The plaque and cerebrovascular amyloid of Alzheimer’s disease is composed of 42 or 43 amino acid residue protein designated A4 (Masters et al., 1985) or /?-protein (Glenner & Wong, 1984), which has been shown to be derived from a 695 amino acid residue precursor protein (Kang et al., 1987). More recently, alternative forms of the precursor containing an additional region, homologous to Kunitz protease inhibitors, have also been demonstrated to occur (Kitaguchi et al., 1988; h n t e et al., 1988; Tanzi et al., 1988). All of the precursors have amino acid sequence similarities to glycosylated cell-surface receptor proteins (Kang et al., 1987), but evidence for A4-related synthetic peptide binding sites in a number of tissues and structural parallels between the A4 precursor and epidermal growth factor precursor led Allsop et al. (1988) to put forward a ligand-releasing role for A4 precursor. However, the function or functions of the precursor molecules remain undefined at present, as do precise relationships between variant precursors and the A4 product. Here we have used a monoclonal antibody, 1G10/2/3, raised against a synthetic peptide representing residues 8-1 7 of the A4 sequence. A full description of the production and use of 1G10/2/3 which recognizes plaque and cerebrovascular amyloid in tissue sections has been given (Allsop et al., 1986). The monoclonal antibody, in ascites fluid, was purified by anion-exchange fast protein liquid chromatography (Pharmacia Ltd., Milton Keynes, U.K.) and a 5 mg amount was coupled to a HEMA-cart vinyl-sulphoneactivated affinity matrix cartridge (Anachem, Luton, U.K.) by following the protocol of the supplier. Experimental conditions for the binding and release of specific immunoaffinity-bound materials were assessed by using the same synthetic peptide conjugated to myoglobin (Allsop et al., 1986); it was found that the conjugate was retained by the column when dissolved in phosphatebuffered saline (0.01 M-sodium phosphate buffer, pH 7.5, in 0.15 M-NaCI) and was released in 1 M-NH, but not in 0.2 Mglycine (pH 2.2). In experiments aimed at isolating A4-related material released from plaques during the first part of the procedure (enriched neuronal fraction preparation) employed for the isolation of plaque cores (Allsop et al., 1983); we used that method to produce an initial supernatant obtained by centrifugation at 400 g for 5 min. The brain tissue source was cerebral cortex from a case of Alzheimer’s disease with abundant senile plaques and neurofibrillary tangles. Particulate material was removed from the initial supernatant by a second centrifugation at 200000 g for 2 h. This second supernatant was treated with an equal volume of saturated ammonium sulphate and the precipitated proteins were collected by centrifugation and redissolved in phosphatebuffered saline. Solutions containing the redissolved material were passed through the affinity cartridge at room temperature overnight by recycling. After this binding step the cartridge was sequentially washed with phosphate-buffered saline, 0.2 Mglycine (pH 2.2) and 1 M-NH,. The material released in 1 MNH, was dried in vacuo and analysed by SDS/polyacrylamide-gel electrophoresis and Western blotting procedures 350
Read full abstract
Sep 15, 2023
Sukjeong Jeon + 7
Open Access