| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1988: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
Human alpha_1-T glycoprotein (alpha_1-T) was a glycoprotein initially isolated from serum by Heide and Haupt in 1964 by rivanol-ammonium sulfate precipitation. The present author successfully purified this protein from serum with the use of modern chromatographic techniques and determined some physicochemical properties as well as partial amino acid sequence. Using established assays of single radial immunodiffusion and qnzyme immunoassay, clinical significance of this protein was investigated. The outline of the present study was summarized as follows.
I. Establishment of alpha_1-T Purification: Pooled normal sera were precipitated with 45% ammonium sulfate and its supernatent was further saturated to 75%, precipitate of which was used as a starting material. In an initial purification alpha_1-T was isolated by 5 steps of column chromatographies including ConA affinity, anti-albumin attached affinity, Blue Sepharose, gel-filtration on Sephadex G-200, and cation exchange on CM Sephadex
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C-50. After the preparation of mouse monoclonal antibody these 5 steps were reduced to 3 steps while keeping high yield and purity. Purified alpha_1-T was demonstrated to be a molecular weight of 77,500 daltons, PI 4.5, and tryptophan poor content in amino acid composition. A partial amino acid sequence was determined which showed no any homology exsisting proteins so far reported.
II. Clinical Application: The serum level in normal individuals aged from 10 to 60 years old was 71.8 12.3 mg/l without age and sex differences, while that in various diseases was low in cases with hepatic dysfunction and CRF. Of note worth is that the value was rather elevated in acute hepatitis with convalescent stage and drug-induced hepatitis with recovery. These results indicated that this protein is mainly produced in the liver and the measurement of this protein is useful for the evaluation of parenchymal liver cell damage and its repair. The study on low molecular proteins was also facilitated by the present project. Less
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