Abstract
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.
Highlights
High mobility group (HMG)1 proteins are a family of nonhistone components in chromatin [1]
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
We showed that the HMG proteins play an important role in nutritional modulations of chick liver RNA synthesis [3] and isolated the HMG 2a cDNA from a gt11 expression library of chick liver using polyclonal antibodies, which encodes a protein of 201 amino acids [4]
Summary
Extraction of PSP—Livers from male Wistar rats were immediately homogenized in two volumes of cold 0.25 M sucrose in buffer (50 mM Tris-HCl (pH 7.5), 25 mM KCl, and 10 mM MgCl2) with a PotterElvehjem type homogenizer. For the isolation of the N-terminal peptide, the peptides eluted from the above agarose column were further digested at 37 °C overnight in 0.1 M sodium phosphate buffer (pH 7.0) with aminopeptidase M at a 1:100 ratio of aminopeptidase M to the protein. The purified peptide was eluted at a retention time of 6.90 min, collected, and subjected to ESI mass spectrometry, amino acid analysis, and automated Edman degradation. After hydration and rinsing in PBS (10 mM phosphate buffer (pH 7.2) containing 0.85% NaCl), the sections were treated with a normal goat serum at 1:60 dilution for 20 min at room temperature to reduce nonspecific staining and incubated at 4 °C overnight with 3 g/ml of polyclonal antibodies against PSP in PBS containing 0.1% bovine serum albumin in a moist chamber. The alignment of homologous proteins was carried out by the software
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