ABSTRACT125I-Ap812s (specific activity, 3-6 x 106 dpm/;.g) in a final volume of 20 pI of phosphate-buffered saline (PBS) at pH 7.4 at 37TC. Samples were analyzed by SDS/PAGE. A 12% Tris-glycine gel was used for analysis of complexes AP3 with CSF proteins and a 13% gel in Tricine buffer was used for analysis of A( aggregation. The gel was then dried and exposed to an x-ray X-Omat film from Kodak. The purification of Ad binding activity was monitored using 'mI-A1i_28 and electrophoresis in SDS/PAGE gel and included three steps.Step 1: 5 ml of CSF was subjected to ion-exchange chromatography on a 5-ml column with DEAE-Sepharose equilibrated with 50 mM Tris-HCl (pH 7.4). The peak of AP3 binding activity was eluted at 0.4 M NaCl.Step 2: the peak fractions were combined, diluted five times, and further passed through a heparin-Sepharose column in 50 mM NaCl/50 mM Tris HCl, pH 7.4. The AP binding activity appeared in unbound fractions.Step 3: the combined fractions containing A(3 binding activity were chromatographed on a FPLC-mono Q column with a gradient of0.1-0.3 M NaCl in 50 mM Tris HCl (pH 7.4 iTo whom reprint requests should be addressed. 8368The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Intraneuronal accumulation of ubiquitin conjugates in inclusion bodies and neurofibrillary tangles is a pathological feature of neurodegenerative disorders such as Alzheimer's disease and Down's syndrome and of normal aging of the brain. Amyloid beta-protein (A beta) and its precursor are found in neurofibrillary tangle-containing neurons. A beta is the major component of extracellular plaques. We showed that A beta acts as an inhibitor of the ubiquitin-dependent protein degradation in vitro. We examined the effect of A beta on the steps of this proteolytic pathway that contribute to the level of ubiquitin conjugates in the cell. Neither conjugate formation nor conjugate deubiquitination was affected by the presence of A beta. However, A beta significantly reduced the rate of conjugate degradation. Our results indicate that A beta interacts with the proteolytic step of the ubiquitin degradative pathway. Since this step is performed by the 26 S proteasome, the effect of A beta on the catalytic core of this proteolytic complex, the 20 S proteasome, was determined. We found that A beta selectively inhibits the chymotrypsin-like activity of the 20 S proteasome. Under pathological conditions in the affected neuron, A beta could interfere with ubiquitin-dependent degradation by inhibiting the 26 S proteasome activity. This finding may explain the origin of the accumulation of ubiquitin conjugates.