Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis.
We found a novel protein in the postmitochondria supernatant fraction of rat liver, which is soluble in 5% perchloric acid and strongly inhibits protein synthesis in a rabbit reticulocyte lysate system. The protein extracted from the supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The protein was shown to consist of two identical subunits with a molecular mass of 14 kDa. By immunoscreening with the rabbit antisera against the protein, a cDNA encoding the protein was cloned and sequenced. The cDNA contained an open reading frame of 411 base pairs encoding a 136-amino acid protein with a molecular mass of 14,149 Da. The deduced amino acid sequence was completely identical with that constructed from all of the above peptides. Interestingly, the perchloric acid-soluble protein inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner from RNase A. The protein is likely to inhibit an initiation stage of cell-free protein synthesis. Among the rat tissues tested, the protein was located only in liver and kidney. These findings are the first report on a new inhibitor that may be involved in the regulation of protein synthesis in those tissues.
Department of Nutrition, School of Medicine, University of Tokushima, Japan.
T
T
Oka
H
H
Tsuji
C
C
Noda
K
K
Sakai
Y M
YM
Hong
I
I
Suzuki
S
S
Muñoz
Y
Y
Natori
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003600:Cytosol / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010472:Perchlorates; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D011817:Rabbits / Q000276:immunology; D051381:Rats; D017208:Rats, Wistar; D012156:Reticulocytes / Q000378:metabolism; D012260:Ribonucleases; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D010472:Perchlorates; D011500:Protein Synthesis Inhibitors; D011506:Proteins; C097211:Rida protein, rat; D012260:Ribonucleases