Tài liệu Báo cáo khoa học: A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action docx

A role for the intersubunit disulfides of seminal RNase
in the mechanism of its antitumor action
Aurora Bracale
1,
*, Francesco Castaldi
1,
*, Lucio Nitsch
2
and Giuseppe D’Alessio
1
1
Dipartimento di Chimica Biologica and
2
Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’,
Universita
`
di Napoli, Italy
The dimeric structure of seminal ribonuclease (BS-RNase)
is maintained by noncovalent interactions and by two
intersubunit disulfide bridges. Another unusual feature of
this enzyme is its antitumour action, consisting in a cyto-
toxic activity selective for malignant cells. This cytotoxic
action is exerted when the protein reaches the cytosol of
the affected cells, where it degrades ribosomal RNA, thus
blocking protein synthesis and leading cells to death. The
current model proposed for the mechanism of antitumour
action of BS-RNase is based on the ability of the protein
to resist the neutralizing action of the cytosolic RNase
inhibitor, a resistance due to the dimeric structure of the
enzyme. Monomeric RNases, and monomeric derivatives
of BS-RNase, are strongly bound by the inhibitor and

inactive as antitumor agents. Here we report on mono-
meric derivatives of BS-RNase that, although strongly
inhibited by the cytosolic RNase inhibitor, are cytotoxic
towards malignant cells. These monomers are produced
by reductive cleavage of the intersubunit disulfides of the
native, dimeric protein followed by linking the exposed
sulfhydryls to small thiols through formation of mixed
disulfides. We found that sulfhydryls from cell monolayers
and cell membranes can attack these mixed disulfides in the
monomeric derivatives, and reconstitute, through sulfhyd-
ryl-disulfide interchange reactions, the native dimeric pro-
tein, which is internalized as such, and displays its
antitumour action.
Keywords: antitumor; BS-RNase; disulfides; RNase.
Seminal RNase from bovine seminal vesicles (BS-RNase)
(reviewed in [1]) is a dimeric RNase in which two identical
subunits are held together by noncovalent interactions
and by two intersubunit disulfide bonds bridging Cys31 and
Cys32 of one subunit with the corresponding Cys32¢ and
Cys31¢ of the partner subunit. BS-RNase is an antitumour
agent, as it is strongly and selectively cytotoxic for
malignant cells in vitro and in vivo,withnoeffectson
normal cells [2].
Since the early studies on the antitumor action of BS-
RNase, it has been recognized that the dimeric structure of
the enzyme is essential for its display of cytotoxic activity
[3]. This conclusion was based on the lack of cytotoxic
activity in a monomeric derivative of the protein obtained
by selective, reductive cleavage of the intersubunit disul-
fides followed by alkylation of the exposed sulfhydryls.

Such conclusion has been subsequently confirmed through
different experimental approaches [4], and explained [4–6]
by the resistance of the enzyme in its dimeric state to the
inhibitory action of CRI (the cytosolic RNase inhibitor).
When the structure of CRI [7] and CRI complexed to
RNases [8,9] were elucidated, it became clear how native,
dimeric BS-RNase cannot fit into the horseshoe cavity of
the inhibitor, whereas a monomeric form of the enzyme
can, and is fully inhibited by CRI. Indeed, monomeric
RNases lacking cytotoxic activity, such as bovine pancre-
atic RNase and monomeric BS-RNase, could be engine-
ered into cytotoxic agents by rendering them resistant to
CRI [6,10].
In a survey of monomeric derivatives of BS-RNase, we
found that some of them, although fully inhibited by CRI,
were active as cytotoxic agents, and selective for malignant
cells. Further investigation revealed that monomeric deri-
vatives of BS-RNase are cytotoxic only when they conserve
the intersubunit cystine residues, so that they can be
re-converted into dimers, an event primed by cell sulfhydryls.
These results indicate that the intersubunit disulfide bonds
of BS-RNase have a key role in the mechanism of
antitumour action of the enzyme.
Materials and methods
Materials
Iodoacetic acid (IAA), iodoacetamide (IAM), 2-bromo-
ethylamine hydrobromide, 5,5¢-dithio-bis(2-nitrobenzoic
Correspondence to G. D’Alessio, Dipartimento di Chimica Biologica,
Universita
`

di Napoli ‘Federico II’, Via Mezzocannone 16,
80134 Napoli, Italy.
Fax: + 39 081 5521217, Tel.: + 39 081 2534731,
E-mail:
Abbreviations:BS-RNase,bovineseminalRNase;MCM,monomeric
bis-Cys31,Cys32-S-carboxymethylated-BS-RNase; MCA, mono-
meric bis-Cys31,Cys32-S-carboxyamidomethylated-BS-RNase;
MAE, monomeric bis-Cys31,Cys32-S-aminoethylated-BS-RNase;
MSSAE, monomeric bis-Cys31,Cys32-S-ethylamine-BS-RNase;
MSSG, monomeric bis-Cys31,Cys32-S-glutathione-BS-RNase;
CRI, cytosolic RNase inhibitor; PM, plasma membrane; IAM,
iodoacetamide.
*Note: These authors contributed equally to this work.
(Received 23 January 2003, revised 5 March 2003,
accepted 13 March 2003)
Eur. J. Biochem. 270, 1980–1987 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03567.x
acid) and alkaline phosphatase-conjugated anti-rabbit
secondary Ig were purchased from Sigma. Reagents for
Western blotting detection (SuperSignalÒ West Dura
Chemiluminescent Substrate, and Immobilon
TM
-P mem-
branes were purchased from Celbio, Milan, Italy). Poly-
clonal antibodies against BS-RNase, obtained from rabbits
as described previously [11], were used at a dilution of
1 : 1000. Fluorescein-tagged goat anti-rabbit secondary Ig
was obtained from Jackson ImmunoResearch (West Grove,
PA, USA). BS-RNase and its monomeric derivatives were
prepared as described [12]. All monomers were homogene-
ous upon SDS/PAGE, and catalytically active by RNase

assay [13].
Other methods
The monomeric derivative MSSAE (monomeric bis-
Cys31,Cys32-S-ethylamine-BS-RNase, 100 lg) was labelled
with 1 mCi carrier-free Na
125
I (Amersham) using IODO-
BEADS (Pierce) following the manufacturer’s instructions,
and desalted on PD10 Sephadex G-25 M columns (Phar-
macia), equilibrated with NaCl/P
i
. The specific activity of
labelled MSSAE was approximately 1 lCi per mg of protein.
Sulfhydryl content was determined as described by [14].
RNase inhibition by the cytosolic RNase inhibitor was
determined as described previously [15].
Cell cultures
SV40-transformed mouse fibroblasts and the parental
nontransformed Balb/C 3T3-line were obtained from
American Type Culture Collection (USA) and grown in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco-Life
Technology) supplemented with 10% fetal bovine serum
(Gibco-Life Technology) and Penicillin-Streptomycin-Glu-
tamine 1X (Gibco-Life Technology). Cell lines were main-
tained at 37 °C in a humidified incubator containing 10%
CO
2
mixedwithair.
Cytotoxicity assay
Cells were seeded in 24- or 96-well plates (1.2 · 10

4
cellsÆ
cm
)2
) in the presence of the RNase to be tested. After a
48-h incubation, cells were trypsinized, resuspended in
growth medium, mixed with trypan blue solution (Sigma)
(1 : 1, v/v), and counted. Cell viability was determined in
triplicate as the percentage of trypan blue-excluding cells
with respect to the total cell count.
Preparation of cell lysates
Cells treated with the RNase under test were washed first
with 1
M
Hepes pH 7.5 containing 0.1
M
NaCl (Hepes/NaCl
buffer) for 5 min, then three times with NaCl/P
i
, scraped
from plates with a rubber policeman, collected by centrifu-
gation at 1000 g, and resuspended in lysis buffer (1% NP-40
in 50 m
M
Tris/HCl at pH 8.0) in the presence of a protease
inhibitors cocktail (CØMPLETE
TM
, Roche). Cells were
lyzed by vortexing, incubated on ice for 30 min, and
centrifuged at 16 000 g for 30 min. The final supernatant

was assayed for protein concentration and frozen at )80 °C,
or processed immediately. All steps were performed at 4 °C.
Preparation of the membrane fraction
Cells were grown to confluency in 150 mm plates, washed
twice with NaCl/P
i
and scraped with a rubber policeman in
homogenization buffer (10 m
M
Tris/HCl, pH 7.5, 0.25
M
sucrose containing the protease inhibitors cocktail). Cells
were homogenized by 25 strokes with the tight pestle of a
Dounce homogenizer. The homogenate was centrifuged at
1000 g for 10 min and the supernatant was centrifuged at
16 000 g for 30 min. The pellet, representing the plasma
membrane enriched fraction (PM), was resuspended in
NaCl/P
i
, assayed for protein concentration and frozen at
)80 °C or processed immediately. All steps were performed
at 4 °C.
Immunofluorescence studies
Immunofluorescence experiments were performed as previ-
ously described [11]. Briefly, mouse fibroblasts were incu-
bated with the RNase under test and fixed with 3.7%
formaldehyde in NaCl/P
i
for 15 min at room temperature.
RNases were detected with the BS-RNase antiserum. To

test the immunofluorescence of internalized proteins, cells
were washed with Hepes/NaCl for 5 min and permeabilized
with 0.1% Triton X-100 in NaCl/P
i
for 5 min at room
temperature. Fluorescein-conjugated secondary antibody
was used at a dilution of 1 : 50. Cells were visualized by
epifluorescence using an Axiophot microscope (Zeiss).
Results and discussion
The monomeric derivatives of BS-RNase employed in this
study, illustrated in Table 1, were prepared following
established procedures [12] for the derivatization of Cys31
and Cys32, the cysteine residues that form the intersub-
unit disulfides of BS-RNase. Briefly, MCM (monomeric
bis-Cys31,Cys32-S-carboxymethylated-BS-RNase), MCA
(monomeric bis-Cys31,Cys32-S-carboxyamidomethylated-
BS-RNase), and MAE (monomeric bis-Cys31,Cys32-S-
aminoethylated-BS-RNase) were obtained by selective
reduction of the protein intersubunit disulfides followed
by alkylation of the exposed sulfhydryls with iodoacetate,
iodoacetamide or 2-bromoethylamine hydrobromide,
respectively. The MSSAE monomer was obtained by
reaction with methyl aminoethanethiosulfonate of the
sulfhydryls exposed by selective reduction of the
Table 1. Monomeric derivatives of BS-RNase. LC
50
is the protein
concentration producing 50% of cell death.
Mixed
disulfide

LC
50
(lgÆmL
)1
)
M–(CH
2
–S–CH
2
–COO
–
)
2
(MCM) No >200
M–(CH
2
–S–CH
2
–CONH
2
)
2
(MCA) No >200
M–(CH
2
–S–CH
2
–CH
2
–NH

3
+
)
2
(MAE) No >200
M–(CH
2
–S–S–CH
2
–CH
2
–NH
3
+
)
2
(MSSAE) Yes 47 ± 6
M–(CH
2
–S–S–CH
2
–cGlu
–
)
2
(MSSG) Yes 31
|
Gly
M–(CH
2

–S–S–CH
2
)
2
–M (BS RNase) 25 ± 4
Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1981
intersubunit disulfides. The MSSG monomer (mono-
meric bis-Cys31,Cys32-S-glutathione-BS-RNase) was a by-
product of the preparation of recombinant BS-RNase, in
which Cys31 and Cys32 residues form mixed disulfides with
glutathione moieties.
All monomeric derivatives retained full RNase activity, in
fact they were more active than the parent dimeric enzyme,
as previously reported [12]. As for their sensitivity to the
inhibitory action of the cytosolic RNase inhibitor (CRI), it
is known that MCM is fully inhibited by CRI [16]. We
tested MCA and MAE with increasing concentrations of
CRI and found that they were inhibited by approximately
90% with a 2–4 molar excess of CRI. Monomers MSSAE
and MSSG could not be tested as such for inhibition by
CRI, because the strongly reducing conditions of the assay
produce the cleavage of their mixed disulfides. This in turn
generates, from either MSSAE or MSSG, M(SH)
2
mono-
mers, i.e. BS-RNase monomers with exposed sulfhydryls at
Cys31 and Cys32, and free thioethylamine or glutathione,
respectively. As M(SH)
2
has been shown to be fully

inhibited by CRI [5], all monomers investigated in the
present study can be considered as highly sensitive to the
inhibitory action of CRI.
We tested the cytotoxic activity of the monomeric
derivatives described above on malignant SVT2-3T3 fibro-
blasts by measuring cell survival after 48 h of growth in the
presence of increasing concentrations of each monomeric
derivative. The data illustrated in Fig. 1 show that some
monomers (MCM, MCA, MAE) have no cytotoxic activity
on malignant SVT2 cells, whereas others (MSSG and
MSSAE) are surprisingly cytotoxic. This cytotoxic action
was selective for malignant cells, as when the latter, active
monomers were tested on nonmalignant 3T3 fibroblasts,
they were found to be as devoid of toxicity as native, dimeric
BS-RNase (data not shown).
It is noteworthy that in the inactive MCM, MCA and
MAE monomers Cys31 and Cys32, the cysteine residues
originally involved in the intersubunit disulfide bonding
of BS-RNase, are irreversibly blocked through S-alkylation.
In the active MSSAE and MSSG monomers, instead, the
two Cys residues still form (mixed) disulfide bonds with
thioethylamine or glutathione moieties, respectively
(Table 1). This led us to hypothesize that the cytotoxic
activity of the latter monomers was due to the presence in
these proteins of disulfide bonds, with their potential
chemical instability. It is well known that in the presence
of thiolates, disulfides can undergo sulfhydryl-disulfide
interchange reactions. Thus, at difference with the mono-
mers bearing stable, S-alkylated Cys residues, MSSAE and
MSSG monomers could, when delivered to growing cells,

undergo reactions with cell thiolates, which could lead to
their transformation into dimers, as described below:
where M is a BS-RNase monomer, R is the thioethylamine
or the glutathione moiety, CELL-S
–
are cell thiolates
present in n molar excess, and M-(S-S)
2
-M is a reconsti-
tuted dimer, in fact indistinguishable from native
BS-RNase.
The presence of sulphydryls on the surface of SVT2
cells was tested with 5,5¢-dithio-bis(2-nitrobenzoic acid) a
reagent impermeable to cell membrane [17]. We found
63 nmol of reactive, surface sulphydryls per 10
6
SVT2
cells. In a typical experiment, this would give a molar
excess of cell thiol groups of approximately 50-fold over
the disulfides introduced in the cell culture upon treatment
with the RNase monomers. It should be added that the
intersubunit disulfides of BS-RNase are hyper-reactive to
reduction, even to mild reducing agents, with respect to
intrachain disulfides [18,19], and are completely cleaved by
a 10-fold molar excess of dithiothreitol [19]. This hyper-
reactivity is a feature also of the mixed disulfides formed
by Cys31 and Cys32 with glutathione [12], and of the
mixed disulfides of MSSAE (unpublished results).
To verify the hypothesis described above, SVT2 fibro-
blasts were grown at 37 °C in the presence of 20 lgÆmL

)1
of
radioactively labelled
125
I-labelled MSSAE in binding buffer
(DMEM containing 1 mgÆmL
)1
BSA and 25 m
M
Hepes at
pH 7.5). At increasing time intervals, cells were washed
repeatedly with NaCl/P
i
andthentreatedwith0.6
M
NaCl
in NaCl/P
i
for 5 min at 4 °C to detach labelled monomers
bound to the cell surface [20]. The detached labelled protein
was then analyzed by SDS/PAGE followed by autoradio-
graphy. The results shown in Fig. 2 indicate that MSSAE
monomers upon binding to the cell surface associate into a
dimeric protein, with the molecular size of BS-RNase. A
quantitation of the dimeric bands identified on the gel
(Fig. 2) shows that MSSAE undergoes dimerization into
native-like BS-RNase in a time-dependent manner, and is
Fig. 1. Dose–response effects on BALB/C 3T3-SVT2 cells of mono-
meric derivatives of BS-RNase. Cells were treated for 48 h at 37 °C
with MCA (h), MCM (j), MAE (e), MSSAE (r), MSSG (m)or

BS-RNase as a positive control (d).
2M-ðS-S-RÞ
2
þ n CELL-S
À
! M-ðS-S-CELLÞ
2
þ M-ðS
À
Þ
2
þ 4RS
À
þ n-2 CELL-S
À
ð1Þ
M-ðS-S-CELLÞ
2
þ M-ðS
À
Þ
2
þ n-2CELL-S
À
! M-ðS-SÞ
2
-M þ n CELL-S
À
ð2Þ
1982 A. Bracale et al.(Eur. J. Biochem. 270) Ó FEBS 2003

almost totally dimeric after a 24-h contact with growing
cells. When
125
I-labelled MSSAE was incubated in binding
buffer in the absence of cells no dimerization occurred (data
not shown).
These results indicate that a monomeric derivative of BS-
RNase in which disulfide bonds are conserved at Cys31 and
Cys32 residues can reconstitute into native-like BS-RNase
when administered to growing fibroblasts. Such a transfor-
mation can be explained by sulfhydryl-disulfide interchange
reactions occurring between cell sulfhydryls and the mixed
disulfide bonds present in the monomeric derivative.
We further investigated whether the MSSAE and MSSG
monomers conserved the acquired dimeric structure upon
cell internalization. This was considered a necessary condi-
tion to attribute to the dimerization event a role in the
antitumour action of BS-RNase, as BS-RNase monomers
would be neutralized in the cytosol by the action of CRI.
SVT2 cells were grown with MSSAE, MSSG, or native BS-
RNase at a concentration of 50 lgÆmL
)1
. After 24 h cells
were washed at 4 °CwithNaCl/P
i
, then with 0.6
M
NaCl to
remove proteins from the cell surface. Washed cells were
then lysed and analyzed by SDS/PAGE followed by

immunoblotting with an anti-BS-RNase serum. The results
of these experiments, illustrated in Fig. 3, show that inside
the cells BS-RNase, MSSG and MSSAE are all present as
dimers. These dimers are covalent, as when the electro-
phoresis run was performed under reducing conditions,
most of the dimeric proteins dissociated into monomers
(Fig. 3). Identical results were obtained when cell lysis was
carried out in the presence of 2 m
M
iodoacetamide (IAM) to
block any free sulfhydryls (Fig. 3). This indicates that dimer
formation through disulfide bonding did not occur as an
artifact during lysis.
These results led us to conclude that indeed BS-RNase
monomers linked through disulfides to thioethylamine or
glutathione moieties are reconstituted in the presence of
growing fibroblasts into the parent dimeric protein, which
is internalized as a native-like dimeric RNase. They also
indicate for the first time that when BS-RNase is internal-
ized by malignant cells, it maintains its dimeric structure.
We have previously demonstrated by immunofluores-
cence studies that BS-RNase binds to the surface of SVT2
cells and is internalized inside the cells, whilst the MCM
monomer does not bind and is not internalized [11]. We
repeated these experiments with the MSSAE monomer and
treated exponentially growing SVT2 cells with 50 lgÆmL
)1
of
MSSAE for 75 min at 37 °C. When treated cells were tested
with anti-BS-RNase serum MSSAE was found to bind

effectively to their surface (Fig. 4A). SVT2 fibroblasts were
then treated with MSSAE, then stripped of surface bound
proteins with a high salt solution made up of 1
M
Hepes
pH 7.5 containing 0.1
M
NaCl [11], and permeabilized with
0.1% Triton X-100. The results of this experiment, illustra-
ted in Fig. 4B, show that BS-RNase immunoreactivity is
localized inside the cells in endosome-like vesicles throughout
the cytoplasm (Fig. 4B). These results are identical to those
obtained under identical conditions with native BS-RNase
[11]. Together with the results described above, they confirm
that MSSAE monomers dimerize outside the cells, and are
internalized as dimeric BS-RNase.
As the dimerization event occurs outside the cells, before
internalization, we investigated the role of plasma mem-
branes (PM) in the transformation of MSSAE into a
dimeric protein.
125
I-labelled MSSAE was incubated with
isolated membranes from SVT2 fibroblasts (0.45 mgÆmL
)1
of total protein) for 16 h at 37 °Cin0.2mLNaCl/P
i
. The
membranes were either washed with 0.6
M
NaCl in NaCl/P

i
,
or washed with NaCl and then, after removal of the
supernatant by centrifugation for 20 min at 16 000 g,
treatedwith2m
M
dithiothreitol in NaCl/P
i
. Labelled
proteins extracted from plasma membranes and membrane
pellets were then analyzed by SDS/PAGE and autoradio-
graphy. Figure 5 shows that after incubation with labelled
MSSAE, membranes contained radioactive protein both
monomeric and dimeric (lane 1). This indicates that under
the conditions employed a substantial fraction of MSSAE
was dimerized. In the fraction extracted from PM by the salt
treatment (lane 2), most (approximately 80%) of the protein
was dimeric. Clearly, monomers remained entrapped in the
PM pellet, which upon electrophoresis in SDS was found to
contain almost all monomeric protein (lane 3). When
membranes were extracted with 0.6
M
NaCl and the
Fig. 2. Time-course of dimerization of the labelled monomeric derivative
of BS-RNase
125
I-labelled MSSAE added to growing SVT2 cells.
125
I-
labelled MSSAE was detached by high salt from SVT2 cells at

increasing time intervals. In the insert, autoradiographic scans of
the SDS/PAGE runs. D and M mark the electrophoretic mobilities
of BS-RNase and monomeric BS-RNase, respectively.
Fig. 3. Immunoblots of SVT2 cell lysates. Lysates were from cells
treated for 24 h with BS-RNase (lane 1), monomeric MSSAE (lane 2),
monomeric MSSG (lane 3), MSSAE from a lysate performed in the
presence of 2 m
M
iodoacetamide (lane 4), BS-RNase from a lysate
performed in the presence of 2 m
M
iodoacetamide (lane 5), MSSAE as
in lane 2 after electrophoresis under reducing conditions (lane 6) and
BS-RNase as in lane 1 after electrophoresis under reducing conditions
(lane 7).
Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1983
membrane pellet was treated with dithiothreitol, the mem-
brane entrapped monomers could be released (lane 4), albeit
not completely, as some of them were still found to remain
entrapped by PM (lane 5).
The dimerization effect of PM on MSSAE was dependent
on PM concentration. As shown in Fig. 6, at approximately
0.25 mgÆmL
)1
of PM protein concentration, dimerization
reached a plateau.
These data indicate that the cell sulfhydryls responsible
for the exchange with the protein disulfides are located in
the plasma membrane. Furthermore, they show that, as
proposed in the hypothesis above, the RNase monomers are

linked through disulfides to the cell membrane, and are
released only when additional sulfhydryl–disulfide exchange
reactions occur, which eventually lead to their association
into dimers.
These results were confirmed when the separation of
monomeric and dimeric RNase species produced by treating
membranes with
125
I-labelled MSSAE was performed by
gel filtration. In these experiments the role of membrane
sulfhydryls in MSSAE dimerization was further verified by
testing the effect on dimerization of iodoacetamide (IAM).
125
I-labelled MSSAE (20 lgÆmL
)1
)wasaddedtocell
membranes in the presence or the absence of 10 or 50 m
M
Fig. 4. Fluorescence studies of SVT2 fibroblasts treated with the MSSAE monomeric derivative of BS-RNase. Cells were treated with 50 lgÆmL
)1
MSSAE for 75 min at 37 °C and fixed without permeabilization (A) or after a high-salt washing and permeabilization with Triton X-100 (B). The
RNase was detected with anti-BS-RNase serum followed by incubation with fluorescein-tagged anti-rabbit secondary Ig. The bar represents 10 lm.
1984 A. Bracale et al.(Eur. J. Biochem. 270) Ó FEBS 2003
IAM and incubated for 16 h at 37 °C. The labelled protein
extracted from PM by 0.6
M
NaCl in NaCl/P
i
was
gel-filtered on a Superdex-75 column. As shown in Fig. 7A,

after 16 h of incubation with PM, MSSAE was found to be
totally converted into dimers. When the incubation was
carried out in the presence of 10 m
M
IAM, the product of
Fig. 5. Autoradiography of SDS/PAGE runs of the labelled monomeric
derivative of BS-RNase
125
I-labelled MSSAE incubated with plasma
membranes (PM) from SVT2 cells. Lane 1, plasma membranes treated
for 16 h with
125
I-labelled MSSAE; lane 2, labelled proteins extracted
from PM with high salt; lane 3, labelled proteins still bound to
extracted PM; lane 4, labelled proteins extracted from the PM pellet
with 2 m
M
dithiothreitol; lane 5, proteins from the PM pellet after
treatment with dithiothreitol.
Fig. 6. Dimerization effect of PM isolated from SVT2 cells on the
labelled monomeric derivative of BS-RNase
125
I-labelled MSSAE trea-
ted with increasing concentrations of PM. Inset, autoradiographic scans
of the SDS/PAGE runs of
125
I-labelled MSSAE detached by high salt
from PM. D and M mark the electrophoretic mobilities of BS-RNase
and monomeric BS-RNase, respectively.
Fig. 7. Gel-filtration analysis of the labelled monomeric derivative of

BS-RNase
125
I-labelled MSSAE after a 16-h incubation with isolated
PM from SVT2 cells. The incubation was performed (A) in the absence
of iodoacetamide (IAM), (B) in the presence of 10 m
M
IAM, or (C) of
50 m
M
IAM. D and M mark the elution volumes of BS-RNase and
monomeric BS-RNase, respectively.
Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1985
dimerization decreased to 60% (Fig. 7B); at the higher IAM
concentration (50 m
M
), only 30% of dimer was produced
(Fig. 7C).
The data from the experiments on plasma membranes
indicate that the cell sulfhydryls responsible for the inter-
changes with disulfides, the reactions that reconstitute
native-like BS-RNase, belong to the plasma membranes.
They also show that BS-RNase monomers derived from
MSSAE bind covalently through disulfide bonds to the
membranes, as they can be released from the membranes as
monomers only through the action of a reducing agent, such
as dithiothreitol. The labelled RNase monomer, when
added to PM, is released from the membranes as a dimeric
protein, apparently produced by a sulfhydryl–disulfide
interchange occurring on the membranes. These are exactly
the events described in Eqns (1 and 2) of the hypothesis

proposed above.
It has been reported [21–23] that protein-disulfide iso-
merase (PDI) is present and active at the plasma membrane
surface of many types of cells. We thus considered the
possibility that PDI had a role in the dimerization reaction
of BS-RNase M(SSR)
2
monomers. However, we did not
detect any effects of 1–10 m
M
concentrations of bacitracin
(Sigma), a known inhibitor of PDI [21], on the dimerization
reaction. Likewise, an anti-PDI serum (Stressgen) had no
inhibitory effects on the reaction. These data suggest that
PDI has no role in the reconstitution of dimeric BS-RNase
from M(SSR)
2
monomers.
Conclusion
The results reported here reveal a new, significant event in
the mechanism of cytotoxic action of BS-RNase on
malignant cells. The event consists in the interactions,
through sulfhydryl–disulfide interchange reactions, between
surface cell sulfhydryls and the intersubunit disulfides that
link the two subunits of BS-RNase. Monomeric derivatives
of the protein are inactive as cytotoxic agents when they are
prepared by reductive cleavage of the intersubunit disulfides
and the resulting free sulfhydryls are blocked through
alkylation. Monomers of BS-RNase are instead active when
obtained by linking to small thiol compounds the sulfhy-

dryls exposed after reductive cleavage. The latter monomers
are found to reconstitute into disulfide linked dimers when
they interact with malignant cells, or with isolated cell
membranes, and are recovered as covalent dimers in treated
cell lysates. Also native BS-RNase is found to be a covalent
dimer inside the cells. These data lead us to conclude
that the same interchange reactions occur when the native
BS-RNase dimer binds and penetrate cells, with the protein
undergoing a double transition from dimer to monomers
linked to cell sulfhydryls, to covalent dimer again. Thus, the
reported results provide a first clue to the mechanism by
which BS-RNase is endocytosed by cells.
Acknowledgement
This work was financed by grants from the Associazione Italiana per la
Ricerca sul Cancro (AIRC), Ministero dell’Universita
`
e della Ricerca
(Progetti di Rilevante Interesse Nazionale 2001) and Consorzio
Interuniversitario Biotecnologie. Aurora Bracale was supported by a
fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC).
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