Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase.
Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of lambdagt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE') technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.
Gómez-Fabre
P M
PM
Departamento de Biología Molecular y Bioquímica, Laboratorio de Química de Proteínas, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, 29071 Málaga, Spain.
Aledo
J C
JC
Del Castillo-Olivares
A
A
Alonso
F J
FJ
Núñez De Castro
I
I
Campos
J A
JA
Márquez
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D005972:Glutaminase / Q000096:biosynthesis / Q000235:genetics*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D005972:Glutaminase