|Budget Amount *help
¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
In the previous study, we found that calcium binding proteins, including alpha2-HS glycoprotein, prothrombin and its degradation products, and osteopontin, were exclusively associated with calcium phosphate crystals (CAPs) precipitated from normal human urine. Applying this phenomenon, we have developed a method which can isolate calcium binding proteins efficiently from mixed protein solutions.
A minimum electrolyte solution of the following composition was developed ; 5mM CaCI_2, 30mM NaH_2PO_4, 4mM Na_2HPO_4, 2.7mM C_6H_5Na_3O_7 and pH of 6.0. CAPs were precipitated from this solution by alkalifying up to pH 7.4, after adding a mixed protein solution. The presence of citrate was indispensable to avoid nonspecific adsorption of proteins to the CAPs.
When human plasma or serum were applied to the system, an unidentified protein(M.W.more than lOOkD) was coprecipitated with CAPs in addition to alpha2-HS glycoprotein, prothrombin and its degradation products. When cow milk were applied to the system, a protein of 3lkD M.W.was exclusively obtained. This protein was homologous to bovine alpha-S1 casein precursor by amino acid analysis. But no exclusive protein was obtained when skim milk or human milk were applied to the system, owing to a large amount of nonspecific protein coprecipitation.
Addition of mixed protein solutions after precipitation of CAPs in the minimum electrolyte solution may also be effective to isolate calcium binding proteins.