Identification of zeta-crystallin/NADPH:quinone reductase as a renal glutaminase mRNA pH response element-binding protein

J Biol Chem. 2001 Jun 15;276(24):21375-80. doi: 10.1074/jbc.M101941200. Epub 2001 Apr 9.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Crystallins / isolation & purification
  • Crystallins / metabolism*
  • Cytosol / metabolism
  • Glutaminase / genetics*
  • Hydrogen-Ion Concentration
  • Kidney / enzymology*
  • Male
  • Molecular Sequence Data
  • NAD(P)H Dehydrogenase (Quinone) / isolation & purification
  • NAD(P)H Dehydrogenase (Quinone) / metabolism*
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / isolation & purification
  • RNA-Binding Proteins / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Transcription, Genetic
  • zeta-Crystallins

Substances

  • Crystallins
  • RNA, Messenger
  • RNA-Binding Proteins
  • zeta-Crystallins
  • NAD(P)H Dehydrogenase (Quinone)
  • Glutaminase