From the Department of Biology, University Roma Tre,
Viale Marconi 446, 00146 Rome, Italy and
Consiglio Nazionale
delle Ricerche Center of Molecular Biology, University La Sapienza,
Piazzale Aldo Moro 5, 00185 Rome, Italy
Received for publication, August 8, 2000, and in revised form, October 18, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A fully active recombinant human ceruloplasmin
was obtained, and it was mutated to produce a ceruloplasmin stable to
proteolysis. The stable ceruloplasmin was further mutated to perturb
the environment of copper at the type 1 copper sites in two different
domains. The wild type and the mutated ceruloplasmin were produced in
the yeast Pichia pastoris and characterized. The mutations
R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the
spectroscopic and catalytic properties of ceruloplasmin, supporting the
hypothesis that this site is reduced and locked in a rigid frame. In
contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving
a small fraction endowed with oxidase activity. This result, while
indicating the importance of this site in stabilizing the overall
protein structure, suggests that another type 1 site is competent for
dioxygen reduction. During the expression of ceruloplasmin, the yeast
maintained a high level of Fet3 that was released from membranes of
yeast not harboring the ceruloplasmin gene. This indicates that
expression of ceruloplasmin induces a state of iron deficiency in yeast
because the ferric iron produced in the medium by its ferroxidase
activity is not available for the uptake.
Ceruloplasmin (Cp)1 is a
130-kDa multicopper protein widely distributed in vertebrates.
It occurs mainly in the plasma and plays an important role in iron
homeostasis (1, 2). Other roles include its participation in the
antioxidant defense (3-6) or in oxidative damage mechanisms (7, 8) and
its involvement in a number of metabolic processes related to the
metabolism of copper (9), biogenic amines (10), and nitric oxide (11).
Ceruloplasmin supports such diverse functionalities by different
catalytic activities. It has long been known that Cp is an oxidase and
belongs to the class of multicopper "blue" oxidases (12). These
enzymes couple the four-electron reduction of dioxygen to water to the
four sequential one-electron oxidations of a variety of
substrates. Other enzymes of the same group are ascorbate
oxidase, laccase, and Fet3, a plasma membrane protein of yeasts,
recently discovered (13). Among these enzymes, only Cp (14, 15) and Fet3 (16) are able to oxidize an inorganic substrate (i.e.
Fe(II) ions) in addition to organic substrates such as diamines. While the precise physiologic function of Cp associated to its amine oxidase
activity has not yet been defined, the ferroxidase activity is now
considered the main function of ceruloplasmin. The complete absence of
Cp, because of inherited mutations (17) or targeted disruption of the
Cp gene (18), leads to a long term accumulation of parenchyma iron due
to an impaired iron efflux from cells. The oxidation of the Fe(II)
released from cells and its subsequent incorporation into
apotransferrin would be the mechanism whereby Cp is involved in
mediating iron release from cellular stores (19, 20). Analogously, the
ferroxidase reaction performed by Fet3 is also an essential reaction
for the high affinity iron uptake in yeasts (13, 21).
Multicopper oxidases contain three distinct spectroscopic types of
copper sites: at least one type 1 (T1), or "blue," site in addition
to one type 2 (T2), or "normal," copper site and to one type 3 (T3), or binuclear, copper site lacking an EPR signal (12). The three
copper atoms of these two last sites comprise a trinuclear cluster,
observed in the x-ray structure of ascorbate oxidase (22), Cp (23), and
laccase (24), which serves as the oxygen binding and reducing site
during the catalysis. The T1 is the site of entry of electrons from
substrate. The T1 sites of multicopper oxidases exhibit different
geometry and different redox potential, due to differences in their
coordination sphere (25). With the exception of Cp, there is generally
one T1 site per trinuclear cluster in these enzymes. A conserved
structural motif of the polypeptide chain, His-Cys His, where the Cys
is a ligand of the T1 site and the two His residues are ligands of two
copper atoms of the cluster, facilitates the intramolecular electron
transfer (ET) between the two sites, ~13 Å apart (26, 27).
Ceruloplasmin contains multiple T1 sites. This is a long recognized
peculiarity of Cp that has been outlined by numerous spectroscopic and
catalytic studies and it has been confirmed by the x-ray structural studies on the human protein (23). Human ceruloplasmin (HCp) is a
single chain of 1046 amino acids (28), with a carbohydrate content of
7-8% and a copper content of six integral copper atoms. The x-ray
structure has shown that the molecule of HCp is composed of six compact
domains, with large loop insertions, and that the six integral copper
atoms are distributed in one trinuclear cluster, located at the border
between domains 1 and 6 and possessing ligands from each domain and in
three mononuclear T1 sites. Those located in domains 4 and 6 are
typical T1 sites such as that of ascorbate oxidase (22) and have a set
of four ligands, two histidines, one cysteine, and one methionine,
arranged in a distorted tetrahedral geometry. The one located in domain
2 is a tricoordinate T1 site in that it lacks the axial Met ligand,
which is replaced in the amino acid sequence by Leu, as the single blue
site of some laccases (26), and Fet3 (29). These latter sites
have redox potential values higher than those of typical T1 sites.
Whether and how all three T1 sites take part in the turnover reaction
of Cp is presently a major question (27). Only the T1 site of domain 6 is connected by the cysteine-histidine linkages to the trinuclear cluster (23). Binding sites for iron (15) and amine substrates (10)
have been identified in domains 4 and 6 by x-ray studies, which did not
evidence equivalent structures on domain 2. This latter finding has
been related to the peculiar redox properties of the T1 site of domain
2. A redox potential value of at least 1 V has been calculated for this
site, which is apparently the highest one among T1 sites of blue
proteins (30). On this basis, it has been postulated that the copper at
this center stays permanently reduced, thus spectroscopically silent
and not able to take part in the catalytic cycle of the oxidase.
Mutation studies have been used to clarify the influence of ligands and
the contribution of other elements of the protein matrix to the
properties of each type of copper (31-33). Although a recombinant
human ceruloplasmin has been expressed in mammalian cells and it has
been partially purified (34), the physicochemical properties of the
recombinant protein were not assessed. In this paper, we report the
expression of human ceruloplasmin in Pichia pastoris and the
use of site-directed mutagenesis to explore its structure to function
relationships. The residues responsible for the proteolytic
susceptibility of the protein, Arg481, Arg701,
and Lys887 had to be mutated in tandem ahead of targeted
mutations at the copper sites. Mutation of Leu329 to Met
and of Cys1021 to Ser in the coordination sphere of the T1
sites of domains 2 and 6, respectively, demonstrated a different role
for these two sites. During the expression of the heterologous oxidase, the yeast maintained its endogenous oxidase Fet3, although a soluble, fully active, derivative of Fet3 was found in the medium.
Chemicals and Biologicals--
Superdex 200, Sephacryl S-200,
Sephadex G-75, and Sepharose 4B were from Amersham Pharmacia Biotech.
DE52 was from Whatman. Enzymes for DNA manipulation were from New
England Biolabs or Promega. Anti-HCp polyclonal antibodies were from
Sigma. Peptide:N-glycosidase was from Roche Diagnostic
GmbH. Media for yeast cultures were from Difco. The Pichia
Expression Kit was from Invitrogen. All other reagents were purchased
from Sigma unless otherwise noted and used without further purification.
Strains, Culture, and Media--
The P. pastoris
strain used for the etherologous expression of HCp was GS115
his4 and was routinely grown in buffered glycerol complex
medium; buffered methanol complex medium was used for the induction.
The 10F' Escherichia coli was grown in Luria-Bertani medium.
Integration of HCp Expression Cassette in P. pastoris Genome and
Site-directed Mutagenesis--
The cDNA of HCp, encoding the
precursor protein with a 19-amino acid signal peptide, was kindly
provided by Professor J. D. Gitlin (Washington University, St.
Louis, MO). Site-directed mutagenesis was performed on the cDNA
cloned into the Bluescript II KS plasmid (pBS) with the following
mutagenic primers: 5'-GAAAAAGGCTTGCATACCGGC-3' for the mutation L329M;
5'-CCCAGAGCGCAAGTGTGCCTC-3' for the mutation R481A;
5'-CTCAGACTGCGCCCTGCATTG-3' for the mutation R701A;
5'-ACCTTACTTGGCAGTATTCAATCC-3' for the mutation K887A;
5'-GTTACTCCACTCCCATGTGACC-3' for the mutation C1021S. The
megaprimer technique (35) was used to obtain the K887A mutant first,
and then the mutations of R481 and R701
to Ala were introduced in a second round of mutagenesis according to
Ref. 36 to obtain the triple mutant R481A/R701A/K887A. The mutations
L329M and C1021S were introduced on the triple mutant with the
megaprimer technique. All mutations were verified by automated
sequencing at the ENEA-BIOGEN sequencing facility.
The cDNA encoding each HCp (wild type or mutant) with its own
signal peptide was subcloned from pBS into the expression vector pHIL-D2, which has a single EcoRI cloning site adjacent to
the alcohol oxidase 1 gene promoter, and used to transform E. coli (TOP 10F') cells. Plasmid DNA was purified from selected
colonies, linearized by digestion with NotI, and used to
transform GS115 his4 cells by spheroplasting according
to the manufacturer's instructions. Histidine-independent
transformants were selected and subsequently screened for their slow
methanol utilization phenotypes. Positive clones were induced with
methanol and screened for HCp production by Western blotting of the
culture medium.
Yeast Cultivation and Ceruloplasmin Production--
Yeast
colonies that had undergone the appropriate recombination events to
incorporate the Cp gene into the P. pastoris genome were
used to inoculate the buffered glycerol complex medium (800 ml). The
flasks (2 liters) were incubated at 30 °C in a shaking incubator
(250 rpm) until the cells attained an A600 of
~6. The cells were harvested by centrifugation at 3000 × g, and the cell pellet was washed extensively and
resuspended in the buffered methanol complex medium containing 300 µM CuSO4 and 30 µM
FeSO4 to an approximate A600 of 100. These conditions were settled by monitoring the effect of various
concentrations of these salts on the level of Cp secreted. The baffled
flasks were shaken at 30 °C. The cultures were monitored for up to 7 days. The daily addition of methanol to a final concentration of 0.5%
maintained the induction conditions of the alcohol oxidase 1 promoter.
Aliquots of cultures of induced P. pastoris cells were
collected at various times during the induction period, and cells were removed by centrifugation. The supernatants were analyzed either by
SDS-PAGE followed by Western blot analysis probed with anti-HCp polyclonal antibodies or by native PAGE followed by staining with o-dianisidine at pH 5, to monitor the oxidase activity. In
this case, the samples (5 ml) were fractionated on small columns of Sephadex G-75, and the high molecular weight fraction was collected and
concentrated to ~50 µl. Total membrane extracts from cell pellets
were obtained according to Ref. 21 and were analyzed by non denaturing
SDS-PAGE (37) followed by staining with o-dianisidine for
oxidase activity.
Ceruloplasmin Purification and Analysis--
The purification of
rHCp was routinely performed after a 72-h induction. At variance with
authentic HCp, which was isolated from plasma by a single step on a
column of activated Sepharose 4B (38), the isolation of recombinant
ceruloplasmin required multiple steps due to the abundance of brownish
components in the induction medium. The amount of this material was
somehow variable, and any effort to limit its presence in the medium
was in vain. It was adsorbed by DE52 added in batch (60 g/500 ml) to
the supernatant obtained from induced cultures. After 15 min, the resin
was filtered off, and the medium was depleted of low molecular weight
components and brought to low ionic strength by gel filtration on a
column of Sephadex G-75 (10 × 60 cm) equilibrated at pH 7 with 50 mM phosphate buffer containing 0.5 mM EDTA and 5 mM amino caproic acid. The fractions containing
ceruloplasmin were loaded on a bed of the activated Sepharose (2.5 × 20 cm). Extensive washings with 120 mM phosphate buffer
were necessary to completely elute Fet3. Nearly 50% of the original
content of rHCp was found in these fractions. The remaining rHCp was
eluted at 200 mM phosphate buffer. This fraction was
diluted and applied again to a small column of activated Sepharose to
concentrate ceruloplasmin. The procedure was accomplished in 10 h,
and the yield was nearly 1 mg of rHCp from 500 ml of induction medium, which represented ~20% of the recombinant protein secreted in the
medium. Very often, however, the concentrated sample of the recombinant
protein still contained yellow component(s) responsible for a huge
absorption below 500 nm. These samples required treatment with DE52,
added in batch, and gel filtration on either Superdex 200 by fast
protein liquid chromatography or Sephacryl S-200, that, however,
lowered the yield to ~0.6 mg.
Deglycosylation was performed on ceruloplasmin denatured at 90 °C in
50 mM Tris-Cl buffer, pH 8, containing 0.5% SDS, 0.1 M 2-mercaptoethanol, by the addition of 5 units of
peptide:N-glycosidase F in the presence of 20 mM
1,10-phenanthroline, 2% Triton X-100. The mixture was incubated for
2 h at 30 °C.
The amine oxidase activity was measured at pH 6 and pH 7, 0.2 M phosphate buffer, by a coupled NADH/pPD assay
(39). The ferroxidase activity was measured at pH 6 in 0.3 M acetate buffer by monitoring the appearance of ferric
ions at 315 nm (40).
Characterization of Fet3--
The limited proteolysis
experiments and the purification of the soluble derivative of Fet3 were
carried out as already reported (41). To test the susceptibility of the
purified soluble Fet3 to proteases, the protein, in 0.1 M
phosphate buffer, pH 7.4, was incubated with 2 µM trypsin
and 2 µM chymotrypsin, for 10 h. Oxidase activity
measurements and SDS-PAGE analyses were performed on aliquots from the
mixture at various times.
To isolate full-length Fet3 from transformed P. pastoris,
the cells were collected 48 h after the induction with methanol. The same purification procedure (41) applied to the membranes of
untransformed GS115 cells, grown in the presence of bathophenanthroline disulfonate, was used. Nondenaturing SDS-PAGE (37) was used to monitor
the electrophoretic heterogeneity of Fet3 during the purification; the
fractions of Fet3 at the various steps reproduced the pattern of
membranes, although a decrease of the slower component was noticed
along the entire procedure. For the N-terminal sequence determination,
the gels were soaked in the SDS buffer, containing 2.5%
mercaptoethanol, after the electrophoretic run and incubated for 10 min
at 90 °C prior to the transfer to the polyvinylidene difluoride membranes.
Analytical Methods--
Protein concentration was determined by
the copper/bicinchoninic acid assay (Pierce). Copper was determined by
flameless atomic absorption spectrophotometry on a PerkinElmer Life
Sciences model 3030 instrument equipped with graphite furnace. The
N-terminal sequence analyses of electrophoretic bands transferred on
polyvinylidene difluoride membranes were performed on an Applied
Biosystems model 475A sequencer. EPR spectra were obtained using a
Bruker ESP300 spectrometer equipped with a variable temperature
controller. Samples were run at 100 K, 9.43-GHz microwave frequency,
10-milliwatt power, and 10-Gauss modulation amplitude. Paramagnetic
copper content was estimated by double integration of the sample signal by using a copper-EDTA standard. Absorbance spectra were recorded on a
PerkinElmer Life Sciences Expression of Human Ceruloplasmin in P. pastoris--
Expression
and recovery of recombinant human ceruloplasmin was accomplished by
subjecting P. pastoris cells to an induction time of 3 days
in complete medium containing iron and copper salts. The behavior of
Fet3, the endogenous multicopper oxidase of yeast, was carefully
monitored over this time. As shown in Fig.
1A, the electrophoretic
analyses of the culture medium, performed under nondenaturing
conditions to detect the oxidase activity, revealed the presence of two
bands, both exhibiting a different mobility with respect to the
authentic human serum Cp. The two bands exhibited an opposite behavior.
The one with higher electrophoretic mobility was very intense at
24 h from the induction, and then it gradually decreased at longer
times. Although it was not recognized by the anti-Cp antibodies, this
oxidase mimicked the behavior of authentic HCp during purification
(38). It was retained by activated Shepharose, and it was found in the
high ionic strength fractions when the isolation of recombinant HCp was
afforded at 24 h postinduction. The SDS-PAGE analysis (Fig.
1D) showed that the principal component of these fractions
had an apparent molecular mass of ~100 kDa. Its N-terminal sequence,
ETHTWNFTTGFV, turned out to be identical to that determined for the
soluble derivative of Fet3 obtained by limited proteolysis of membrane
suspensions of P. pastoris (41).
Since Fet3 was not found in the media of cultures of uninduced cells,
this suggested that proteolytic attack(s) at the extracellular portion
of membrane-bound Fet3 could release its catalytic domain, upon
exposing yeasts to the conditions of the induction. The susceptibility of this domain to massive proteolytic attacks once in the medium can
explain the time-dependent decrease of the higher mobility band shown in Fig. 1A. This hypothesis was supported by the
observation that the soluble derivative of Fet3, purified from P. pastoris cells subjected to limited proteolysis as already
described (41), was more than 90% inactivated after 10 h of
incubation at room temperature with the same proteases. After this
time, the electrophoretic analyses showed that not only the
oxidase-active band but also the protein band had nearly vanished
(results not shown).
To verify that a decrease of the membrane-bound Fet3 was occurring,
concomitant to the appearance of Fet3 in the medium, the amine oxidase
activity of membrane extracts was assayed by using pPD as
substrate (41). Transformed cells showed only a modest decrease of the
specific oxidase activity, which accounted for ~90% that of
uninduced cells at 24 h postinduction and remained at these levels
over the induction period. In contrast, the parent GS115 strain,
subjected to same conditions, showed a variable batch-to-batch, but
important (50-80%), decline of its oxidase activity, checked at
different times postinduction. The electrophoretic analyses of the
membrane fraction from cell pellets harvested before and after the
induction to monitor the membrane-bound Fet3 confirmed these results
(Fig. 1C), thus suggesting that coding for a secreted
heterologous oxidase was responsible for the behavior of the
transformed yeast. It should be noted that the electrophoretic pattern
of transformed cells at 36 h postinduction showed a more pronounced heterogeneity due to an increased intensity of the faster
migrating of the bands with slower mobility with respect to the
principal band of Fet3. This behavior appears to be typical of P. pastoris subjected to low iron conditions in that this band (and
occasionally other components) is well resolved also in the electrophoretic pattern of the membranes isolated from cells grown in
the presence of 80 µM bathophenanthroline disulfonate
(41). Fet3, purified from transformed cells induced with methanol,
retained this component, which gave the same N-terminal sequence
(ETHTWNFTTGFVNANPDG) as the principal band of Fet3.
This analysis was also performed on the oxidase-active bands of the
fractions obtained at early steps of the purification and revealed that
Fet3 was the major component of the slower band. The other sequences
found varied batch-to-batch and did not match any known sequence. Since
the anomalous electrophoretic mobility of Fet3, as well as that of Cp
(37), in nondenaturing gels strongly correlates with the overall
protein conformation, a plausible explanation is that the slower band
represents a fraction of Fet3 locked in a different conformation or,
more simply, an aggregated form of the protein.
The oxidase-active band, much broader and slower than authentic HCp on
native gels (Fig. 1A), was recognized by anti-HCp antibodies (Fig. 1B), and the parallel enhancement of these bands at
longer times of induction indicated the gradual accumulation of an
active rHCp in the medium. The presence of multiple bands on Western blot (Fig. 1B), however, indicated that rHCp might be
suffering proteolytic attack(s), probably from the same proteases
responsible for the decline of the soluble Fet3 in the medium. Thus,
the induction was usually stopped after 72 h to avoid
excessive degradation of recombinant Cp.
Molecular Properties of Recombinant Ceruloplasmin--
As
described under "Experimental Procedures," the isolation of rHCp
from the culture medium was quite laborious with respect to the
single-step method employed to isolate HCp from plasma (38). The
electrophoretic analyses, performed on the purified protein, confirmed
the results obtained on the medium. rHCp as isolated appeared as a
broad band on native gels and dissociated under denaturing conditions
in two principal and heterogeneous components (Fig. 1C). The
N-terminal sequence determinations gave the following results. The
cluster of bands at 130-170 kDa gave only one sequence, that of the
mature HCp, KEKHYYIGIIETTW. The sequence SVPPSASHVA was obtained from
the major component of the cluster at around 80 kDa, and this result
indicated that a proteolytic attack at Arg481 had cut the
polypeptide chain of rHCp nearly in two halves. The sequence of the
other fragment, i.e. the N-terminal sequence of HCp, was
found in the multiple bands scattered in the region above and below 80 kDa. The heterogeneity of these components was undoubtedly due to the
carbohydrate moiety, since treatment of denatured samples of rHCp with
peptide:N-glycosidase F induced a mobility shift of these
bands consistent with a decrease in the molecular weight. In
particular, the high molecular weight bands were reduced to a more
homogeneous species that migrated with an apparent mass of ~120 kDa
(Fig. 1E), corresponding to that obtained from authentic HCp.
Different preparations of rHCp were analyzed for copper content taking
into account a molecular mass value of 120 kDa. A value of 5.9 ± 0.2 g atoms of copper/120 kDa was obtained, which indicated the
full occupancy of the integral copper sites. The specific oxidase
activity of rHCp as isolated was, at pH 6, 0.11 ± 0.03 µmol/min/mg of protein with pPD as substrate, a value
consistent with that of authentic HCp run as control, 0.14 ± 0.02 µmol/min/mg of protein. At pH 7, the activity decreased nearly 1 order of magnitude. Same results were obtained in assays of the
ferroxidase activity (see below).
Storage induced a decrease of the enzymatic activity, ~50% decrease
after two months, which was more pronounced with respect to that of HCp
and that was paralleled by changes in the electrophoretic pattern. The
recombinant protein gradually shifted from the pattern in Fig.
1D to that shown in Fig. 1F, which was typically
obtained from samples of rHCp purified at the longer induction times
and/or higher cellular density in an attempt to raise the yield
of the recombinant protein. The fragmentation pattern now reproduced that usually found in HCp (28), with fragments at ~116, 19, and 50 kDa, produced by the cleavage of the polypeptide chain at
Lys887.
Catalytic and Spectroscopic Properties of Recombinant
Ceruloplasmin--
Optical and EPR spectroscopies were used to analyze
the state of copper sites (Fig. 2). The
EPR spectrum of rHCp was superimposable to that of HCp isolated by the
single-step method from serum, reproducing the peculiar features of
this form of protein (Fig. 2B), namely a low content of T2
copper, as detected by the low field hyperfine line, and a low content
of paramagnetic copper, which accounted for ~40% of the total copper
content. According to these findings, a low absorption intensity of the
blue sites was also found (Fig. 2A); the molar extinction
coefficient at 610 nm was determined to be ~7000
M
The peculiar spectroscopic properties of HCp are due to the tendency of
a fraction of its T1 sites to stay reduced in the resting protein (30,
43). Binding of Cl
Altogether, these results confirmed the identity of recombinant
ceruloplasmin with authentic ceruloplasmin. They also indicated that
the varied carbohydrate moiety was not affecting the functional properties of the protein.
Site-directed Mutagenesis at the Proteolytic Sites of
Ceruloplasmin--
As shown in Fig.
3A, the three residues
Arg481, Arg701, and Lys887,
responsible for the lability of HCp to proteases, appear to be excluded
from the compact six-domain core of the molecule harboring the copper
sites. On this basis, site-directed mutagenesis of these residues was
attempted to protect the recombinant protein against degradation. A
mutant was then produced where each residue of the three sites was
replaced by alanine. The triple mutant, R481A/R701A/K887A rHCp (R,R,K/A
rHCp), turned out to be stable to proteolysis. It was free of the low
molecular weight bands in the SDS-PAGE analysis under denaturing
conditions (Fig. 3B). Storage did not induce the appearance
of fragments. Its spectroscopic and catalytic properties matched
exactly those of wild type rHCp already described. This mutant was then
used to afford the mutagenesis at the copper sites.
Site-directed Mutagenesis at the Copper Sites of
Ceruloplasmin--
Two different mutations, L329M and C1021S, were
performed to modify the coordination sphere of the copper of the sites
of domains 2 and 6.
The T1 site of domain 2 is the most critical metal binding site of HCp.
This site is considered unable to engage redox reaction with known
ceruloplasmin substrates, due to its high redox potential value (30).
It is a tricoordinate site, and it is characterized by the lack of the
axial Met ligand, which is replaced in the amino acid sequence by a
leucine residue unable to coordinate copper (Fig.
4A). It is not clear how this
difference in geometry, with respect to the typical T1 sites of domains
4 and 6, and/or the nature of surrounding protein matrix are
responsible for its properties. As shown in Fig. 4A, a
methionine residue can replace the leucine 329, without destroying the
site, and the mutated site assumes a geometry more similar to that of a
typical T1 site (25).
The mutation of Leu329 to Met was introduced into the
triple mutant; the resulting quadruple mutant was produced to the same extent as the wild type rHCp and the triple mutant. The spectroscopic properties of (R,R,K/A)L329M rHCp were carefully analyzed; however, no
qualitative and quantitative differences of either the optical absorption or of the EPR signal were found (Fig. 4B). The
measurements of the enzymatic activities, both the ferroxidase activity
(Fig. 4C) and the amine oxidase activity (0.10 ± 0.03 µmol/min/mg versus pPD), indicated that the
presence of methionine was also ineffective in producing significant
modifications of the catalytic properties. Upon the addition of
Cl
Cys1021 is a ligand of the typical T1 site of domain 6, together with His975, His1026, and
Met1031 (23). Its flanking His residues are ligands for two
of the copper atoms of the trinuclear cluster so to furnish a bifurcate pathway to the electron donated to the copper center by a substrate. The mutation of Cys1021 to Ser was afforded to destroy the
catalytic activity pertinent to this site (10, 15) to ascertain whether
the other T1 site(s) could sustain the enzymatic activity of the
mutated protein. The mutant C1021S rHCp proved to be very difficult
with respect to the other recombinant ceruloplasmins in that it was
produced at lower levels. Furthermore, it exhibited an anomalous
behavior during purification, since only part of it bound to activated Sepharose and the fraction retained by the resin eluted at low ionic
strength, with the 100 mM buffer. During the purification, small fragments of ~40 kDa, were noticed in all fractions. The overall result was that only a minute amount of this mutant, a few
hundred µg, could be obtained.
The electrophoretic analyses revealed the extreme heterogeneity of this
mutant (Fig. 5). The copper content of
these samples was exceedingly low, ~0.1 g atom of copper/120 kDa,
with respect to the expected stoichiometric value of 5/6; therefore,
the spectroscopic characterization was not attempted, nor were careful
activity measurements done. However the electrophoretic analyses
performed under nondenaturing conditions showed a quite interesting
finding, since they revealed the presence of an oxidase-active band
(Fig. 5, right) among the multiple inactive bands, which are
likely to be various forms of the recombinant protein, completely or partially depleted of copper. The active band is clearly the only form
of the recombinant protein retaining a full complement of copper at the
trinuclear cluster accompanied by the occupancy of at least one of the
two other T1 copper sites. As to the amount of this active form, it
should be noted that the gel had to be overloaded with 10 µg of
protein to observe the active band.
We succeeded in obtaining a recombinant human ceruloplasmin
displaying all of the properties of the authentic protein as purified from plasma. Unfortunately, the recombinant protein retained also the
susceptibility of HCp to proteolysis, a property that has hampered all
studies on this protein (45). The stability of the triple mutant that
we obtained by substituting Arg481, Arg701, and
Lys887 with alanine demonstrates that only these residues
are responsible for the fragmentation pattern that affects all
ceruloplasmin preparations.
It should be recalled that the lability of human ceruloplasmin is such
that it is found fragmented even in the plasma (38). The fragments
initially do not dissociate and mimic the intact molecule, but the
protein suffers changes of its redox properties that lead to a
redistribution of electrons within its copper sites (46). In fact, the
antioxidant or the prooxidant activities of Cp are specific to the
cleaved and to the intact molecule, respectively (8). Thus, it was of
outmost importance to have a stable Cp to work with before attempting
mutagenesis targeted to the copper sites.
The mutagenesis has been focused on the two T1 copper sites more
critical to the function of ceruloplasmin (i.e. those on domains 2 and 6). The aim of these experiments was not only studying the role of the ligands on the properties of the sites, as it is the
case of current studies on multicopper oxidases containing a single T1
site. In the case of ceruloplasmin, the major problem concerns the
reasons for having multiple T1 sites with different disposition with
respect to a single trinuclear cluster (23). This is a peculiarity of
ceruloplasmin that cannot be simply related to the necessity of
increased potentiality of electron capture from substrates. Fet3 has
only one T1 site (29) and is as able as Cp to oxidize both Fe(II) ions
and amines. Structural and catalytic studies are trying to find
plausible electron transfer pathways in the molecule to disclose the
functional relations of the three sites, if any (27), and their
interaction with the cluster during catalysis (15).
The mutation of Cys1021 to Ser was performed for this
purpose. Since it has been ascertained that mutation of Cys at
the T1 site is fatal to multicopper oxidases having a single T1 site,
this mutation in the T1 site of domain 6 of Cp should clarify whether this site is the entrance to the bifurcate ET pathway to the cluster also for electrons (possibly) released to the other T1 sites. The same
mutation in Fet3 succeeded in producing an inactive T1-depleted protein
that conserved unaltered the structure of the T2 and T3 sites (33). An
analogous mutation in bilirubin oxidase produced an inactive protein
that did not, however, conserve the trinuclear cluster intact (31). Our
results on the C1021S mutant, its anomalous behavior during
purification, and its much less than stoichiometric copper content
indicate that the occupancy of the T1 site of domain 6 contributes to
stabilizing the other copper binding sites and/or the protein
structure. The observation of a species of this mutant retaining the
enzymatic activity indicates that another T1 site is able to engage a
redox reaction with the cluster. This site is most likely that of
domain 4, since x-ray experiments locate on it the binding site for
amine substrates (10).
Since the substitution of Cys abolishes the character of the T1 site,
the mutation of Cys1021 is also important to shed some
light on the spectroscopic properties of HCp. Our results indicate that
future studies aimed at producing rHCp mutated at domain 6 that is
suitable for spectroscopic studies have to be carefully planned. In
fact, the mutation C1021D failed to produce transformants with
meaningful expression.2
HCp typically occurs in a partially reduced state (30, 44). The protein
treated with oxidants still contains reduced copper, and its molar
absorptivity does not exceed the value of 9000 M Methionine is not an indispensable residue to produce the peculiar
nature of the T1 site, which seems rather to be related to the ligation
of Cys within the trigonal core ligation His-Cys-His (25). Its more
likely role is to tune the redox potential and the spectroscopic
properties of the site, as indicated by extensive site-directed
mutagenesis of blue proteins (47-50) and also by results on
multicopper oxidases (31). That the mutation of leucine 329 to
methionine in the polypeptide chain of human ceruloplasmin is silent
under all aspects is an intriguing result that can, however, shed some
light on the properties of tricoordinate T1 sites of multicopper oxidase.
Assuming that Met329 is able to bind the copper atom of the
site of domain 2, a conclusion would be that Met329 binds
copper without any effect on the structure of the site. The recent
study on Trametes villosa laccase shows that all properties of the enzyme are affected by the mutation of Phe463 to Met
(32). The changes of the spectroscopic properties observed in this
enzyme, a 13-nm shift in the maximal absorption wavelength and the
variation of magnetic parameters of the EPR spectrum, are such that
they had to be detected even when masked by the contribution of other
T1 sites, as in rHCp. A decrease of 0.1 V of the redox potential value
accompanies the variation of the spectroscopic properties in mutated
laccase, resulting in an EPR signal intermediate between that of the
wild type laccase and of the typical T1 site of plastocyanin. As to the
variation of the redox potential expected for mutated T1 site of HCp,
it has to be considered that the value calculated for this site in
authentic HCp exceeds 1 V (30) so that even when lowered by
hundreds of mV, according to the results on laccase and those
obtained in model systems (51), it would remain an exceedingly high
value, the highest among T1 sites. Assuming that it is in the reduced state in the wild type protein, it will probably remain in this state
also in the L329M mutant, explaining why the mutation fails to affect
spectroscopic and catalytic properties of HCp. The fact that the
introduction of Met is unable to render this site more similar to the
other two T1 sites confirms that other elements of the protein matrix
play an important role beyond ligands in tuning the structure of T1
sites (32, 52, 53). In other words, it is locked in a definite frame,
and this should confer a specialized function to it. It might either
play an active role in the reactions of the oxidase, under the specific
control of effectors affecting its redox properties, or be
responsible to other functions, specific to the human protein.
This consideration comes from the observation that the three T1 sites
of other mammalian ceruloplasmin (54) do not differ between each other
to the extent that characterizes human ceruloplasmin.
An additional finding of this work was that secreted ceruloplasmin can
affect the iron metabolism of the yeast. The fact that the transformed
yeast cells tend to maintain a constant level of Fet3 on their
membranes balancing the leakage of the protein indicates that they are
in a condition of iron deficiency (13, 21). The only way to explain
this result is to consider that recombinant ceruloplasmin is under
turnover conditions as a ferroxidase in the medium of induced cells, as
demonstrated by its partially reduced state upon isolation. This would
shift the redox state of iron to an oxidized ferric form not available
to cells, confirming that uptake of iron by yeast requires Fe(III) ions
produced by a member (Fet3) of its transport system (55). The
ferroxidase reaction of ceruloplasmin, which decreases the level of
free Fe(II) in the plasma, favoring release of iron from cells, results
in a condition of iron deficiency for yeast with consequent induction of Fet3.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
14 model instrument. Figures representing views of the crystal structure of HCp and of the copper site were created by using crystallographic coordinates taken from the Protein Data Bank (Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ) with the entry code 1KCW. The
two loop regions and the C-terminal portion of the polypeptide chain
not clearly defined in the electron density map were modeled as
described previously (42).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (46K):
[in a new window]
Fig. 1.
Expression of human ceruloplasmin in P. pastoris. Top, analysis of proteins secreted
following methanol induction. Aliquots of culture media of P. pastoris were removed at the indicated times postinduction and
centrifuged. The supernatants were fractionated on Sephadex G-75 and
concentrated 100× before loading on gels for PAGE and in-gel assay
activity by using o-dianisidine (A) and loaded as
such on gels for denaturing SDS-PAGE and Western blot analysis by using
anti-HCp (B). C, membrane extracts from cell
pellets from either uninduced cells grown in buffered glycerol complex
medium (0), and from cells collected 36 h postinduction
were analyzed by nondenaturing SDS-PAGE. The behavior of untransformed
GS115 is compared with the transformed yeast. Bottom,
SDS-PAGE analyses of preparations of wild type (wt)
recombinant human ceruloplasmin. D, the 200 mM
fraction from activated Sepharose of a preparation at 24 h
postinduction is compared with recombinant HCp purified at 72 h
postinduction; the arrows indicate the band corresponding to
the sequence of Fet3. E, the effect of
peptide:N-glycosidase treatment on the sample at 72 h
postinduction and on authentic Cp. Samples were denatured at 90 °C
and deglycosylated with 5 units of
peptide:N-glycosidase at 30 °C for 24 h.
F, electrophoretic pattern of samples purified 7 days
postinduction showing the proteolytic fragments. HCp and
Fet3 in both A and B refer to the
purified proteins run in each case as control.
1 cm
1.
For reference, this value in other multicopper oxidases, having a
single T1 site, is ~5000 M
1
cm
1 (26, 29). Below 450 nm, the optical
spectra of purified rHCp samples invariably exhibited a slightly higher
intensity with respect to HCp. The presence of extra bands in the
region of the 330-nm chromophore (12) of rHCp could, however, be
excluded on the basis of differential spectra, obtained by subtracting the absorption of authentic Cp from rHCp spectra. Transitions occurring
at lower wavelengths, due to traces of the strongly absorbing
pigment(s) of the induction medium, are the most reasonable explanation
for this difference.
View larger version (12K):
[in a new window]
Fig. 2.
Spectroscopic analyses of wild type
(wt) recombinant HCp. The visible spectrum
(A) and the EPR spectrum (B) of the recombinant
protein purified at 72 h postinduction are compared with those of
authentic human ceruloplasmin. The EPR spectrum of a concentrated
sample of the 200 mM fraction from activated Sepharose
(C) is shown before and after the addition of 100 mM Cl . EPR spectra were recorded at 100 K.
to this protein activates
intramolecular ET to the cluster with a resulting increase of the
absorbance at 610 nm (44). The addition of 100 mM
Cl
to purified rHCp samples to probe the oxidation level
of their T1 sites did not affect the spectroscopic properties. In
contrast, a considerable increase of the optical absorption at 610 nm
(results not shown) and of the intensity of the EPR signal (Fig.
2C) was noticed when, still impure, the recombinant protein
was tested immediately after elution from the activated Sepharose. The
extent of the spectroscopic changes varies considerably among samples of HCp, ranging between 10 and 60%. The reasons of these differences have never been investigated. Thus, to understand the behavior of the
recombinant protein, the reactivity of authentic Cp was carefully
investigated, and it was found that samples of serum Cp that exhibited
a 40% increase of the visible absorption, immediately after the
isolation, became nearly unresponsive to chloride when tested after
they were subjected to the same chromatographic steps employed to
obtain rHCp samples shown in Fig. 2, A and B.
View larger version (39K):
[in a new window]
Fig. 3.
Site-directed mutagenesis at the proteolytic
sites of ceruloplasmin. A, view of the HCp molecule
(top) and scheme of the polypeptide chain
(bottom) showing the location of the proteolytic
sites. The molecule is oriented to present its flat surface to the
observer. B, electrophoretic analyses of the
R481A/R701A/K887A triple mutant by SDS-PAGE (left) and by
native PAGE (right). In both cases purified HCp was run as a
control.
View larger version (15K):
[in a new window]
Fig. 4.
Site-directed mutagenesis at the
tricoordinate copper site of domain 2 of human ceruloplasmin.
A, view of the site showing in addition to the three
copper ligands (two histidines and one cysteine) the leucine 329 (top), replaced by methionine (bottom). The
distances (Å) of these two latter residues from copper are
indicated. Spectroscopic (B) and catalytic (C)
properties of (R,R,K/A)L329M rHCp are shown. The kinetics of the
ferroxidase activity of the L329M mutant ( ) as a function of iron
concentration are shown in comparison with authentic HCp (
)
and wild type rHCp (
). All proteins were 0.05 µM in the assay. mT, millitesla.
to a partially purified sample of this mutant,
i.e. to the 200 mM fraction from activated
Sepharose, the behavior of wild type rHCp shown in Fig. 2C
was again observed in that the anion elicited the spectroscopic
properties of this mutant, but this did not entail the appearance of
new species (results not shown).
View larger version (92K):
[in a new window]
Fig. 5.
Properties of recombinant ceruloplasmin
mutated at the copper site of domain 6. The native PAGE analyses
of C1021S mutant were performed on 2 µg of protein for Western blot
(left) and 10 µg of protein for both protein staining
(middle) and activity staining with o-dianisidine
(right). In each case, HCp was run as control in the
following amounts (from left to right):
250 ng, 1 µg, and 0.5 µg.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 cm
1,
which is consistent with only two T1 sites bearing Cu(II) ions (26).
The EPR spectra of the resting, as isolated, and of the oxidized forms
of HCp can, however, be simulated on the basis of two (30) as well as
of three (43, 56) nonequivalent T1 components. This problem is related
to the state and the function of the T1 site of domain 2 which has been
the target of the other mutation, L329M.
![]() |
FOOTNOTES |
---|
* This work was supported by Consiglio Nazionale delle Ricerche "Progetto Finalizzato Biotechnologie."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Inst. of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr. Bohrgasse 9/4, A-1030 Vienna, Austria. Tel.: 43-1-427754614; Fax: 43-1-42779546; E-mail: pamela@gem.univie.ac.at.
¶ Present address: Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.
** This work was in partial fulfillment of a Ph.D.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007176200
2 P. Bielli, G. C. Bellenchi, and L. Calabrese, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Cp, ceruloplasmin; HCp, human ceruloplasmin; rHCp, recombinant human ceruloplasmin; T1, T2, and T3, type 1, 2, and 3 copper, respectively; PAGE, polyacrylamide gel electrophoresis; pPD, p-phenylenediamine; ET, electron transfer.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Frieden, E., and Hsieh, H. S. (1976) Adv. Enzymol. 44, 187-236[Medline] [Order article via Infotrieve] |
2. | Gitlin, J. D. (1998) Pediatr. Res. 44, 271-276[Abstract] |
3. | Gutteridge, J. M. C. (1983) FEBS Lett. 157, 37-40[CrossRef][Medline] [Order article via Infotrieve] |
4. | Miyajima, H., Takahashi, Y., Serizawa, M., Kaneko, E., and Gitlin, J. D. (1996) Free Radical Biol. Med. 20, 757-760[CrossRef][Medline] [Order article via Infotrieve] |
5. | Cha, M. K., and Kim, I. H. (1999) Biochemistry 38, 12104-12110[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Inoue, K.,
Akaike, T.,
Miyamoto, Y.,
Okamoto, T.,
Sawa, T.,
Otagiri, M.,
Suzuki, S.,
Yoshimura, T.,
and Maeda, H.
(1999)
J. Biol. Chem.
274,
27069-27075 |
7. | Swain, J., and Gutteridge, J. M. C. (1995) FEBS Lett. 368, 513-515[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Mukhopadhyay, C. K.,
Mazumder, B.,
Lindley, P. F.,
and Fox, P. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11546-11551 |
9. | Harris, E. D. (1993) Prog. Clin. Biol. Res. 380, 163-179[Medline] [Order article via Infotrieve] |
10. | Zaitsev, V., Zaitseva, I., Papiz, M., and Lindley, P. F. (1999) J. Biol. Inorg. Chem. 4, 579-587[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Bianchini, A.,
Musci, G.,
and Calabrese, L.
(1999)
J. Biol. Chem.
274,
20265-20270 |
12. | Malmstrom, B. G. (1982) Annu. Rev. Biochem. 51, 21-59[CrossRef][Medline] [Order article via Infotrieve] |
13. | Askwith, C., Eide, D., Van Ho, A., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell 76, 403-410[Medline] [Order article via Infotrieve] |
14. |
Osaki, S.
(1966)
J. Biol. Chem.
241,
5053-5059 |
15. | Lindley, P., Card, G., Zaitseva, I., Zaitsev, V., Reinhammar, B., Selin-Lindgren, E., and Yoshida, K. (1997) J. Biol. Inorg. Chem. 2, 454-463[CrossRef] |
16. |
de Silva, D. M.,
Davis-Kaplan, S.,
Fergestad, J.,
and Kaplan, J.
(1997)
J. Biol. Chem.
272,
14208-14213 |
17. | Harris, Z. L., Takahashi, Y., Miyajima, H., Serizawa, M., MacGillivray, R. T. A., and Gitlin, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2539-2543[Abstract] |
18. |
Harris, Z. L.,
Durley, A. P.,
Man, T. K.,
and Gitlin, J. D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10812-10817 |
19. |
Osaki, S.,
Johnson, D. A.,
and Frieden, E.
(1966)
J. Biol. Chem.
241,
2746-2751 |
20. | Chidambaram, M. V., Barnes, G., and Frieden, E. (1983) FEBS Lett. 159, 137-140[CrossRef][Medline] [Order article via Infotrieve] |
21. | Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T., and Klausner, R. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2632-2636[Abstract] |
22. | Messerschmidt, A., Ladenstein, R., Huber, R., Bolognesi, M., Avigliano, L., Petruzzelli, R., Rossi, A., and Finazzi Agrò, A. (1992) J. Mol. Biol. 224, 179-205[Medline] [Order article via Infotrieve] |
23. | Zaitseva, I., Zaitsev, V., Card, G., Moshkov, K., Bax, B., Ralph, A., and Lindley, P. (1996) J. Biol. Inorg. Chem. 1, 15-23[CrossRef] |
24. | Ducros, V., Brzozowski, A. M., Wilson, K. S., Brown, S. H., Ostergaard, P., Schneider, P., Yaver, D. S., Pedersen, A. H., and Davies, G. J. (1998) Nat. Struct. Biol. 5, 310-316[Medline] [Order article via Infotrieve] |
25. | Holm, R. H., Kennepohl, P., and Solomon, E. I. (1996) Chem. Rev. 96, 2239-2314[CrossRef][Medline] [Order article via Infotrieve] |
26. | Solomon, E. I., Sundaram, U. M., and Machonkin, T. E. (1996) Chem. Rev. 96, 2563-2605[CrossRef][Medline] [Order article via Infotrieve] |
27. | Farver, O., Bendahl, L., Skov, L. K., and Pecht, I. (1999) J. Biol. Chem. 274, 36135-36140 |
28. | Ortel, T. L., Takahashi, N., and Putnam, F. W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4761-4765[Abstract] |
29. |
Hassett, R.,
Kosman, D. J.,
Yuan, D. S.,
and Kosman, D. J.
(1998)
J. Biol. Chem.
273,
23274-23282 |
30. | Machonkin, T. E., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1998) Biochemistry 37, 9570-9578[CrossRef][Medline] [Order article via Infotrieve] |
31. | Shimizu, A., Know, J. H., Sasaki, T., Satoh, T., Sakurai, N., Sakurai, T., Yamaguchi, S., and Samejima, T. (1999) Biochemistry 38, 3034-3042[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Xu, F.,
Palmer, A. E.,
Yaver, D. S.,
Berka, R. M.,
Gambetta, G. A.,
Brown, S. H.,
and Solomon, E. I.
(1999)
J. Biol. Chem.
274,
12372-12375 |
33. | Blackburn, N. J., Ralle, M., Hassett, R., and Kosman, D. J. (2000) Biochemistry 39, 2316-2324[CrossRef][Medline] [Order article via Infotrieve] |
34. | Rafiq, M., Suen, C. K. M., Choudhury, L., Joannou, C. L., White, K. N., and Evans, R. W. (1997) FEBS Lett. 407, 132-136[CrossRef][Medline] [Order article via Infotrieve] |
35. | Sarkar, G., and Sommer, S. S. (1990) BioTechniques 4, 404-407 |
36. | Aiyar, A., and Leis, J. (1993) BioTechniques 3, 366-369 |
37. |
Sato, M.,
and Gitlin, J. D.
(1991)
J. Biol. Chem.
266,
5128-5134 |
38. |
Musci, G.,
Bonaccorsi di Patti, M. C.,
Fagiolo, U.,
and Calabrese, L.
(1993)
J. Biol. Chem.
268,
13388-13392 |
39. | Musci, G., Carbonaro, M., Ariani, A., Lania, A., Galtieri, A., and Calabrese, L. (1990) Arch. Biochem. Biophys. 279, 8-13[Medline] [Order article via Infotrieve] |
40. | Bonomi, F., Kurtz, D. M., and Cui, X. (1996) J. Biol. Inorg. Chem. 1, 67-72[CrossRef] |
41. | Bonaccorsi di Patti, M. C., Bellenchi, G. C., Bielli, P., and Calabrese, L. (1999) Arch. Biochem. Biophys. 372, 295-299[CrossRef][Medline] [Order article via Infotrieve] |
42. | Musci, G., Polticelli, F., and Calabrese, L. (1999) Adv. Exp. Med. Biol. 448, 175-182[Medline] [Order article via Infotrieve] |
43. | Musci, G., Bonaccorsi di Patti, M. C., and Calabrese, L. (1993) Arch. Biochem. Biophys. 306, 111-118[CrossRef][Medline] [Order article via Infotrieve] |
44. | Musci, G., Bonaccorsi di Patti, M. C., and Calabrese, L. (1995) J. Protein Chem. 14, 611-619[Medline] [Order article via Infotrieve] |
45. | Rydèn, L. (1984) in Copper Proteins and Copper Enzymes (Lontie, R., ed), Vol. 3 , pp. 37-100, CRC Press, Inc., Boca Raton, FL |
46. | Calabrese, L., and Musci, G. (1997) in Molecular Properties of Ceruloplasmin from Different Species in Multi-Copper Oxidase (Messerschmidt, A., ed) , pp. 307-354, World Scientific Publications, Singapore |
47. | Canters, G. W., and Gilardi, G. (1993) FEBS Lett. 325, 39-48[CrossRef][Medline] [Order article via Infotrieve] |
48. | Romero, A., Hoitink, C. W., Nar, H., Huber, R., Messerschmidt, A., and Canters, G. W. (1993) J. Mol. Biol. 229, 1007-1021[CrossRef][Medline] [Order article via Infotrieve] |
49. | Libeu, C. A. P., Kukimoto, M., Nishiyama, M., Horinouchi, S., and Adman, E. T. (1997) Biochemistry 36, 13160-13179[CrossRef][Medline] [Order article via Infotrieve] |
50. | Hall, J. F., Kanbi, L. D., Strange, R. W., and Hasnain, S. S. (1999) Biochemistry 39, 12675-12680[CrossRef] |
51. | Olsonn, M. H. M., and Ryde, U. (1999) J. Biol. Inorg. Chem. 4, 654-663[CrossRef][Medline] [Order article via Infotrieve] |
52. | Wittung-Stafhede, P., Hill, M. G., Gomez, E., Di Bilio, A. J., Karlon, B. G., Leckner, J., Winkler, J. R., Gray, H. B., and Malmstrom, B. G. (1998) J. Biol. Inorg. Chem. 3, 367-370[CrossRef] |
53. | Malmstrom, B. G., and Leckner, J. (1998) Curr. Opin. Chem. Biol. 2, 286-292[CrossRef][Medline] [Order article via Infotrieve] |
54. | Musci, G., Fraterrigo, T. Z. L., Calabrese, L., and McMillin, D. R. (1999) J. Biol. Inorg. Chem. 4, 441-446[CrossRef][Medline] [Order article via Infotrieve] |
55. | Askwith, C., and Kaplan, J. (1998) Trends Biochem. Sci. 23, 135-138[CrossRef][Medline] [Order article via Infotrieve] |