detector but not by the mass spectrometer. Those compounds account for the four unidentified peaks appearing between 15 and 30 min in the absorbance chromatogram (Figure 3).The utility of reconstructed mass chromatograms is evident in Figure 4. BHT and Irganox 1076 are easily detected in the reconstructed mass chromatogram, but in the total ion current chromatogram, Irganox 1076 is obscured by the peak at scan 557.

A major pre-P-amyloid proteinsps (APP,,,) processing activity from Alzheimer's disease brain extracts was identified and found to be indistinguishable from the activity of cathepsin D.APP,, processing activity cleaved APP,,, into a series of fragments that reacted on immunoblots to a monoclonal antibody (C286.8a) against p-amyloid-( 1 -7)-peptide and cleaved N-dansyl-APP-(591-601)-amide at the Glu-Val and Met-Asp bonds. Fragments of 5.5 kDa and 10-12 kDa were formed from the cleavage of APP,,, by cathepsin D at the Glu593-Val594 bond, and had the same Nterminus as a minor form of P-amyloid released by cells. Abbreviations. APP,,,, pre-P-amyloid protein,,, isoform; [N5", L5'6]APP,,,, APP,,, with lysine and methionine at positions 595 and 596 replaced by asparagine and leucine, respectively; dansyl, 5-(dimethylamino)-napthalene-1-sulfonyl; N-dansyl-APP-(591-601)-amide, N-dansyl-ISEVKMDAEFR-amide; C286.8a, IgG,, mAb recognizing P-amyloid residues at positions 1-7; PhMeSO,F, phenylmethylsulfonyl fluoride; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; P-2, post-I5000 g pellet.Enzymes. Cathepsin D (EC 3.4.23.5); type I1 site-specific deoxyribonucleases (EC 3.1.21.4).-~ processing enzyme, the activity of which is influenced by a mutation associated with the Swedish pedigree of familial Alzheimer's disease. MATERIALS AND METHODSHuman cathepsin D from Calbiochem was pure by SDS/ PAGE, 93% accurate by amino acid analysis, and contained only cathepsin D derived N-termini, the majority of which were from equimolar amounts of the mature light (GPI-PEVLKNY-), and heavy (GGVKVERQVF-) chains. Pepstatin A and heparin agarose were from Sigma. Mono-Q columns were from Pharmacia. Rabbit antiserum to human cathepsin D was from Dako Corp. IgG was purified from rabbit sera using Avid AL [23] and coupled to CnBr-activated Sepharose 4B [24]. The methods and instrumentation for peptide synthesis [25], amino acid analysis [26] and mass spectrocopy [26] were as cited previously. The protein content of crude extracts was determined by the Bradford assay [27].

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Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.