Expression of functional human glutaminase in baculovirus system: affinity purification, kinetic and molecular characterization.
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.
Campos-Sandoval
José A
JA
Departamento de Biología Molecular y Bioquímica, Laboratorio de Química de Proteínas, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain.
López de la Oliva
Amada R
AR
Lobo
Carolina
C
Segura
Juan A
JA
Matés
José M
JM
Alonso
Francisco J
FJ
Márquez
Javier
J
D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002384:Catalysis / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000494:pharmacology; D005972:Glutaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D000641:Ammonia; D005972:Glutaminase