In vitro gamma-secretase cleavage of the Alzheimer’s
amyloid precursor protein correlates to a subset
of presenilin complexes and is inhibited by zinc
David E. Hoke, Jiang-Li Tan, Nancy T. Ilaya, Janetta G. Culvenor, Stephanie J. Smith,
Anthony R. White, Colin L. Masters and Genevie
`
ve M. Evin
Department of Pathology, The University of Melbourne and the Mental Health Research Institute, Parkville, Victoria, Australia
Gamma-secretase is an aspartyl protease that cleaves
type I integral membrane proteins intramembranously.
The amyloid precursor protein (APP) undergoes
sequential cleavages by beta-site APP cleaving enzyme
and c-secretase to form amyloid-b (Ab). The beta-site
APP cleaving enzyme cleavage releases an APP ecto-
domain leaving a 99-amino acid membrane spanning
C-terminal fragment (CTF), C99. C99 then undergoes
intramembranous cleavage to form Ab peptides of
different lengths. c-secretase also releases an APP
intracellular domain (AICD or e-CTF) by cleaving 9–7
amino acids from c 40 and 42 sites at the e site [1–3].
Several lines of evidence support the pathogenic role
of c-cleavage of APP in Alzheimer’s disease (AD). The
genes encoding presenilin 1 and 2 (PS) are essential for
c-secretase activity and 150 mutations in the PS genes
have been found associated with autosomal dominant
early onset familial AD [4]. Although only 16 muta-
tions have been found in the APP gene, the majority
linked to early onset familial AD occur in the
Keywords
Alzheimer’s disease; amyloid precursor
protein; gamma-secretase; amyloid beta

fractionated cellular and tissue-derived c-secretase activity is dependent
upon detergent concentration and that activity correlates to a subset of
high molecular mass presenilin complexes. We also show that by changing
the solvent environment with dimethyl sulfoxide, detection of e-C-terminal
fragments can be elevated. Lastly, we show that zinc causes an increase in
the apparent molecular mass of an amyloid precursor protein c-secretase
substrate and inhibits its cleavage. These studies further refine our know-
ledge of the complexes and biochemical factors needed for c-secretase
activity and suggest a mechanism by which zinc dysregulation may contrib-
ute to Alzheimer’s disease pathogenesis.
Abbreviations
AD, Alzheimer’s disease; APP, amyloid precursor protein; CTF, C-terminal fragment; NTF, N-terminal fragment; PS, presenilin.
5544 FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS
transmembrane region near c and e cleavage sites (as
reviewed in [5]). Gamma-secretase activity is attributed
to an integral membrane complex of the four trans-
membrane proteins: PS, nicastrin (Nct), anterior pha-
rynx-defective, and presenilin enhancer 2 (as reviewed
in [6]).
In vitro c-secretase assays have been essential in elu-
cidating the mechanism of inhibitors [7–9], the struc-
ture of active c-secretase complexes [10,11], and have
aided in the finding of activity-modulating factors
[12,13]. These assays have shown that peripheral mem-
brane proteins are not necessary for activity as car-
bonate washing retains activity [14]. Additionally,
detergent solubilization has allowed solution-based
biochemical manipulation to show that all four of the
genetically determined c-secretase components interact
to form high molecular mass, enzymatically active

standard) and 49 (epsilon-3FLAG standard) were also
made to aid in the identification of 3FLAG-tagged
CTFs (Fig. 1A).
Ab-like and e-CTF-like products are present in
extracts from E. coli expressing MC99-3FLAG
Upon expression of MC99-3FLAG in E. coli,we
observed three predominant anti-FLAG immunoreact-
ive peptides. The major product migrated at  18 kDa
and was also detected by anti-Ab antibody, WO2.
From its apparent molecular mass and its immunore-
activity, it can be concluded that it corresponds to
MC99-3FLAG (Fig. 1B and C). There were also sev-
eral higher molecular mass and degraded species iden-
tified by both antibodies. The higher molecular mass
forms may correspond to aggregated MC99-3FLAG.
One anti-FLAG immunoreactive peptide migrated
similarly to the gamma and epsilon standards at
9 kDa (Fig. 1B) and the corresponding N-terminal
fragment resembling Ab was identified by WO2 west-
ern blot analysis (Fig. 1C). Further identification of
the anti-FLAG-immunoreactive CTFs was made by
coelectrophoresis with gamma and epsilon standards.
Co-electrophoresis obviates subtle lane-to-lane varia-
tions that may occur for these low molecular mass
proteins. The  9-kDa peptide comigrated with the
e-3FLAG standard (Fig. 1D) but faster than the
c-3FLAG standard (Fig. 1E). No anti-FLAG immuno-
reactivity was detected in lysates of mock-transformed
cells (Fig. 1E). Collectively, these data indicate that
upon expression or during purification, a small frac-

confirm that the fragment detected was produced by
c-secretase activity. L-685,458 inhibited formation of
this e-CTF-3FLAG in a dose-dependent manner, with
an effect still observed at concentrations as low as
3.3 nm, consistent with previous reports [17]. The
preparation of MC99-3FLAG contained additional
anti-FLAG immunoreactive peptides but these did not
interfere with the assay (Fig. 2A, long exposure).
Assay sensitivity was tested by varying the enzyme
amount, enzyme dilution, and CHAPSO concentra-
tion. For this experiment, c-secretase activity was pre-
pared by extracting carbonate-washed guinea pig brain
membranes with 1% CHAPSO. A 2 mgÆmL
)1
extract
was diluted to obtain a final concentration of 0.5%
CHAPSO, and dilutions were made in 0.5% CHAPSO
to 20 lgÆmL
)1
. Incubation of these dilutions with sub-
strate showed c-secretase activity to be enzyme dose-
dependent, and that signal was detectable using the
40 lgÆmL
)1
dilution of extract. Therefore, as little as
1 lg of membrane extract was sufficient to obtain a
signal (Fig. 2B). Secondly, the 2 mgÆ mL
)1
extract was
A

performed by spiking MC99-3FLAG-trans-
formed E. coli lysate with c-3FLAG stand-
ard. This standard migrated slower than the
E. coli product. Mock-transformed E. coli
had no background anti-FLAG immuno-
reactivity.
Characterization of in vitro c-secretase activity D. E. Hoke et al.
5546 FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS
diluted to 0.25% CHAPSO and subsequently diluted
in 0.25% CHAPSO to 40 lgÆmL
)1
. In contrast to the
0.5% CHAPSO dilution, 0.25% dilution did not show
a dose-dependent relation of enzyme amount to prod-
uct formed (Fig. 2C). Rather the highest concentration
and amount showed little activity while the least con-
centrated sample (1.2 lgof40lgÆmL
)1
) showed the
highest activity and greater than the corresponding
dilution in 0.5% CHAPSO. Perhaps this 0.25% con-
centration was not enough to keep high concentrations
of extract solubilized leading to an apparent loss of
activity. Collectively, these data show that two-step
purified MC99-3FLAG is an appropriate substrate to
study tissue-derived c-secretase activity that is inhibited
by a specific c-secretase inhibitor and is detergent con-
centration-sensitive.
Mammalian expression and proteolytic
processing of SPC99-3FLAG

calculated molecular mass of 11.1 kDa migrating
between a- and c-3FLAG standard proteins that may
correspond to a minor a-secretase cleavage product [1]
(Fig. 3C). Therefore, SPC99-3FLAG is expressed in
mammalian cells as a C101-3FLAG protein that
undergoes the expected processing by a- and c-cleav-
ages to produce a CTF corresponding to cleavage at
the e-site.
AB
C
Fig. 2. Detection of c-secretase activity in tissue extracts using exogenous MC99-3FLAG substrate. (A) Purified MC99-3FLAG substrate was
added to 0.5% CHAPSO soluble c-secretase from PS1 A246E transgenic mouse brain and incubated at 37 or 4 °C for 15 h with or without
L-685,458 inhibitor. These reactions were analysed by anti-FLAG western blot analysis. Gamma-secretase activity was defined as the gen-
eration of e-CTF-3FLAG (e) signal upon incubation at 37 °C over background 4 °C levels; this was inhibited by L685,458 in a dose-dependent
manner. Longer exposures showed a contaminating CTF (indicated by *) that migrated slightly faster than the e-CTF. (B) Sensitivity of exo-
genous substrate c-secretase assay in the presence of 0.5% CHAPSO. Guinea pig brain soluble c-secretase was diluted to 0.5% CHAPSO
and further dilutions in 0.5% CHAPSO were incubated with MC99-3FLAG at 37 or 4 °C for 15 h. The reactions were analysed by anti-FLAG
Western blot. Gamma-secretase activity was detected using as little as 1 lg of membrane extract. (C) A similar experiment to that in (B)
except that guinea pig brain soluble c-secretase was diluted to 0.25% CHAPSO with further dilutions in 0.25% CHAPSO incubated with
MC99-3FLAG. The most highly concentrated reaction shows little activity while the lowest concentration shows the greatest activity.
D. E. Hoke et al. Characterization of in vitro c-secretase activity
FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS 5547
In vitro c-secretase assay with COS7-SPC99-
3FLAG solubilized membranes
To complement our MC99-3FLAG based in vitro
c-secretase assays, an in vitro assay using COS7-
SPC99-3FLAG CHAPSO extracts was developed. The
substrate in this assay is synthesized, processed, and
trafficked in the cell and would theoretically be presen-
ted to the c-secretase complex in a more native state

need of overexpressing the c-secretase complex compo-
nents and are highly sensitive under diluting conditions,
we set out to determine the molecular mass of activity
by size exclusion chromatography. Unlike blue native
PAGE, this method allows the simultaneous determin-
ation of c-secretase complexes size and activity. A 1%
CHAPSO extract from COS-7-SPC99-3FLAG cells
A
DEF
BC
Fig. 3. Expression of SPC99-3FLAG in COS-7 cells and detection of c-secretase activity in whole-cell and cell-free assays. (A) Schematic of
the SPC99-3FLAG protein. (B) Anti-FLAG Western blot analysis of extracts from COS7-SPC99-3FLAG cells. Lane 1, spiked with e-3FLAG
standard; Lane 2, lysate sample alone. Note that the intensity of the e-CTF-3FLAG band was greater in lane 1 spiked with e-3FLAG standard.
(C) A similar experiment to that in (B) was performed except that lane 1 is a sample spiked with c-3FLAG standard indicated by the arrow
marked ‘c std’. Lane 2, lysate sample alone. Note that a separation between c-3FLAG standard and e-CTF-3FLAG was achieved in lane 1.
An uncharacterized anti-FLAG immunoreactive protein migrating between the a and c peptides was detected on long exposures (indicated
by the arrow on the right of panel C). (D) In vitro assay with CHAPSO-solubilized c-secretase from COS7-SPC99-3FLAG cells. 1% CHAPSO
extracts from COS7-SPC99-3FLAG cells were diluted to 0.5% and incubated at 37 or 4 °C for 16 h with dimethylsulfoxide or the c-secretase
inhibitor L-685,458 at the concentrations indicated. The reactions were analysed by anti-FLAG Western blot. Note that activity was abolished
in a dose-dependent manner upon addition of L-685,458. (E) Sensitivity of COS-7-SPC99-3FLAG soluble c-secretase assay in 0.5% CHAPSO:
4, 2, or 1 lg soluble c-secretase was diluted to 0.5% CHAPSO, incubated at 37 or 4 °C and analysed by anti-FLAG Western blot. Note that
e-CTF production was detected from 2 lg of membrane extract. (F) COS7-SPC99-3FLAG soluble c-secretase was diluted to 0.25% CHAPSO
and 2, 1, and 0.5 lg of extract tested for activity. Note that faint activity was seen with 1 lg extract.
Characterization of in vitro c-secretase activity D. E. Hoke et al.
5548 FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS
was diluted to 0.5% CHAPSO and chromatographed
on a Superose 6 column equilibrated with 0.5%
CHAPSO. This CHAPSO concentration was chosen as
it is compatible with c-secretase activity (as shown in
Fig. 3E) and it results in a lesser dilution of sample

chromatography. As we observed that the CHAPSO
concentration had an effect on c-secretase activity in
unseparated materials, we repeated the size exclusion
experiment in the presence of 0.25% CHAPSO. Immu-
noblots for PS1 NTF showed elution at the void vol-
ume (Fig. 4B), a result in contrast to the 669-kDa
peak obtained with chromatography in presence of
0.5% CHAPSO. Because most of C101-3FLAG
immunoreactivity was again found in fractions
between 440 and 25 kDa, separate from the fractions
containing PS1, c-secretase activity acting upon trans-
fected C101-3FLAG was not tested. Rather, fractions
were tested by adding exogenous MC99-3FLAG and
phospholipids (Fig. 4C). Under these conditions, the
fractions eluting at the void volume were able to pro-
duce a strong e-CTF signal upon incubation at
37 °C. It was noted that activity did not directly cor-
relate to the amount of PS1-NTF present in these
pooled fractions. These results indicate that endo-
genous c-secretase activity from COS-7 cells is associ-
ated with a CHAPSO concentration-sensitve complex
A
B
C
Fig. 4. Superose 6 size fractionation of COS7-SPC99-3FLAG soluble
c-secretase. (A) Sixty-seven micrograms of 1% CHAPSO cell mem-
brane extract was diluted to 0.5% CHAPSO and loaded onto a
Superose 6 column equilibrated in 0.5% CHAPSO. Arrows at the
top indicate elution of molecular mass standards. PS1 NTF fractio-
nates in 669-kDa fractions while C101-3FLAG fractionates between

CHAPSO extract was diluted to 500 lgÆmL
)1
in 0.25%
CHAPSO and 400 lL (200 lg) loaded onto the col-
umn. Gamma-secretase activity was detected in high
molecular mass fractions in duplicate column runs
(Fig. 5A). The c-secretase complex components Nct,
PS1, and PS2 were likewise found primarily in high
molecular mass fractions but not exactly overlapping
with c-secretase activity (Fig. 5B). Aph1a, Aph1b, and
Pen2 could not be detected in any fraction due to
sample dilution during chromatography. Similarly, a
2mgÆmL
)1
1% CHAPSO extract was diluted to
1mgÆmL
)1
in 0.5% CHAPSO and 400 lL (400 lg)
loaded onto a Superose 6 column. Gamma-secretase
activity was not detected in any fraction (Fig. 5C),
confirming that column chromatography in the pres-
ence of 0.5% CHAPSO resulted in a loss of c-secretase
activity. Interestingly, when Nct, PS1, and PS2 immu-
noreactivity was tested in these 0.5% CHAPSO frac-
tions it was found that a significant amount of these
proteins were present in high molecular mass fractions
A
BD
C
Fig. 5. Size fractionation of guinea pig brain soluble c-secretase by Superose 6 column chromatography. (A) Guinea pig brain soluble

5%, and decreased by 10% dimethylsulfoxide when
compared to non-dimethylsulfoxide control reactions
(Fig. 6). These data show that dimethylsulfoxide can
enhance or decrease detection of c-secretase activity
depending on the concentration used.
Zinc treatment of COS7-SPC99-3FLAG CHAPSO
extracts causes C101-3FLAG to elute at a high
molecular mass
Zinc binding to Ab has been shown to promote Ab
oligomerization [22–25]. Since a functioning zinc-bind-
ing domain may be present in the Ab sequence of C99,
we hypothesized that zinc may affect the oligomeriza-
tion state of C101-3FLAG. Therefore, the molecular
mass of C101-3FLAG before and after zinc treatment
was determined by size exclusion chromatography
(Fig. 7). Without the addition of zinc, C101-3FLAG
eluted as a peak in the 67–43-kDa molecular mass
range. After treatment with ZnCl
2
, C101-3FLAG eluted
as a high molecular mass peak corresponding to the
void volume of this column. These data show that zinc
can alter the apparent molecular mass of an APP-
derived c-secretase substrate.
Zinc inhibits c-secretase activity in COS7-SPC99-
3FLAG and MC99-3FLAG based assays
We hypothesized that zinc-induced substrate oligomeri-
zation may affect its ability to be cleaved. Therefore,
the effect of zinc on the two in vitro c-secretase assays
was determined. Firstly CHAPSO extracts from COS7-

buffer with or without 234 l
M Zn before loading onto a column
equilibrated in the same buffer with or without zinc. The fractions
were analysed by anti-FLAG western blot for C101-3FLAG immuno-
reactivity with the resulting C101-3FLAG signal quantified by image
densitometry. This data (y-axis) was plotted according to fraction
number (x-axis). The elution points for blue dextran (void), BSA
(67 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa) are
indicated by arrows.
Fig. 6. Effects of dimethylsulfoxide on the detection of in vitro
c-secretase cleavage of C101-3FLAG. CHAPSO-solubilized (0.5%)
c-secretase from COS7-SPC99-3FLAG cells was incubated in the
absence or presence of dimethylsulfoxide at the concentrations
indicated. Adding 2.5% dimethylsulfoxide significantly increased
e-CTF signal compared to 0% and 10% dimethylsulfoxide reactions.
D. E. Hoke et al. Characterization of in vitro c-secretase activity
FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS 5551
strategy for the prevention of AD. An initial step in
discovering c-secretase inhibitors is the development of
assays that monitor c -secretase activity. This paper
describes two novel in vitro c-secretase assays. During
the development of these assays we identified an
Ab-like NTF and e-like CTF from extracts of MC99-
3FLAG-transformed E. coli. Secondly, we show that
detergent concentration can affect the apparent size of
the c-secretase complex components and affect c-secre-
tase activity which correlates to a subset of PS com-
plexes. Thirdly, dimethylsulfoxide can modulate the
detection of in vitro c-secretase activity. Lastly we
show that zinc causes a change in the apparent

phospholipids. Calculations allowing for a 50% theor-
etical loss during chromatography, and the fact that
only an aliquot of each fraction was assayed still
placed the theoretical yield well within the detection
limits of our assay which showed that activity could be
detected with 2 lg of extract regardless of enzyme
source, dilution, or CHAPSO concentration. There-
fore, the reason for a lack of c-secretase activity can-
not be attributed to low assay sensitivity.
Size-separation of c-secretase using 0.25% CHAPSO
as the column buffer allowed detection of c-secretase
activity despite using less starting material than for
0.5% CHAPSO columns. Analysis of fractions from
COS7-SPC99-3FLAG separations showed a shift for
PS1 NTF to low molecular mass fractions after chroma-
tography in the presence of 0.5% CHAPSO as com-
pared to elution at the void volume of the column in the
presence of 0.25% CHAPSO. Fractionation of guinea
pig brain membrane extracts did not show as dramatic a
decrease in the c-secretase complex molecular mass
upon 0.5% CHAPSO chromatography as all of the
AB C
Fig. 8. Zinc inhibits in vitro c-secretase activity. (A) 0.5% CHAPSO-solubilized c-secretase from COS7-SPC99-3FLAG cells was incubated
with ZnCl
2
. This resulted in a dose-dependent inhibition of activity. (B, C) CHAPSO-solubilized (0.5%) c-secretase from guinea pig brain
acting upon the MC99-3FLAG substrate was incubated with ZnCl
2
(B), and ZnSO
4

activity was always present in the highest molecular
mass fractions before PS levels had peaked. These data
suggest that a subset of PS involved in the highest com-
plexed state yields significant activity as has been sugges-
ted by other methods previously [30] and by inhibitor
binding assays [31,32]. A restrospective analysis of the
work by Li et al. [21] also indicates an imperfect rela-
tionship between activity and PS NTF ⁄ CTF levels. The
successful recovery of native activity after size-exclusion
chromatography, described in this work, is an important
step in identifying the factors that enable c-secretase
cleavage in these highest molecular mass fractions.
Our data show that detection of e-CTFs from
in vitro c-secretase activity can be increased two- to
fivefold by the addition of 2.5% dimethylsulfoxide.
Three hypotheses for this effect can be made. Firstly,
dimethylsulfoxide can alter c-secretase enzyme kinet-
ics through its ability to interact with the phospho-
lipid bilayer [33–36]. Secondly, dimethylsulfoxide
could stabilize the c-secretase complex making it act
longer without altering the rate of proteolysis. As di-
methylsulfoxide affects the phase behaviour of bilay-
ers it probably affects the c-secretase complex which
is composed of at least 18 transmembrane domains
and its interaction with transmembrane substrates.
This is supported by our work and by other studies
showing its activity is highly sensitive to factors that
modulate membrane structure and stability, inclu-
ding detergent type [21], detergent concentration
[21,28,37], and phospholipid content [12,28]. How-

oligomerization to a noncleavable state. An alternate
explanation for the effect of zinc and copper inhibition
is an interaction between metals and phospholipid
bilayers. Zinc has been shown to be the most potent
metal in dehydrating lipid bilayers with copper being
the second most potent [39,40]. As water molecules are
necessary for most proteoytic processes, zinc and cop-
per modulation of the hydration state of lipid bilayers
may control c-secretase activity regardless of substrate.
Future experiments with c-secretase substrates that do
not bind metals will clarify the mechanism by which
zinc and copper inhibit in vitro c-secretase activity.
A universal characteristic of AD pathology is the
post-mortem detection of Ab plaques, thus confirming
the pathological relevance of c-secretase cleavage of
APP. Since only a small subset of AD cases are linked
to mutant PS or APP proteins, it has been hypothes-
ized that disease modifying genes and environmental
factors account for the common pathology of Ab pla-
que formation in sporadic cases. Here we have shown
that dimethylsulfoxide, and detergent concentrations
alter in vitro c-secretase activity. While these experi-
mental manipulations could not be compared to envir-
onmental factors they do show that agents known to
D. E. Hoke et al. Characterization of in vitro c-secretase activity
FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS 5553
modify phospholipid bilayers can modulate in vitro
c-secretase activity positively or negatively. Likewise,
high cholesterol levels have been shown to increase the
risk of AD [41] and some reports have suggested that

SPC99-3FLAG vector: forward, 5¢-GGGGGGCCAT
GGCGACAGTGATCGTC-3¢; reverse, 3FLAG HindIII
creating the plasmid c-3FLAG standard. Finally, the pri-
mer pairs forward, 5¢-GGGGGGCCATGGTGATGCTGA
AGAAGAACAG-3¢ and reverse 3FLAG HindIII were
used to generate the plasmid e-3FLAG standard.
Preparation of MC99-3FLAG
Escherichia coli was grown, induced, and harvested as in
[49]. Eshcherichia coli pellets were then sonicated in Hepes
buffer (50 mm Hepes, 5 mm MgCl
2
,5mm CaCl
2
, 0.15 m
KCl) +1% (w ⁄ v) protease inhibitor cocktail and centri-
fuged at 100 000 g to create a soluble and membrane frac-
tion. The 100 000 g pellet was homogenized in Hepes
buffer + 1% (v ⁄ v) CHAPSO by repeated passage through
a 25-G needle and incubated with end-over-end rocking for
1 h. This mixture was then centrifuged at 18 000 g and the
supernatant transferred to a separate tube. This supernatant
was brought up to 10% glycerol (v ⁄ v) and loaded onto a
Superdex-75 (Pharmacia, Fairfield, CT, USA) column
equilibrated with 0.5% (v ⁄ v) Triton X-100 in NaCl ⁄ P
i
.
Fractions were analysed by anti-FLAG western blot analy-
sis. Fractions rich in MC99-3FLAG but depleted in lower
molecular mass cleavage products were pooled. These
pooled fractions were then applied to an anti-FLAG, M2

3
pH 11.2) and centrifuged at 100 000 g for
1h 4°C. The final carbonate-washed pellet was washed
twice with Hepes buffer before resuspension in Hepes buf-
fer + 1% (v ⁄ v) CHAPSO and mixing end-over-end at 4 °C
for 1 h. The suspension was centrifuged at 18 000 g for
5 min at room temperature and the supernatant, named
‘soluble c-secretase’, was aliquotted and stored at )80 °C.
MC99-3FLAG c-secretase assay with soluble
c-secretase from mouse brain extracts, guinea
pig brain membrane extracts and column
fractions
MC99-3FLAG was added to soluble c-secretase or size-
fractionated soluble c-secretase at a 1 : 60 dilution. Experi-
ments in which the CHAPSO content was not indicated
Characterization of in vitro c-secretase activity D. E. Hoke et al.
5554 FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS
were performed in 0.5% CHAPSO. Dimethylsulfoxide or
L685,458 in dimethylsulfoxide, were added in equivalent
volumes to make control and inhibitor reactions. Metal
inhibition assays were performed by incubating soluble
c-secretase activity from guinea pig brain membrane
extracts with equal volumes of glycine buffer (0.1 m glycine
pH 7.0), or ZnCl
2
⁄ ZnSO
4
dissolved in glycine buffer to
make control and experimental reactions that were incuba-
ted at 37 °C for 9 h. 3-sn-Phosphatidylethanolamine from

proteins as 11.1 kDa.
In vitro c-secretase assays with COS7-SPC99-
3FLAG cells
Assays were performed by thawing soluble c-secretase, dilu-
ting to 0.5% or 0.25% (v ⁄ v) CHAPSO in Hepes buffer and
incubating at 4 °Cor37°C for 2–16 h. Experiments in
which the CHAPSO content was not indicated were per-
formed in 0.5% CHAPSO. Inhibitor assays were incubated
with equivalent volumes of dimethylsulfoxide or L-685,458
diluted in dimethylsulfoxide. Metal inhibition assays were
performed by incubation with equal volumes of Hepes
buffer or ZnCl
2
dissolved in Hepes buffer to make con-
trol ⁄ experimental reactions that were incubated at 37 °C
for 9 h. The assays were stopped by adding SDS sample
buffer and the reactions were separated on tricine gels [50].
Size exclusion chromatography
A1· 30 cm column was packed with Superose 6 resin and
calibrated with blue dextran (void volume), ferritin (880-
kDa dimer eluted at the void volume and 440-kDa mono-
mer), thyroglobulin (669 kDa), and chymotrypsinogen A
(25 kDa). A 1 · 30-cm column was packed with Superose
12 resin and calibrated with blue dextran (void volume),
BSA (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen
(25 kDa). All solutions were filtered through a 0.2-lm filter
prior to the addition of CHAPSO. CHAPSO solutions were
then filtered through Whatman paper. Glycerol was added
to soluble c-secretase (10% glycerol, v ⁄ v) plus Hepes buffer
making the final CHAPSO concentration 0.5% or 0.25%

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