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.
Department of Biochemistry, Colorado State University, Fort Collins 80523.
R A
RA
Shapiro
L
L
Farrell
M
M
Srinivasan
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NP
Curthoys
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