(Received for publication, July 7, 1995; and in revised form, November 14, 1995)
From the
The copper-glutathione complex (Cu(I)-GSH) efficiently acted in vitro as the source of Cu(I) in the reconstitution of
apoceruloplasmin. Copper was found to reinstate in the various sites in
a multistep process, with metal entry into the protein in a first
phase, and a second step involving conformational changes of the
protein leading to the recovery of the native structural and functional
properties. This latter phase was found to be strongly facilitated by
Mg or Ca
and by ATP. Both
Mg
and ATP had to be present for optimal
reconstitution.
These results may shed some light on the mechanisms
governing the biosynthesis of ceruloplasmin in vivo. Cu(I)-GSH
was the only complex able to reconstitute ceruloplasmin at neutral pH.
Glutathione may thus function to shuttle the metal from the membrane
copper pump, as the Wilson disease ATPase, and ceruloplasmin in the
secretory compartments of the cell. The finding that ceruloplasmin
acquires the native conformation after metal entry through a complex
pathway triggered by Mg and ATP suggests that they
may act as physiological modulators of this process in vivo.
Ceruloplasmin (CP), ()an
-glycoprotein found in the plasma of all vertebrates,
is synthesized mainly in the liver as an apoprotein and secreted into
plasma as a holoprotein associated to 5-6 tightly bound copper
atoms. The role of the prosthetic metal in the physiologic activity of
CP is unclear. The peculiar spectroscopic and functional properties of
the copper atoms are typical of multicopper blue oxidases and suggest
that CP acts as an enzyme (Rydén, 1984). The
metal ions of blue oxidases are divided into three spectroscopically
distinguishable types of centers which are referred to as the Type 1,
or ``blue'' copper, with an intense optical absorbance around
600 nm and a very small A
hyperfine
splitting in the EPR spectrum; Type 2 copper, with no visible
absorbance and large A
values; and Type 3
copper, absorbing at 330 nm, and not detectable by EPR. This latter
site is constituted by two antiferromagnetically coupled Cu(II) ions in
close proximity to the type 2 site (Calabrese et al., 1988,
1989) as in laccase (Allendorf et al., 1985) and ascorbic
oxidase (Messerschmidt et al., 1989). The resulting trinuclear
cluster is the oxygen activating site during the catalytic cycle of the
enzyme. The oxidase activity of CP is exerted toward a number of
substrates, including ferrous ion and aromatic amines (Frieden, 1980).
As a ferroxidase, the protein is implicated both in iron metabolism
(Frieden, 1980; Harris et al., 1995) and in the antioxidant
defense (Halliwell and Gutteridge, 1986; Fleming et al.,
1991). A severely impaired iron metabolism has been found in a case of
aceruloplasminemia due to a homozygotic mutation of the CP gene (Harris et al., 1995), where the enzymatic and copper kinetics had
been found to be normal (Miyajima et al., 1987). These
findings seem at odd with the long time proposal that CP serves as a
copper transporter (Cousins, 1985). As a matter of fact, the protein
binds most of the plasma copper (
90-95%), and dismetabolic
copper pathologies like Wilson disease are associated to low levels of
circulating CP and to abnormal accumulation of copper in tissues
(Danks, 1989).
CP incorporates copper within the secretory compartments of hepatocytes (Sato and Gitlin, 1991; Terada et al., 1995). The exact pathway for CP biosynthesis and the mechanism of copper insertion remain, however, to be clarified. Little is known about multicopper proteins biosynthesis, namely the mechanism controlling the proper molecular architecture for a correct incorporation of copper, and the involvement of other biochemical factors in the process.
Studies of copper reinsertion into CP deprived in vitro of the metal have been attempted only in few cases, and it has been generally accepted that CP takes up copper in an all-or-none fashion (Aisen and Morell, 1965). A recovery of the enzymatic and spectroscopic properties has been reported using Cu(I)-thiourea as the metal donor at acidic pH values (Schechinger et al., 1988). The complex was chosen to mimic the metal sites of metallothioneins, since these proteins had in turn been showed to be able to transfer copper into apoCP in the presence of activated leukocytes (Schechinger et al., 1986). The results were assumed as evidence for a possible role of metallothioneins as copper donors for CP.
The recent identification of the gene product defective in Wilson disease as a membrane-bound P-type ATPase with a putative copper-transport role (Yamaguchi et al., 1992; Bull et al., 1993; Tanzi et al., 1993), may suggest that this pump is involved in copper donation to nascent CP. Whether a direct interaction between ATPase and apoCP takes place, or rather some unidentified low mass molecule serves as copper carrier between the pump and the newly synthesized CP chain, is an unaddressed issue.
Several studies have pointed out a role for glutathione in the intracellular traffic of copper (Freedman et al., 1986, 1989). Copper associated to glutathione as Cu(I)-GSH may be directly transferred to different copper-dependent proteins like apo-Cu,Zn-superoxide dismutase (Ciriolo et al., 1990) and apohemocyanin (Brouwer and Brouwer-Hoexum, 1992), as well as to thioneins (Freedman et al., 1989; Da Costa Ferreira et al., 1993) and to phytochelatins (Mehra and Mulchandani, 1995).
In this study, we have addressed the problem of the processes governing copper reincorporation into apoCP. A complex pathway involving, after metal entry, conformational changes of the protein stimulated by effectors like divalent cations and ATP has been devised.
The Cu(I)-GSH was aerobically prepared shortly before use as described previously (Ciriolo et al., 1990). A GSH:copper stoichiometric ratio of 3:1 was used. The Cu(I)-thiourea complex was prepared according to Minder and Stocker(1936). All solutions were prepared with water pretreated with Chelex 100 resin, to remove traces of metal ions. The absence of any oxidized copper in the complexes was assessed by EPR.
Reconstituted holo and apoCP were chromatographically
separated on either DEAE-cellulose or Mono-Q. The mixture loaded on a
2.5 30-cm column of DEAE-cellulose equilibrated with 50 mM phosphate buffer, pH 7.0, was eluted with linear 50-150
mM gradient of the buffer. The holoprotein eluted at ionic
strengths corresponding to 85 and 120 mM phosphate buffer, pH
7.0, for the sheep and the human protein, respectively, while the apo
component was recovered at higher ionic strength. Alternatively,
selective elution of the holo- and apo- components was obtained by FPLC
(Pharmacia Biotech) on Mono-Q by a linear gradient from 0 mM NaCl at pH 7.5 to 500 mM NaCl at pH 8.5, in 20 mM triethanolamine buffer.
Samples of CP, both sheep and human, were depleted of copper
immediately after isolation from plasma. The reduction of the metal by
ascorbate and the rise of pH, up to 9, which occurred after
addition of CN
(see ``Experimental
Procedures'') were found to be necessary in order to completely
and rapidly labilize the metal binding sites. At neutral pH, several
days were required to remove most, but not all, of the copper, in
agreement with a recent report on rat CP (Terada et al.,
1995).
Upon metal release, sheep and human CP exhibited a different
electrophoretic behavior. The sheep apoprotein migrated with slower
mobility than the holo form on PAGE. In these conditions, apo and holo
forms of human CP had the same mobility, while in non-denaturing
SDS-PAGE, human holoCP ran as a doublet of bands with apparent M of 78,000 and 84,000, and the apoprotein
invariably ran as a 130-kDa band, as already reported (Sato and Gitlin,
1991).
A temporary increase of the absorption
at 610 nm was achieved by adding Cu(II)SO to apoCP (either
sheep or human) at neutral pH in 50 mM Mops, 150 mM KCl buffer in the presence of air (Fig. 1, trace
a). The recovery of the blue color took place within the mixing
time. However, the band at 610 nm diminished in intensity within a few
minutes (Fig. 1, trace b) and eventually converted into
a band centered at higher wavelength (Fig. 1, trace c).
The phenomenon could be observed at various Cu(II)/CP ratios and a
maximum effect (
15% of the expected A
)
was obtained at a ratio around 10. The derivative was completely
inactive versus paraphenylenediamine. Attempts to stop the
decay by rapidly chromatographing the apoCP-Cu(II) mixture on G-25 were
unsuccessful. Treatment with EDTA followed by G-25 resulted in the
complete loss of the optical features, thus suggesting that
Cu
could not correctly reinstate in the native copper
sites.
Figure 1:
Optical spectra of sheep
apoCP 1 min (a), 10 min (b), and 30 min (c)
after the addition of CuSO [10 Cu(II)/CP]. The
protein was 6
10
M in 50 mM Mops, 150 mM KCl, pH 7.0.
Cu(I)-thiourea was found to partially restore the spectroscopic properties of apoCP at neutral pH. However, although a spectroscopically stable adduct was obtained, the maximum yield, achieved by anaerobically incubating the protein with the complex for 45 min before reopening to air, was very poor (<15%), and even lower when the incubation was carried out directly in aerobiosis (data not shown).
The complex Cu(I)-GSH, when assayed at neutral pH in
different buffers, including phosphate, Mes, and Mops, failed to
restore the native spectroscopic properties of CP, which maintained the
electrophoretic mobility of the apo form. However, copper
stoichiometry, determined after prolonged, i.e. 3 h,
incubation of the protein with the complex and gel filtration on G-25,
revealed the presence of tightly bound metal atoms, 6
ions/protein. Of this, about 50% was EPR-detectable, with parameters
typical of type 2 copper sites. A chemical assay for reduced copper
revealed that the remaining copper atoms were associated to the protein
as Cu(I).
In the presence of Mg, a divalent cation
that binds to CP (Musci et al., 1995), a different result was
observed. Addition of Cu(I)-GSH to apoCP (6 Cu(I)/CP molecule) in 50
mM Mops, 150 mM KCl buffer (pH 7) containing 5 mM Mg
led to a progressive recovery of the
absorption at 610 nm, which reached a plateau after 4 h (Fig. 2). Sheep and human apoCP behaved similarly. Within the
same species, some variability was observed among different batches of
apoCP, with a recovery of the intensity at 610 nm ranging between 50%
and 70%. The sheep was slightly more efficient than the human protein.
Increasing the Cu(I)/CP ratio did not improve the reconstitution yield.
Electrophoretic analyses run, as mentioned before, with non-denaturing
SDS-PAGE for the human, and with PAGE for the sheep protein, indicated
the presence of only two components in the mixture at the various times
of incubation. As shown in the case of sheep CP (Fig. 3A), the intensity of the band with mobility
corresponding to the native protein grew up at the expense of the band
with R
typical of the apoprotein. Only the
component corresponding to the holo form, the intensity of which was
consistent with the recovered absorbance at 610 nm, stained positively
for oxidase activity (data not shown). The results clearly indicated
that a fraction of apoCP never regained the spectroscopic and
electrophoretic properties of holoCP, even after several hours of
incubation with the Cu(I)-GSH complex.
Figure 2:
Time course of the recovery of the
absorbance at 610 nm of 1.2 10
M sheep apoCP incubated at 25 °C with 7.2
10
M Cu(I)-GSH in 50 mM Mops, 150
mM KCl, containing 5 mM Mg
.
Figure 3: Electrophoretic analyses of apoCP reconstituted with Cu(I)-GSH. Panel A, PAGE of sheep apoCP incubated for 0 min (lane 1), 30 min (lane 2), 180 min (lane 3), and 270 min (lane 4) with Cu(I)-GSH under the experimental conditions reported in Fig. 2. Panel B, PAGE of sheep apoCP incubated for 270 min with Cu(I)-GSH and then chromatographed on DE52. Lanes 1 and 3 are the peak of the reconstituted holo fraction, stained for proteins and for oxidase activity, respectively; lanes 2 and 4 show the other peak. Panel C, denaturing (lanes 1 and 3) and non-denaturing (lanes 2 and 4) SDS-PAGE of human apoCP incubated for 270 min with Cu(I)-GSH and then chromatographed on DE52. Lanes 1 and 2 are from the peak of the reconstituted holo protein; lanes 3 and 4 are from the other peak.
The presence of
Mg was found not to improve the reconstitution yield
of apoCP by Cu(I)-thiourea or the stability of the protein treated with
CuSO
.
The EPR spectrum of the protein reconstituted with Cu(I)-GSH showed, even after gel filtration on G-25, the prevailing presence of resonances with parameters typical of type 2 copper, which obscured the signals due to type 1 copper (Fig. 4, trace a). Treatment with EDTA (at a final concentration of 50-100 mM), performed at the end of the incubation either by dialysis, or by direct addition of EDTA and passage on G-25, gave a protein with spectroscopic properties similar to those of native CP, although the EPR spectrum (Fig. 4, trace b) showed a content of type 2 copper still slightly higher than that expected.
Figure 4: X-band EPR spectra of sheep apoCP incubated for 180 min with Cu(I)-GSH under the experimental conditions of Fig. 2and then chromatographed on G-25 without (spectrum a) or with (spectrum b) treatment with EDTA. Experimental settings: microwave power, 20 milliwatts, modulation amplitude, 1 millitesla, temperature, 100 K.
Comparable results were obtained in samples incubated for 3 h with
Cu(I)-GSH in the absence or in the presence of air. The sample
incubated in anaerobiosis recovered the blue color within 10 min after
admission of air into the optical cuvette, a time consistent with
reoxidation of copper at the native sites (Calabrese et al.,
1989). Substitution of Ca for Mg
did not vary the extent of the recovery of the 610 nm absorption,
which proceeded, however, with noticeably slower kinetics.
In order
to evaluate the copper stoichiometry of the holo fraction, this was
separated from the mixture, at the end of the incubation, by
ion-exchange chromatography on DE52 or, alternatively, on Mono-Q by
FPLC (see ``Experimental Procedures''). With both methods,
and for both sheep and human CP, only two peaks were resolved. The peak
eluting at lower ionic strength contained a protein with a copper
content of 5 and
6 copper ions/CP for sheep and human CP,
respectively, and with spectroscopic properties (Fig. 5) and
catalytic parameters, K
and V
, indistinguishable from those of the
respective native CP. The electrophoretic behavior was also that of the
corresponding native protein (Fig. 3, panel B, lanes 1 and 3; panel C, lanes 1 and 2). The other peak was due to a protein that, although with
the electrophoretic mobility typical of apoCP (Fig. 3, panel
B, lane 2; panelC, lanes 3 and 4) and lacking any oxidase activity (Fig. 3B, lane 4), nevertheless contained
2 copper ions/molecule. These copper ions had no optical features
and were in the oxidized state, with an EPR spectrum typical of type 2
copper ions. Quantitative measurements carried out by HPLC techniques
(Reed et al., 1980) revealed that no glutathione, either
reduced or oxidized or as a mixed disulfide, had remained associated to
the fraction of holoCP or to the protein that had not recovered the
spectroscopic properties. Therefore, an irreversible modification of
apoCP by glutathione could not be invoked as the cause of the only
partial reconstitution of the protein.
Figure 5: Optical (panels A and C) and EPR (panels B and D) spectra of sheep (panels A and B) and human (panels C and D) CP after reconstitution with Cu(I)-GSH and separation on DE52. Dotted curves are the native holoproteins shown for comparison.
Figure 6:
Change of the optical absorbance at 610 nm
of sheep apoCP incubated with Cu(I)-GSH for 15 min and then
chromatographed on G-25. All steps were performed both in the presence (closed circles) and in the absence (open circles) of
Mg. See text for details.
To
sustain this hypothesis, the behavior of the copper sites during the
process was analyzed in deeper detail. Taking advantage of the ability
of EDTA to remove both Mg and copper loosely bound to
the protein (cf.Fig. 4), aliquots of the sample a` (Fig. 6) were treated with EDTA at different times after
the gel filtration on G-25 performed in the presence of
Mg
. The optical and the EPR spectra were measured
after an additional passage on G-25 equilibrated with Mops/KCl buffer
to remove free and EDTA-complexed metal ions. The optical spectra
showed that the absorbance at 330 nm was totally recovered in the
sample treated with EDTA 1 min after G-25, at variance with the
absorbance at 610 nm, suggesting that the copper atoms of the
trinuclear cluster were already oxidized at this stage. The EPR
spectra, shown in Fig. 7(panel A) for the samples
treated 1 and 150 min after G-25, had a completely different lineshape,
mostly due to type 2 copper at shorter times and to type 1 copper at
longer times. Fig. 8(upper panel) graphically reports
the content of paramagnetic, type 1, and type 2 copper atoms of the
samples quenched with EDTA at different times. Type 1 copper was
evaluated from the absorbance at 610 nm, while type 2 copper was
obtained by subtracting the contribution of type 1 copper to the
paramagnetic copper content of the samples. The amount of paramagnetic
copper remained essentially stable, suggesting that a modification of
type 2 centers into type 1 copper sites was at the base of the regain
of the spectroscopic properties. PAGE analysis of the aliquots revealed
again the presence of the two bands, the oxidase-inactive one, with
mobility corresponding to that of apoCP, prevailing at shorter times,
and the oxidase-active one, with the mobility of the holoprotein,
preponderant at longer times.
Figure 7:
Panel A, X-band EPR spectra of sheep
apoCP incubated with Cu(I)-GSH for 15 min, chromatographed on G-25 and
then incubated for 1 min (spectrum a) and 150 min (spectrum b) before treatment with EDTA. All steps were in 50
mM Mops, 150 mM KCl, pH 7.0, containing 5 mM Mg. Panel B, X-band EPR spectra of
sheep apoCP incubated with Cu(I)-GSH for 1 min, chromatographed on
G-25, and then incubated for 1 min (spectrum a) and 150 min (spectrum b) before treatment with EDTA. All steps were in 50
mM Mops, 150 mM KCl, pH 7.0, containing 5 mM Mg
and 10 mM ATP. Experimental settings
are described in Fig. 4.
Figure 8:
Content of paramagnetic (open
circles), type 1 (closed squares), and type 2 copper (open squares) of sheep apoCP incubated with Cu(I)-GSH for 15
min, chromatographed on G-25 and then treated with EDTA at different
times. All steps were in 50 mM Mops, 150 mM KCl, pH
7.0, containing 5 mM Mg. Upper
panel, in the absence of ATP; lower panel, in the
presence of 10 mM ATP.
Fig. 9shows the elution
profiles on Mono-Q of the samples quenched at 6, 22, and 47 min after
the gel filtration, compared to those of the apoprotein and the native
protein. Two peaks were obtained, and their relative contribution to
the profile progressively changed with time, with the peak matching
that of the holoprotein growing up at the expense of the second peak.
Measurement of the copper stoichiometry on the separated peaks showed
that the reconstituted holoprotein had a content of 5 copper
ions/CP at all times of incubation. The peak eluting at the position of
apoCP had instead a progressively decreasing copper content, from 1.8
to 0.7 copper ions/CP. The fact that the copper content of the second
peak was, at longer times, lower than that reported above (
2) is
not surprising, since these samples had been incubated with Cu(I)-GSH
for only 15 min.
Figure 9: Elution profiles on Mono-Q of the samples depicted in Fig. 8( upper panel), treated with EDTA 6 min (a), 22 min (b), and 47 min (c) after G-25. The peaks of the genuine holoCP and apoCP are shown by the dotted and the dotted broken lines, respectively.
Most of Mg is complexed in the
cell to a variety of ligands, including ATP. In order to investigate a
possible involvement of this nucleotide, sheep apoCP was incubated with
stoichiometric Cu(I)-GSH in the presence or in the absence of
Mg
and/or ATP. In this case the reaction was carried
out at 30 °C and allowed to proceed until no further changes of the
optical density at 610 nm were observed. Yields and half-times of
reconstitution were evaluated from the kinetic traces and are reported
in Table 1. ATP alone, at 10 mM, was able to facilitate
incorporation of copper into apoCP, although with an efficacy lower
than that exerted by 5 mM Mg
.
Mg
-ATP, on the other hand, was more efficient than
Mg
alone, the difference being mainly kinetic in
nature (t
= 15 min versus
35
min). These results were corroborated by binding studies, which
revealed that ATP could interact with both holoCP and apoCP, with
apparent K
values of 251 ± 9 µM and 318 ± 7 µM, respectively.
The
enhancement exerted by ATP did not reflect a different mechanism. The
experiments described in Fig. 6Fig. 7Fig. 8Fig. 9were repeated in
the presence of both Mg and ATP (5 and 10
mM, respectively). Also in this case, it was possible to
observe the progressive change of the electrophoretic and
chromatographic patterns concomitant to the spectroscopic conversion of
type 2 sites into type 1 copper species after removal of unreacted
Cu()I-GSH (Fig. 8, lower panel). The values of the
amount of type 2 and type 1 copper intersected at an earlier time in
the presence than in the absence of ATP. Consistent with the data of Table 1, a higher content of both type 1 and paramagnetic copper
was attained with the nucleotide in the incubation mixture (Fig. 8). These results confirmed that ATP could enhance the
yield of reconstitution and, in the presence of Mg
,
the kinetics of the process. As a matter of fact, the reconstitution
process could be followed in the presence of ATP and Mg
even when the protein had been incubated with Cu(I)-GSH for as
little as 1 min before chromatography on G-25. It is worth noting that,
under these conditions, the EPR spectrum taken at the shorter time was
almost completely devoid of resonances due to type 1 copper sites (Fig. 7, panel B). The effects exerted by ATP were
specific, as neither a different nucleotide triphosphate, GTP, nor ADP
had an appreciable effect on the reconstitution process (Table 1).
The results presented in this paper show that Cu(I)-GSH is
effective in transferring the metal to ceruloplasmin. A protein with
spectroscopic, enzymatic, and physicochemical properties
indistinguishable from those of the native protein has in fact been
obtained with good yields by incubating apoCP with stoichiometric
amounts of the complex. These results are at variance with those
obtained with a different Cu(I) complex, namely Cu(I)-thiourea, or with
a source of oxidized copper. The Cu(I)-thiourea complex has been used
in the past to reconstitute apoCP, due to its structural analogy with
the metal binding site of metallothioneins (Schechinger et
al., 1988). The experimental conditions were however quite
different, especially as the pH value (6) was concerned, and the
reconstituted protein, although regaining the oxidase activity, showed
a fairly high amount of EPR-detectable type 2 copper. When assayed at
neutral pH, the Cu(I)-thiourea complex has turned out to be rather
inefficient in transferring copper to apoCP, independent of the
presence of other effectors. On the other hand, apoCP could regain the
absorbance at 610 nm when incubated with CuSO
, but the
derivative turned out to be highly unstable, possibly because of
incorrect or missing filling of non-blue sites by Cu(II).
The
Cu(I)-GSH adduct appears the most suitable source of copper so far
investigated to re-establish the native structural and functional
properties of CP at neutral pH. Two different aspects should however be
pointed out: (i) there is a stringent requirement for the presence of
effectors like Mg and ATP, as will be better
discussed later on; (ii) even under these conditions, reconstitution
yields never reach 100%, leaving a fraction of apoCP only partially
saturated with copper and incapable to recover the correct metal
stoichiometry and spectroscopic properties. This latter phenomenon can
be reasonably explained on the basis of some denaturation induced by
the extreme conditions necessary for copper removal, in particular by
the high pH value attained during dialysis of the protein versus cyanide.
The data obtained with Cu(I)-GSH allow the outlining
of a mechanism for copper incorporation into apoCP. GSH is able to
transfer copper to the protein, but cannot promote the recovery of the
spectroscopic properties unless a divalent cation like Mg and/or ATP is present. ApoCP reconstitutes in two distinct steps.
In a first phase, the metal binds to the protein moiety and reoxidizes,
possibly at the right sites, but with incorrect geometries, reflected
in spectroscopic properties different from those of native CP. The
protein recovers the correct optical and EPR features in a second
phase. During this phase, which strictly depends on the presence of a
divalent cation (Mg
or Ca
) or ATP,
the chromophores of CP behave quite differently. The 330 nm absorption
band readily regains its native shape and intensity, suggesting that
the trinuclear cluster promptly recovers its structure. The blue
absorption and the native EPR lineshape of the type 1 copper sites, on
the other hand, are recovered slowly, in a process not involving a
redox phenomenon, as it occurs also in the absence of oxygen.
Therefore, it is likely that the role of Mg
and of
the nucleotide is to induce some conformational rearrangements which
affect the protein organization. Such a structural change is in fact
not confined to the ligands of the blue sites, as it also produces the
abrupt change, in an all-or-none fashion, of the electrophoretic and
chromatographic behavior of the protein.
Cu(I)-GSH has been employed here for the first time with CP. This tripeptide is the most abundant non-protein thiol in mammalian cells, and has been shown to be able to chelate and detoxify metals soon after they enter the cell (Fukino et al., 1986; Andrews et al., 1987; Singhal et al., 1987; Kang and Enger, 1988). GSH can form very stable complexes with Cu(I), and the Cu(I)-GSH complex has been implicated in the incorporation of Cu(I) into metallothionein (Freedman et al., 1989) and phytochelatins (Mehra and Mulchandani, 1995), as well as in copper donation to both intra- and extracellular proteins like Cu,Zn-superoxide dismutase (Ciriolo et al., 1990) and hemocyanin (Brouwer and Brouwer-Hoexum, 1992). Glutathione plays a crucial role in the ER, where the GSH/GSSG couple constitutes the principal redox buffer and has been implicated in the correct folding of nascent proteins (Hwang et al., 1992). Therefore, a role for glutathione in the metal traffic control within this compartment, as in the cytosol, is not unlike.
It is now generally accepted that CP incorporates copper early during biosynthesis of the polypeptide chain, although the exact subcellular localization of the process is not clear, i.e. whether it takes place in the ER or in the Golgi (Sato and Gitlin, 1991; Terada et al., 1995). Our finding that Cu(I)-GSH reconstitutes apoCP at neutral pH is consistent with the ER being the site of copper incorporation (Mellman et al., 1986). The genetic studies carried out on patients with Wilson disease, a metabolic disorders of copper, have allowed to identify an ATPase as the copper pump involved in copper incorporation into nascent apoCP (Yamaguchi et al., 1992; Bull et al., 1993; Tanzi et al., 1993). However, also in this case the subcellular localization remains to be clarified. It can not be excluded that, in vivo, copper incorporation into CP is directly mediated by this membrane-bound ATPase (Bull et al., 1993; Tanzi et al., 1993). This mechanism would imply that the pump can specifically interact with the many different copper-dependent proteins to be processed within the ER, including secretory proteins other than CP (extracellular superoxide dismutase and lysyl oxidase) or membrane proteins like yeast Fet3p (Yuan et al., 1995). It is easier to figure out that a soluble molecule shuttles the metal from the pump to the target. Glutathione is, in this respect, a likely candidate, since, as stated above, its presence in the ER is well documented (Hwang et al., 1992; Young et al., 1993).
A role for divalent cations and for ATP in the ER is well
established, and the effects exerted by these molecules in vitro on apoCP may therefore have a physiological relevance. All these
species exert a common effect, they are able to stimulate recombined CP
to establish the proper spatial relationships at the blue copper sites.
Both Ca and Mg
have been recently
demonstrated to bind to CP, with affinities in the millimolar range
(Musci et al., 1995). Calcium is actively stored in the ER,
where it can reach millimolar levels being bound to specific ER
proteins (Sambrook, 1990). However, Ca
showed to be
less efficient with apoCP than Mg
, which is at
millimolar concentrations in the ER (Gunther, 1990). ATP is required by
different ER systems including chaperones (Hendrick and Hartl, 1993),
and it is involved in a number of crucial phenomena, including
translocation of proteins to the cis-Golgi (Beckers et
al., 1987, 1990). It also binds to CP, with a slightly higher
affinity for the apo form. However, our data do not allow to
unequivocally assess that the Mg
-ATP complex is the
active species, although the observation that the maximum effect was
observed with both Mg
and ATP, with respect to
Mg
or ATP alone, strongly suggests a role for the
complex in assisting the protein during the rearrangement taking place
at the metal sites.
Finally, our results demonstrate that neither the electrophoretic nor the chromatographic mobility are diagnostic for apoCP, and that the copper-depleted status needs to be assessed by direct measurement of the metal stoichiometry and/or by spectroscopic techniques. This is particularly relevant when we consider that several studies on the pathway of copper incorporation into apoCP within the cell rely on the different electrophoretic mobility of apo and holoCP (Sato and Gitlin, 1991; Terada et al., 1995). Rat CP, when analyzed in the secretory compartments, displays the holo electrophoretic mobility in the cis-Golgi fraction, but not in the ER fraction (Terada et al., 1995). Kinetic studies indicate, however, that incorporation of copper into CP takes place in the rough ER immediately following translation (Sato and Gitlin, 1991). Assuming that the events taking place in vivo during the biosynthesis of CP are those of the multistep mechanism that controls the reconstitution of the protein in vitro, one could speculate that CP takes up copper in the ER and assumes the holo conformation by rearranging the copper sites upon translocation to the cis-Golgi.