Effects of thymoquinone on isolated and cellular
proteasomes
Valentina Cecarini, Luana Quassinti, Alessia Di Blasio, Laura Bonfili, Massimo Bramucci,
Giulio Lupidi, Massimiliano Cuccioloni, Matteo Mozzicafreddo, Mauro Angeletti and
Anna Maria Eleuteri
School of Biosciences and Biotechnology, University of Camerino, Italy
Introduction
Black cumin seed (Nigella sativa) oil extracts have been
used for many centuries in the treatment of several
human diseases, and thymoquinone (TQ), its active
component, has recently been tested for its efficacy
against several diseases, including cancer [1–3].
In this regard, TQ was found to inhibit proliferation
in a concentration-dependent manner in numerous cell
lines [4,5]. It has shown significant antineoplastic activ-
ity against multidrug-resistant human pancreatic ade-
nocarcinoma, uterine sarcoma and leukemic cell lines,
with minimal toxicity for normal cells [6].
In a mouse model, the injection of the essential oil
into the tumor site significantly inhibited solid tumor
development as well as the incidence of liver metasta-
sis, thus improving mouse survival [5]. These results
indicate that the antitumor activity or cell growth inhi-
bition could in part be due to the effect of TQ on the
cell cycle [5]. Furthermore, it has been demonstrated
that the growth of prostate cancer cells is highly
sensitive to the inhibitory effect of TQ, and that this
inhibitory action is extremely selective, showing very
little effect on the growth of noncancerous prostate
epithelial cells in culture, and preventing the growth of
human prostate tumors in nude mice [7].

induces selective and time-dependent proteasome inhibition, both in isolated
enzymes and in glioblastoma cells, and suggest that this mechanism could
be implicated in the induction of apoptosis in cancer cells.
Abbreviations
AMC, 7-amino-4-methyl-coumarin; BrAAP, branched chain amino acid-preferring; ChT-L, chymotrypsin-like; ECL, enhanced
chemiluminescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;
pAB, 4-aminobenzoate; PGPH, peptidyl-glutamyl peptide-hydrolyzing; PVDF, poly(vinylidene difluoride);
Suc, succinyl; T-L, trypsin-like; TQ, thymoquinone; Ub, ubiquitin.
2128 FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS
completely clear. Recent findings suggest that TQ has
a strong chemopreventive potential for the inhibition
of carcinogenesis by modulating lipid peroxidation and
the cellular antioxidant milieu [8,9]. In fact, TQ is
reported to possess strong antioxidant properties,
inhibiting free radical generation [10]. Interestingly,
according to Gali-Muhtasib et al., TQ is able to trigger
apoptosis in several cell lines in a p53-independent or
a p53-dependent manner [11,12], and, as recently
shown, its proapoptotic effects are linked to its
pro-oxidant activity [13].
Among the different mechanisms involved in the
induction of apoptotic pathways, the tumor suppressor
protein p53 plays a pivotal role [14]. Under physiologi-
cal conditions, p53 is maintained at low steady-state
levels by the MDM2 protein, an E3 ubiquitin (Ub)
ligase, which ubiquitinates and targets p53 for protea-
some-mediated degradation [15]. Specific stress agents
make p53 and MDM2 undergo different post-transla-
tional modifications, including phosphorylation, thus
disrupting the interaction and leading to activation of

branched chain amino acid-preferring (BrAAP) activ-
ity, b2 is associated with the trypsin-like (T-L) activity,
and b5 is associated with the chymotrypsin-like (ChT-
L) activity. However, mutational analyses have shown
that b5 also has a tendency to cleave after small neu-
tral and branched side chains; therefore, two other
activities, BrAAP and small neutral amino acid-prefer-
ring (SNAAP), can be assigned to this subunit [25]. In
certain conditions, such as in the presence of c-inter-
feron, these three b subunits can be replaced by
homologous subunits, b1i, b2i, and b5i, resulting in a
de novo synthesized proteasomal form, the immuno-
proteasome, which produces mainly immunogenic pep-
tides in association with major histocompatibility
complex class I [19].
Malignant gliomas are the most common and lethal
tumors of the central nervous system [26]. Treatment
outcomes, even with an aggressive approach including
surgery, radiation therapy, and chemotherapy, are dis-
mal. The median survival of treated patients with glio-
blastoma multiforme is less than 1 year, with fewer
than 20% surviving for 2 years [27]. There is therefore
an urgent need to devise alternative therapeutic strate-
gies with which to fight gliomas.
In the present work, the effects of TQ on protea-
some functionality were investigated both in isolated
and in cellular complexes. For this purpose, constitu-
tive and immune-isolated proteasomes and two human
glioblastoma cell lines, U87 MG and T98G, differing
in their p53 gene status, were used. Specifically,

BrAAP activity was not significantly influenced by the
presence of TQ (data not shown).
V. Cecarini et al. Thymoquinone inhibits proteasome functionality
FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2129
Interestingly, the inhibition showed concentration-
dependent behavior only up to 20 lm, when the maxi-
mum detectable rates of inhibition were 30% and 40%
for the ChT-L and T-L components, respectively, of
the immunoproteasome. Thereafter, an increase in TQ
concentrations did not lead to enhanced inhibition.
Supported by the literature [30], we propose that this
U-shaped inhibition depends on the presence of an
additional binding site on the proteasomal complex to
which TQ binds with a lower affinity than it does to
the active site. Our model assumes that TQ preferen-
tially binds to the active site at low concentrations,
resulting in the observed inhibition, whereas at higher
concentrations the binding to the additional site
becomes significant, allosterically restoring the activity.
The fraction of TQ bound to the active site is now
released, allowing the substrate to enter it and be suc-
cessfully degraded, resulting in the activity recovery
observed at TQ concentrations higher than 20 lm.To
verify this hypothesis, we performed an experiment
using the peptide aldehyde Z-LLF-CHO, a selective
and reversible proteasome inhibitor, with the aim of
blocking part of the proteasome active sites [31]. After
1 h of incubation of the 20S immunoproteasome with
Z-LLF-CHO, TQ at different concentrations was
added and the T-L activity was measured (Fig. 3). In

and C
4
carbonyl were found to be nucleophilically attacked by the OH
group of Thr1.
Thymoquinone inhibits proteasome functionality V. Cecarini et al.
2130 FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS
carry, respectively, the wild-type and a mutant p53
gene. This mutation consists of a single G fi A transi-
tion in codon 237, resulting in a missense mutation of
methionine to isoleucine [32,33]. Interestingly, a study
conducted by Van Meir et al. on different glioblastoma
lines and their p53 status revealed that this mutation
in the T98G line results in a transcriptionally inactive
form of p53 [34].
A set of dose–response experiments was performed
to compare the effects of TQ on cell viability in
U87 MG and T98G cells. Cells were incubated in the
presence of TQ at concentrations ranging from 0.0 lm
to 200 lm. Analysis by light microscopy showed that
treatment of glioblastoma cells with increasing
amounts of TQ resulted in significant alterations in
cell morphology and impaired the ability of the cells
to become confluent (Fig. 5A). Data obtained with
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) assay indicated that cell viability
was significantly reduced in a dose-dependent and
exposure time-dependent manner in both cell lines
(Fig. 5B). In both cell lines, almost complete loss of
viability was seen after exposure to 200 lm TQ. At
lower concentrations, TQ exerted a stronger inhibitory

proteasome inhibition, with relevant differences also at
24 h, as evident for the T-L and BrAAP activities.
Generally, in this cell line, TQ induced a global and
stronger decrease in proteasome functionality than that
observed in T98G cells.
We also measured the ChT-L component of the 26S
proteasome, whose proteolytic activity is ATP-depen-
dent, and obtained, at 72 h, similar percentages of inhi-
bition in the two lines. However, at 48 h, a significant
Fig. 3. TQ binding to a secondary site of the proteasome complex.
After Z-LLF-CHO and 20S immunoproteasome preincubation, in
order to partially inhibit the enzyme, the effects of increasing con-
centrations of TQ on the T-L activity were tested. Data are reported
as percentages relative to proteasome activity in the presence of Z-
LLF-CHO (mean values ± standard deviations of five independent
determinations).
A
B
Fig. 4. Detection of quinone adducts. 20S isolated immunoprotea-
somes were treated with different concentrations of TQ and lacta-
cystin (see Experimental procedures), resolved by SDS ⁄ PAGE, and
electroblotted onto PVDF membranes. Adducts were visualized
after 45 min of incubation with Nitro Blue tetrazolium. Lane C rep-
resents 20S proteasome loaded without pretreatment with TQ and
lactacystin. (A) Densitometry related to three different experiments.
(B) A representative membrane after the Nitro Blue tetrazolium
staining.
V. Cecarini et al. Thymoquinone inhibits proteasome functionality
FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2131
difference after TQ exposure was evident for U87 MG

cifically, the former responded in a shorter time, with
significant increases at 48 h and 72 h (1.21-fold and
1.42-fold, respectively, that seen in controls), whereas
the latter presented significant enhancement only at
72 h, with a 1.44-fold increase as compared with the
respective control (Fig. 11).
Discussion
The debate on the use of naturally derived drugs as
coadjuvants in the treatment of cancer is of growing
interest. In fact, owing to concerns about the possible
A
B
Fig. 5. TQ effects on U87 MG and T98G
cells. (A) Morphology of U87 MG and T98G
cells grown under standard conditions and
treated with 50 l
M or 100 lM TQ dissolved
in dimethylsulfoxide. Dimethylsulfoxide con-
centrations in treated and control cells did
not exceed 0.25% per well. Cells were
observed by using an inverted microscope
24 h post-treatment. (B) Dose–response
curve for the effect of TQ on cell viability
after 24, 48 and 72 h of exposure. Cell via-
bility was determined by the MTT assay,
and is reported as the percentage of viable
cells. Each value is the mean ± standard
deviation of three separate experiments
performed in triplicate.
Table 1. Thymoquinone IC

to induce apoptosis in tumor cells opened the possibil-
ity of their use as potential drugs, and numerous stud-
ies have been conducted with the aim of finding
natural, nontoxic and inexpensive compounds [36–38].
Fig. 6. 20S and 26S proteasome functionality in U87 MG cells treated with 20 lM TQ. Activities were assayed as reported in Experimental
procedures. Data are expressed as percentage of activity relative to control cells in each set (mean values ± standard deviations of five inde-
pendent determinations). Fluorescence due to nonproteasomal degradation was subtracted. The asterisks indicate data points that are statis-
tically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01).
V. Cecarini et al. Thymoquinone inhibits proteasome functionality
FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2133
In this scenario, we decided to investigate the possible
interaction between TQ and proteasomes in order to
determine whether TQ could modulate the enzyme
functionality.
Considering the data obtained from computational
analysis, it is reasonable to think that TQ could
behave as a nucleophilic target, resulting in inhibition
of proteasome activity. To confirm this hypothesis, we
tested proteasome functionality after TQ treatment of
both isolated and cellular complexes. Interestingly, we
observed subunit-dependent and composition-depen-
dent inhibition of both the purified enzymes, with the
immunoproteasome being the most sensitive and the
ChT-L and T-L components being the most influenced
activities. We also demonstrated that TQ induces a
U-shaped inhibition in proteasome complexes through
the binding of two distinct sites with different degrees
of affinity.
Exposure of two human glioblastoma cell lines,
U87 MG and T98G, to TQ was able to significantly

some functionality at 24 h. This inhibition was also
confirmed by accumulation of Ub–protein conjugates.
Furthermore, when we tested the 20S expression levels
with specific antibodies, we could not detect any differ-
ences between control and treated cells, demonstrating
the ability of TQ to directly alter proteasome activity
without affecting its synthesis.
Considering our data, it is clear that TQ is able to
modulate proteasome activity, inducing global inhibi-
tion in the studied models, although to different
extents. These results are in line with previously pub-
lished data from our laboratory and others reporting
on the ability of small, naturally derived ligands, e.g.
flavonoids, to inhibit proteasome functionality and
selectively modulate its activity, depending on the
subunit composition [37,39,40].
It has been widely reported that the proteasome,
being responsible for the removal of proapoptotic
A
B
C
Fig. 8. Detection of Ub–protein conjugates in U87 MG and T98G cells. The densitometric analysis from five separate blots, shown as mean
values ± standard deviations, and a representative western blot are shown (A, B). Membranes were reprobed with GAPDH antibody to
ensure equal protein loading (C). Detection was performed with an ECL western blotting analysis system. The asterisks indicate data points
that are statistically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01).
V. Cecarini et al. Thymoquinone inhibits proteasome functionality
FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2135
proteins, is involved in the induction of programmed
cell death [19]. Its inhibition, in fact, triggers the accu-
mulation of proteins such as p53 and Bax [41–43]. For

cellular increases in the levels of apoptotic proteins
such as p53 and Bax, and may be linked to the onset of
apoptotic events. Such findings represent evidence that
this compound, characterized by very low toxicity,
deserves further clinical analysis and investigation,
mostly for its potential application as an adjuvant in
the treatment of cancer and other diseases.
Experimental procedures
Reagents and chemicals
Thymoquinone, substrates for assaying the ChT-L, T-L
and PGPH activities [succinyl (Suc)-Leu-Leu-Val-Tyr-7-
amino-4-methyl-coumarin (AMC), Z-Leu-Ser-Thr-Arg-
AMC, and Z-Leu-Leu-Glu-AMC], proteasome inhibitors
(Z-Gly-Pro-Phe-Leu-CHO and lactacystin), Nitro Blue Tet-
razolium and MTT were purchased from Sigma-Aldrich
S.r.L. (Milan, Italy). The substrate Z-Gly-Pro-Ala-Phe-
Gly-4-aminobenzoate (pAB), for testing BrAAP activity,
and the proteasome inhibitor Z-LLF-CHO (Cbz-Leu-
Leu-Phe-CHO) were kind gifts from M. Orlowski (Depart-
ment of Pharmacology, Mount Sinai School of Medicine,
New York, NY, USA). Aminopeptidase N (EC 3.4.11.2)
for the coupled assay utilized to detect BrAAP activity
[44] was purified from pig kidney as reported elsewhere
[45,46]. TQ was dissolved in dimethylsulfoxide (Sigma
Aldrich S.r.l.). U87 MG and T98G human glioblastoma
cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). All of the
reagents for cell cultures were obtained from Euroclone
(Milan, Italy). Rabbit anti-(human 20S proteasome)
serum, rabbit anti-(human 20S proteasome b5 subunit)

Measurements of isolated 20S proteasome activity
To evaluate the effects of TQ on the 20S constitutive and
immunoproteasome peptidase activities, in vitro assays were
performed with fluorogenic peptides. Suc-Leu-Leu-Val-Tyr-
AMC was used for ChT-L activity, Z-Leu-Ser-Thr-Arg-
AMC for T-L activity, Z-Leu-Leu-Glu-AMC for PGPH
activity, and Z-Gly-Pro-Ala-Phe-Gly-pAB for BrAAP activ-
ity [48–50]. Isolation and purification of the 20S protea-
some from bovine brain and thymus were performed as
previously reported [50,51]. The incubation mixture con-
tained TQ at concentrations ranging from 0.0 to 100.0 lm,
1 lg of the isolated 20S proteasomes, the appropriate sub-
strate, and 50 mm Tris ⁄ HCl (pH 8.0), up to a final volume
of 100 lL. Incubation was performed at 37 °C, and after
60 min the fluorescence of the hydrolyzed 7-amino-
4-methyl-coumarin (AMC) and 4-aminobenzoic acid (pAB)
was detected (AMC, k
exc
= 365 nm, k
em
= 449 nm; pAB,
k
exc
= 304 nm, k
em
= 664 nm) on a SpectraMax Gemini
XPS microplate reader.
To test the presence of a TQ secondary binding site on
the proteasome complex, 1 lg of isolated 20S immunopro-
teasome was preincubated with 3 lm Z-LLF-CHO for 1 h

and 100 lgÆmL
)1
streptomycin, supplemented with 10%
heat-inactivated fetal bovine serum. Cells were maintained
in a 5% CO
2
atmosphere at 37 °C.
Cell viability assay
Cell viability was determined by the standard MTT assay
[53]. Cells were seeded at an initial density of 2 · 10
4
cellsÆmL
)1
in 96-well microtiter plates (Iwaki, Tokyo,
Japan) in 100 lL of growth medium. After incubation for
24 h at 37 °C, cells were exposed to different concentrations
of TQ (0.0–200 lm) containing 0.25% dimethylsulfoxide,
which was applied as a control, for 24, 48 and 72 h in a
humidified atmosphere at 37 °C in the presence of 5%
CO
2
. Cell viability was then quantified by the ability of
living cells to reduce the yellow dye MTT to a purple
formazan product. Cells were incubated with MTT for 4 h,
the medium was replaced with 100 lL of dimethylsulfoxide,
and the attenuance was measured with a Titertek Multiscan
microElisa microplate spectrophotometer reader (Labsys-
tems, Helsinki, Finland) at 540 nm. The IC
50
values were

tested using Suc-Leu-Leu-Val-Tyr-AMC as substrate, and a
50 mm Tris ⁄ HCl (pH 8.0) buffer containing 10 mm MgCl
2
,
1mm dithiothreitol, and 2 m m ATP. In order to evaluate
the effective 20S proteasome contribution to the short pep-
tide cleavage, control experiments were performed using spe-
cific proteasome inhibitors, Z-Gly-Pro-Phe-Leu-CHO and
lactacystin (5 lm in the reaction mixture). Fluorescence val-
ues obtained by analyzing the lysates were then subtracted
from the values of control assays in the presence of the two
inhibitors to find the effective proteasome contribution.
BrAAP activity was determined in a coupled test in the pres-
ence of aminopeptidase N [49]. Incubation was performed at
37 °C for 60 min. The fluorescence of hydrolyzed AMC and
pAB was then measured (AMC, k
exc
= 365 nm,
k
em
= 449 nm; pAB, k
exc
= 304 nm, k
em
= 664 nm) on a
SpectraMax Gemini XPS microplate reader.
Western blot analysis
Cell lysates were resolved by 12% SDS ⁄ PAGE and elec-
troblotted onto PVDF membranes. Membranes with trans-
ferred proteins were incubated with the mouse monoclonal

inhibits tumor angiogenesis and tumor growth through
suppressing AKT and extracellular signal-regulated
kinase signaling pathways. Mol Cancer Ther 7 , 1789–
1796.
3 Padhye S, Banerjee S, Ahmad A, Mohammad R &
Sarkar FH (2008) From here to eternity – the secret of
Pharaohs: therapeutic potential of black cumin seeds
and beyond. Cancer Ther 6, 495–510.
4 Shoieb AM, Elgayyar M, Dudrick PS, Bell JL & Tithof
PK (2003) In vitro inhibition of growth and induction
of apoptosis in cancer cell lines by thymoquinone. Int J
Oncol 22, 107–113.
5 Ait Mbarek L, Ait Mouse H, Elabbadi N, Bensalah M,
Gamouh A, Aboufatima R, Benharref A, Chait A,
Kamal M, Dalal A et al. (2007) Anti-tumor properties
of blackseed (Nigella sativa L.) extracts. Braz J Med
Biol Res 40, 839–847.
6 Worthen DR, Ghosheh OA & Crooks PA (1998) The
in vitro anti-tumor activity of some crude and purified
components of blackseed, Nigella sativa L. Anticancer
Res 18, 1527–1532.
7 Kaseb AO, Chinnakannu K, Chen D, Sivanandam A,
Tejwani S, Menon M, Dou QP & Reddy GP (2007)
Androgen receptor and E2F-1 targeted thymoquinone
therapy for hormone-refractory prostate cancer. Cancer
Res 67, 7782–7788.
8 Badary OA, Al-Shabanah OA, Nagi MN, Al-Rikabi
AC & Elmazar MM (1999) Inhibition of benzo(a)py-
rene-induced forestomach carcinogenesis in mice by
thymoquinone. Eur J Cancer Prev 8, 435–440.

licensed to kill? Operating the assassin. J Cell Biochem
88, 76–82.
16 Saito S, Yamaguchi H, Higashimoto Y, Chao C, Xu Y,
Fornace AJ Jr, Appella E & Anderson CW (2003)
Phosphorylation site interdependence of human p53
post-translational modifications in response to stress.
J Biol Chem 278, 37536–37544.
17 Miyashita T & Reed JC (1995) Tumor suppressor p53
is a direct transcriptional activator of the human bax
gene. Cell 80, 293–299.
18 Jung T & Grune T (2008) The proteasome and its role
in the degradation of oxidized proteins. IUBMB Life
60, 743–752.
19 Jung T, Catalgol B & Grune T (2009) The proteasomal
system. Mol Aspects Med 30, 191–296.
20 Takeuchi J & Toh-e A (1997) [Regulation of cell cycle
by proteasome in yeast]. Tanpakushitsu Kakusan Koso
42, 2247–2254.
21 Balantinou E, Trougakos IP, Chondrogianni N, Marga-
ritis LH & Gonos ES (2009) Transcriptional and post-
translational regulation of clusterin by the two main
cellular proteolytic pathways. Free Radic Biol Med 46,
1267–1274.
22 Voutsadakis IA (2008) The ubiquitin–proteasome sys-
tem in colorectal cancer. Biochim Biophys Acta 1782,
800–808.
23 Vlachostergios PJ, Patrikidou A, Daliani DD &
Papandreou CN (2009) The ubiquitin–proteasome
system in cancer, a major player in DNA repair. Part 1:
Post-translational regulation. J Cell Mol Med 13,

31 Vinitsky A, Michaud C, Powers JC & Orlowski M
(1992) Inhibition of the chymotrypsin-like activity of
the pituitary multicatalytic proteinase complex. Bio-
chemistry 31 , 9421–9428.
32 Ullrich SJ, Mercer WE & Appella E (1992) Human
wild-type p53 adopts a unique conformational and
phosphorylation state in vivo during growth arrest of
glioblastoma cells. Oncogene 7, 1635–1643.
33 Godbout R, Miyakoshi J, Dobler KD, Andison R,
Matsuo K, Allalunis-Turner MJ, Takebe H & Day RS
III (1992) Lack of expression of tumor-suppressor genes
in human malignant glioma cell lines. Oncogene 7,
1879–1884.
34 Van Meir EG, Kikuchi T, Tada M, Li H, Diserens AC,
Wojcik BE, Huang HJ, Friedmann T, de Tribolet N &
Cavenee WK (1994) Analysis of the p53 gene and its
expression in human glioblastoma cells. Cancer Res 54,
649–652.
35 Gali-Muhtasib HU, Abou Kheir WG, Kheir LA,
Darwiche N & Crooks PA (2004) Molecular pathway
for thymoquinone-induced cell-cycle arrest and apopto-
sis in neoplastic keratinocytes. Anticancer Drugs 15,
389–399.
36 Bonfili L, Amici M, Cecarini V, Cuccioloni M, Tacconi
R, Angeletti M, Fioretti E, Keller JN & Eleuteri AM
(2009) Wheat sprout extract-induced apoptosis in
human cancer cells by proteasome modulation. Biochi-
mie 91, 1131–1144.
37 Bonfili L, Cecarini V, Amici M, Cuccioloni M,
Angeletti M, Keller JN & Eleuteri AM (2008) Natural

inducing ability of genistein. Biochem Pharmacol 66,
965–976.
44 Orlowski M & Michaud C (1989) Pituitary multicatalyt-
ic proteinase complex. Specificity of components and
aspects of proteolytic activity. Biochemistry 28, 9270–
9278.
45 Almenoff J & Orlowski M (1983) Membrane-bound
kidney neutral metalloendopeptidase: interaction with
synthetic substrates, natural peptides, and inhibitors.
Biochemistry 22, 590–599.
46 Pfleiderer G (1970) Isolation of an aminopetidase from
kidney particles. In Methods in Enzymology (Perlman
GE & Lorand L, eds), pp. 514–521. Academic Press,
New York, NY.
47 Polik WF & Schmidt JR (2001) WebMO: visualizing
computational chemistry on the World Wide Web.
Abstr Papers Am Chem Soc 222, U181.
48 Wilk S & Orlowski M (1983) Evidence that pituitary
cation-sensitive neutral endopeptidase is a multicatalytic
protease complex. J Neurochem 40, 842–849.
49 Orlowski M, Cardozo C & Michaud C (1993) Evidence
for the presence of five distinct proteolytic components
in the pituitary multicatalytic proteinase complex. Prop-
erties of two components cleaving bonds on the car-
boxyl side of branched chain and small neutral amino
acids. Biochemistry 32, 1563–1572.
50 Eleuteri AM, Angeletti M, Lupidi G, Tacconi R, Bini L
& Fioretti E (2000) Isolation and characterization of
bovine thymus multicatalytic proteinase complex. Pro-
tein Expr Purif 18, 160–168.