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A neuropathological hallmark of Alzheimer disease (AD) is a widespread amyloid deposition. We analyzed the entire amino acid sequences in an amyloid preparation and found, in addition to the major I3/A4-protein (AP) fragment, two unknown peptides. We raised antibodies against synthetic peptides using subsequences ofthese peptides. These antibodies immunostained amyloid in neuritic and diffuse plaques as well as vascular amyloid. Electron microscopic analysis demonstrated that the immunostaining was localized on amyloid fibrils. We have isolated an apparently full-length cDNA encoding a 140-amino-acid protein within which two previously unreported amyloid sequences are encoded in tandem in the most hydrophobic domain. We tentatively named this 35-amino acid peptide NAC (non-A(8 component of AD amyloid) and its precursor NACP. NAC is the second component, after A.8, identified chemically in the purified AD amyloid preparation. Secondary structure predictions indicate that the NAC peptide sequence has a strong tendency to form (structures consistent with its association with amyloid. NACP is detected as a M, 19,000 protein in the cytosolic fraction of brain homogenates and comigrates on immunoblots with NACP synthesized in Escherichia coli from NACP cDNA. NACP mRNA is expressed principally in brain but is also expressed in low concentrations in all tissues examined except in liver, suggesting its ubiquitous and brain-specific functions. The availability of the cDNA encoding full-length NACP should help to elucidate the mechanisms of amyloidosis in AD.Amyloid deposition in the neuritic plaque and blood vessels is the most consistent neuropathology in Alzheimer disease (AD) (1, 2). The major constituent of amyloid has been found to be a 39-to 43-amino acid amyloid 13/A4-protein (AP3) (3,4) derived from its precursor, APP (5-8). The isolation of APP cDNA prompted a burst-of research in AD, culminating in the identification of APP mutations in several familial types of AD (9-12). Thus, APP and AP have been proposed to play a key role in the pathogenesis of this disease (13, 14). Additionally, heparan sulfate proteoglycan, ferritin, immunoglobulins, and many acute-phase proteins, such as a1-antichymotrypsin (ACT), apolipoprotein E, complements, serum amyloid P, and trace peptides were also reported to be associated with plaque core amyloid (15-29), although supportive biochemical data demonstrating their presence in amyloid preparations are not yet available, raising the possibility that those might not be the intrinsic components of amyloid. We have further pursued the biochemical examination of the intrinsic constituents of AD amyloid by purification in SDS and sequencing, and we detected a previouslyThe 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.unrecognized component that we tentatively call NAC (non-AP component of AD amyloid) in this communication...

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Paired helical filaments (PHF), which constitute a distinct type of pathological neuronal fiber, are the principal constituent of neurofibrillary tangles that occur in the brain of patients with Alzheimer's disease. Their insolubility in sodium dodecyl sulfate and urea has prevented the analysis of their subunit composition by gel electrophoresis. A monoclonal antibody (DF2) was isolated that specifically labeled PHF at both the light and electron microscopic levels. It labeled a small polypeptide (5 kilodaltons) that was shown to be ubiquitin in immunoblots of the soluble fraction of brain homogenates. To obtain direct evidence that ubiquitin is a component of PHF, PHF were treated with concentrated formic acid and digested with lysylendopeptidase; ubiquitin-derived peptides were then identified by reversed-phase high-performance liquid chromatography. Two fragments in the PHF digest were identified as derived from ubiquitin by protein sequencing. This procedure should make possible definitive identification of other PHF components.

Hepatocyte growth factor (HGF) activator is a serine protease that is produced and secreted by the liver and circulates in the blood as an inactive zymogen. In response to tissue injury, the HGF activator zymogen is converted to the active form by limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF activator from the conditioned medium of a human MKN45 stomach carcinoma cell line and molecularly cloned its cDNA. The sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, suggesting that the inhibitor is a member of the Kunitz family of serine protease inhibitors. The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence at the COOH-terminal region. Inhibitory activity toward HGF activator was detected in the membrane fraction as well as in the conditioned medium of MKN45 cells. These results suggest that the inhibitor may be produced as a membrane-associated form and secreted by the producing cells as a proteolytically truncated form.