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 lambda gt11 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 68- and 65-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 lambda gt10 library. This segment contains numerous AU-rich 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.
Shapiro
R A
RA
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Farrell
L
L
Srinivasan
M
M
Curthoys
N P
NP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005972:Glutaminase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015183:Restriction Mapping
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D005972:Glutaminase