Abstract
Previously we have shown that the COOH-terminal 100 residues (A4CT) of the amyloid protein precursor (APP), which carry the sequence of the amyloid beta A4 protein of Alzheimer's disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte lysate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Müller-Hill, B., Masters, C. L., and Beyreuther, K. (1988) EMBO J. 7, 949-957). Here we report that aggregation of this COOH-terminal APP fragment A4CT and also of beta A4 itself depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT and beta A4 in the wheat germ expression system resulted in only monomeric forms. We have identified the factors which are capable of transforming both soluble A4CT and beta A4 into insoluble and aggregating molecules. Monomeric A4CT or beta A4 expressed in the wheat germ lysate could be transformed into aggregating molecules by the addition of metal-catalyzed oxidation systems. The addition of radical scavengers such as ascorbic acid, trolox, and amino acids prevented the aggregation process induced by the radical initiators. Thus, the aggregation of amyloidogenic APP fragments if analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis requires amino acid oxidation and protein cross-linking induced by radical generation systems.
Highlights
From the $Center for Molecular Biology, University ofHeidelberg, Im NeuenheimerFeld 282, 0-6900 Heidelberg, Germany, and the VDepartment of Pathology, University of Melbourne, Parkuille, Victoria 3052, Australia
In contrast to the reticulocyte expression system, expression of A4CTand BA4 in the wheat germ expression system resulted in only monomericforms.We have identified the factors which are capable of transforming both soluble A4CT and BA4 into insoluble and aggregating molecules
amyloid protein precursor (APP) Fragments and Amyloidogenicity lysate (Fig. 1, lanes 1 and 7) was alsostrongly reduced by In Vitro Expression ofA4CT"Previously, we reported the expression in the rabbrietticulocyte lysateof a protein termed A4CT, which comprises the COOH-terminal 100 residues of APP (Dyrks et al, 1988).This protein has the methionine residuepreceding the amyloid PA4 sequence atthe NH2 terminus andincludes the complete PA4 sequence, the transmembrane domain, and thecytoplasmic domain of the APP
Summary
In Vitro Expression ofA4CT"Previously, we reported the expression in the rabbrietticulocyte lysateof a protein termed A4CT, which comprises the COOH-terminal 100 residues of APP (Dyrks et al, 1988).This protein has the methionine residuepreceding the amyloid PA4 sequence atthe NH2 terminus andincludes the complete PA4 sequence, the transmembrane domain, and thecytoplasmic domain of the APP. This is in agreement with our previous findings that in thissystem A4CT has a high tendency incubation with RNase A for 30 min at 37 "C (Fig. 1,lane 8). Incubation with Triton X-100results in a change of the RRL contains hemoglobin, hemin, and iron, which have the aggregation pattern (Fig. 1,lanes and 3).In thepresence thepotentialtofunction invivo and in uitro as radical of detergent, the complex band pattern is reduced into one generation systems (Halliwell and Gutteridge, 1984,1988).To with two prominent bandsat 17 and 26 kDa and a weak band study whether the different oxidation potential in the two at 40 kDa. In addition to the strong bandhsi,gher aggregates expression systems was responsible for the different properare present as a smear at the top of the separating gel.
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