epub 2006 Dec 21.
- PubMed ID
- 17267261 [ View in PubMed]
- Abstract
Glutaminase catalyzes the hydrolysis of glutamine yielding stoichiometric amounts of glutamate plus ammonium ions. In mammals, there are two different genes encoding for glutaminase, known as liver (L) and kidney (K) types. The human L-type isoform expressed in baculovirus yielded functional recombinant enzyme in Sf9 insect cells. A novel affinity chromatography method, based on its specific interaction with a PDZ protein, was developed for purification. Kinetic constants were determined for the purified human isozyme, which showed an allosteric behaviour for glutamine, with a Hill index of 2.7 and S(0.5) values of 32 and 64 mM for high and low P(i) concentrations, respectively. Whereas the protein showed a low P(i) dependence typical for L-type glutaminases, the enzyme was unexpectedly inhibited by glutamate, a kinetic characteristic exclusive of K-type isozymes, and was slightly activated by ammonia, unlike the classical liver enzymes which show an absolute dependence on ammonia. Subcellular fractionation demonstrates that recombinant human glutaminase was targeted to both mitochondria and nucleus, and in both locations the protein was catalytically active. This is the first report of the expression of a functional L-type mammalian glutaminase enzyme. The study also provides a simple and efficient method for affinity purification of the recombinant enzyme. Moreover, the data imply that this human enzyme may represent a new isoform different from classical kidney and liver isozymes.
DrugBank Data that Cites this Article
- Drug Enzymes
Drug Enzyme Kind Organism Pharmacological Action Actions L-Glutamine Glutaminase liver isoform, mitochondrial Protein Humans UnknownSubstrateDetails