Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

DNA polymerases a, 8, and e have been purified and characterized from the same HeLa cell extract in order to determine their relationship by comparing them from the same cell type. The catalytic properties and the primary structures of the large subunits of the DNA polymerases as compared by partial peptide mapping with N-chlorosuccinimide are different. Likewise, the small subunit of DNA polymerase e appears to be distinct from the large subunit of the same polymerase and from the smaller subunits of DNA polymerase a. HeLa DNA polymerase 8 is processive only when HeLa proliferating cell nuclear antigen is present, whereas DNA polymerase e is quite processive in its absence. Inhibitor and activator spectra of DNA polymerases a, 8, and E also distinguish the three enzymes. These results and immunologic comparisons published elsewhere support the premise that HeLa DNA polymerases a, 8, and e are distinct enzymes that have common properties with yeast DNA polymerases I, III, and II, respectively.

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␥-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's ␤-amyloid precursor protein (APP), resulting in the release of amyloid ␤-peptide (A␤). The molecular identity of ␥-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that ␥-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of A␤. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the A␤ 42 /A␤ 40 ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of ␥-secretase cleavage. Biogenesis of both A␤ 40 and A␤ 42 was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for ␥-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as ␥-secretase. The results suggest that presenilins may acquire the characteristics of ␥-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.Amyloid ␤-peptide (A␤) 1 is the major molecular component of the cerebral amyloid plaques associated with Alzheimer's disease. The cellular pathways involved in the biogenesis of A␤ have been the subject of intense investigation since the discovery that A␤ originates from intracellular endoproteolytic processing of a type I membrane-spanning glycoprotein termed amyloid precursor protein (APP) (1-4). Extensive studies have established that APP can be processed via two alternative routes, one of which yields the 4-kDa A␤, whereas the other yields a truncated non-amyloidogenic peptide (p3) (5, 6). In most cells the non-amyloidogenic pathway predominates. The first step involves the cleavage of APP within the A␤ domain by a protease termed ␣-secretase (7-9). After release of the Nterminal exodomain, the residual 83-amino acid membranespanning C-terminal fragment is further processed by another protease termed ␥-secretase to remove the cytoplasmic tail and generate p3 (6, 10). Because the latter cleavage occurs within the predicted membrane spanning region of APP (11-13), ␥-secretase is generally thought to be an intramembrane protease. In the alternative amyloidogenic pathway, APP is initially cleaved proximal to the A␤ sequence by ␤-secretase, leaving a 99-amino acid C-terminal fragment (C99) that contains the intact A␤ sequence and the cytoplasmic tail (1,14,15). Thus, when ␥-secretase cuts the latter substrate, A␤ is rel...

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