Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase

Phosphate-activated glutaminase (GA) is overexpressed in certain types of t umour 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 breas t cancer ZR75 cells, by a combination of lambda gt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE') technique. The cDNA of h uman GA is 2408 nt with a 1806-base open reading frame encoding a 602-resid ue protein with a predicted molecular mass of 66309 Da. The deduced amino a cid sequence contains a putative mitochondrial import presequence of 14 res idues at the N-terminal end. Heterologous expression and purification in Es cherichia coli yielded a product of the expected molecular size that was re cognized by using antibodies against the recombinant human GA. Sequence ana lyses showed that human GA was highly similar to the rat liver enzyme. Nort hern gel analysis revealed that the gene is present in human liver, brain a nd pancreas, in which a major transcript of 2.4 kb was demonstrated, but no t in kidney, heart, skeletal muscle, lung or placenta. These results strong ly 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 isoe nzymes, in sharp contrast with the present view that considers the kidney t ype as the isoform expressed in all tissues with GA activity, with the exce ption of postnatal liver.