Rat hepatic glutaminase: identification of the full coding sequence and characterization of a functional promoter

Abstract

Glutamine catabolism in mammalian liver is catalysed by a unique isoenzyme of phosphate-activated glutaminase. The full coding and 5′ untranslated sequence for rat hepatic glutaminase was isolated by screening λ ZAP cDNA libraries and a Charon 4a rat genomic library. The sequence produces a mRNA 2225 nt in length, encoding a polypeptide of 535 amino acid residues with a calculated molecular mass of 59.2 kDa. The deduced amino acid sequence of rat liver glutaminase shows 86% similarity to that of rat kidney glutaminase and 65% similarity to a putative glutaminase from Caenorhabditis elegans. A genomic clone to rat liver glutaminase was isolated that contains 3.5 kb of the gene and 7.5 kb of the 5′ flanking region. The 1 kb immediately upstream of the hepatic glutaminase gene (from -1022 to +48) showed functional promoter activity in HepG2 hepatoma cells. This promoter region did not respond to treatment with cAMP, but was highly responsive (10-fold stimulation) to the synthetic glucocorticoid dexamethasone. Subsequent 5′ deletion analysis indicated that the promoter region between -103 and +48 was sufficient for basal promoter activity. This region does not contain an identifiable TATA element, indicating that transcription of the glutaminase gene is driven by a TATA-less promoter. The region responsive to glucocorticoids was mapped to -252 to -103 relative to the transcription start site.