title: Identification of zeta-crystallin/NADPH:quinone reductase as a renal glutaminase mRNA pH response element-binding protein.
abstract: Increased renal ammoniagenesis and bicarbonate synthesis from glutamine during chronic metabolic acidosis facilitate the excretion of acids and partially restore normal acid-base balance. This adaptation is sustained, in part, by a cell-specific stabilization of the glutaminase mRNA that leads to an increased synthesis of the mitochondrial glutaminase. A direct repeat of an 8-base AU sequence within the 3'-nontranslated region of the glutaminase mRNA binds a unique protein with high affinity and specificity. Expression of various chimeric mRNAs in LLC-PK(1)-FBPase(+) cells demonstrated that a single 8-base AU sequence is both necessary and sufficient to function as a pH response element (pH RE). A biotinylated oligoribonucleotide containing the direct repeat was used as an affinity ligand to purify the pH RE-binding protein from a cytosolic extract of rat renal cortex. The purified binding activity retained the same specific binding properties as observed with crude extracts and correlated with the elution of a 36-kDa protein. Microsequencing by mass spectroscopy and Western blot analysis were used to identify this protein as zeta-crystallin/NADPH:quinone reductase. The purified protein contained eight tryptic peptides that were identical to sequences found in mouse zeta-crystallin and three peptides that differed by only a single amino acid. The observed differences may represent substitutions found in the rat homolog. A second protein purified by this protocol was identified as T-cell-restricted intracellular antigen-related protein (TIAR). However, the purified TIAR neither bound nor affected the binding of zeta-crystallin/NADPH:quinone reductase to the pH RE. Furthermore, specific antibodies to zeta-crystallin, but not TIAR, blocked the formation of the complex between the pH RE and either the crude cytosolic extract or the purified protein. Thus, zeta-crystallin/NADPH:quinone reductase is a pH response element-binding protein.
authors: Department of Biochemistry, Colorado State University, Ft. Collins, Colorado 80523-1870, USA.
authors: A
authors: A
authors: Tang
authors: N P
authors: NP
authors: Curthoys
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005972:Glutaminase / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic; D038226:zeta-Crystallins
chemical_list: D003459:Crystallins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038226:zeta-Crystallins; D016660:NAD(P)H Dehydrogenase (Quinone); D005972:Glutaminase