1998 Fiscal Year Final Research Report Summary
Structural studies of perchloric acid soluble protein (PSP) from rat liver.
| Project/Area Number |
09680594
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Kagoshima University (1998)
The University of Tokushima (1997) |
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Principal Investigator |
OKA Tatsuzo Kagoshima University Department of Veterinay Physiology Professor, 農学部, 教授 (50116795)
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| Co-Investigator(Kenkyū-buntansha) |
NATORI Yasuo University of Tokushima Department of Nutrition Professor, 医学部, 教授 (30035381)
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| Project Period (FY) |
1997 – 1998
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| Keywords | Rat / Perchloric acid-soluble protein / Escherichia coli / Recombinant / Crystallization / Higher structure |
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| Research Abstract |
We have reported isolation and characterization of a novel perchloric acid-soluble protein (PSP) in the cytosolic fraction of rat liver. The PSP is a homodimer, each subunit consisting of 136 amino acid residues with a molecular mass of 14,149 Da. It inhibits cell-free protein synthesis in the lysate of rabbit reticulocyte in a different manner than RNase A.
Recently, a 14-kDa protein which inhibit translation was characterized from human monocyte and mouse liver. The 14-kDa proteins and their mRNAs were reported to be preferentially expressed in the liver and kidney as in the case of PSP.Furthermore, the sequences of complementary DNA (cDNA) encoding the 14-kDa protein and PSP are highly similar to those of cDNA encoding a new hypothetical family (YER057c/YJGF) of small proteins with presently unknown function. Thus the sequences of PSP-like proteins are highly conserved in prokaryotic, cyanobacteria, fungi and eukaryotes. The high degree of evolutionary conservation of these proteins
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suggests that these proteins play an important role in cellular regulation.
Despite the growing body of information gor PSPs, no three-dimensional structure has benn reported. In the following contribution was present the crystallization and preliminary crystallographic characterization of PSP.
At first, an efficient E.coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. cDNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E.coli was expressed by IPTG, the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver.
PSP from rat liver has been crystallized in a form suitable for high resolution X-ray diffraction studies. Octahedral crystals reaching 0.5 mm in size were produced by hangin drop method using polyethylene glycol (Mr=8000 Da) as precipitant. These crystals diffract to 2.44 "A" on an in-house X-ray source and to l.8"A" using a bending, agnet beamline at ESRE Grenoble. The crystals belong to the cubic space group P213 with unit cell parameters a=b=c=89.90"A" and two molecules per asymmetric unit, as indicated from VM value of 2.12"A"3/Da and self-rotation function computation. Screening for heavy-atom derivatives identified a platinum compound that binds to a single site on the protein. Furhter screening for heavy-atom derivatives indeicated a beta sheet flanled by two alpha helice. The structural similarity gave the best hit ftsz (signal recognition particle receptor), chorismate mutase and heat-shock cognate protein 70 kDa. Less
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