Purification by column chromatographies of beta-amyloid precursor proteins and their association with other 95 kDa protein in rat brain.
Beta-amyloid precursor proteins (APPs) in the subcellular fractions of the homogenate of rat brain were detected immunologically. They were found to be localized in both the cytosol and microsome fractions in generally equal amounts. APPs were purified from the cytosol fraction of rat brain by column chromatography in a DEAE-anion-exchanger, Blue-Sepharose, Ni-charged chelating Sepharose, and Sephacryl S-300 columns. They migrated at about 400 kDa or above in a final gel filtration column with trypsin inhibitor activity. They gave two broad protein bands of 80 and 100 kDa and several other protein bands in sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). The 80 and 100 kDa bands were highly concentrated during purification. They gave the same amino terminal sequence and were identified as rat APPs without an amino terminal signal sequence. These results suggest that rat brain APPs form a complex with themselves or with other proteins and contain APP isoforms including a serine protease inhibitor domain, APP770 or APP751, or both. An antibody produced by a rabbit immunized with the final preparation of APPs reacted with a 95 kDa protein band which migrated between the 80 and 100 kDa bands of APPs in SDS-PAGE, but it did not react with the bands of APPs. The 80 and 100 kDa APP bands were coprecipitated with a 95 kDa antigen protein band by reacting this antibody with the partially purified APPs. We conclude that APPs in the rat brain are associated directly or indirectly with another protein to yield the 95 kDa band demonstrated by SDS-PAGE.
Ohshita
T
T
School of Food and Nutrition, Shokei Junior College, Kumamoto, Japan.
Nakatani
A
A
Hiroi
Y
Y
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D000918:Antibody Specificity; D001921:Brain / Q000648:ultrastructure*; D002458:Cell Fractionation; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003600:Cytosol / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D008297:Male; D051379:Mice; D008861:Microsomes / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D051381:Rats
D016564:Amyloid beta-Protein Precursor; D009419:Nerve Tissue Proteins