Isolation, characterization, and in vitro expression of a cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase.

A cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase (pGA) was generated by recombination of two cDNAs that were isolated from a random-primed rat brain Xgtll library.pGA encodes 674 amino acids which includes an N-terminal sequence of 16 residues that should form an amphipathic helix, typical of a mitochondrial targeting sequence.Residues 73-90 correspond to the N-terminal sequence of the more abundant 65-kDa glutaminase peptide.In vitro transcription and translation of pGA yields a 72-kDa peptide that is immunoprecipitated with glutaminase-specific antibodies.Incubation of the glutaminase precursor with isolated mitochondria yields the 68and 66-kDa peptides that are characteristic of the mature glutaminase.Thus, the two mature glutaminase peptides are synthesized from a single precursor.The complete 3' nontranslated region of the GA mRNA was characterized by sequencing a GA cDNA (pGA12) that was isolated from an oligo(dT)-primed rat kidney XgtlO library.This segment contains numerous AUrich regions, four potential stem-loop structures, and a 48 base pair repeat of CA dinucleotides.Such domains may contribute to the increased stability of the GA mRNA that occurs in response to metabolic acidosis.' The abbreviations used are: GA, glutaminase; pBS-SK(-), pBluescriptII-SK(-); kb, kilohase(s); bp, base pair(s).