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INTRODUCTION |
Histidine-proline-rich glycoprotein
(HPRG)1 is an approximately
70-kDa glycoprotein that is present at a relatively high concentration in the plasma of vertebrates. Although the physiological role of this
protein remains unclear, it has been implicated in a number of
processes, including blood coagulation and fibrinolysis, immune response, and transport of metal ions (1). The cellular origin of the
mouse protein has recently been defined by Northern blot analysis
showing that the HPRG mRNA is localized specifically to the liver,
suggesting that the previously described HPRG expression by immune
cells is due to the acquisition of the plasma protein derived from the
liver (2). In a previous paper we reported that denaturation of rabbit
skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) in
acidic medium allows the chromatographic separation from the enzyme of
a peptide with an amino acid composition significantly different from
that derived from the available AMP deaminase cDNAs. N-terminal
sequence analysis of the fragments liberated by limited proteolysis
revealed a striking similarity of the novel protein to rabbit plasma
HPRG although, in comparison with mature HPRG, the AMP
deaminase-associated variant probably contains a unique N-terminal
extension (3). We now report that the AMP deaminase-associated variant
of HPRG can be isolated from rabbit skeletal muscle by a modification
of the protocol used in our laboratory for the purification of AMP
deaminase. The modification allows the partial dissociation of HPRG at
a high degree of purity from the cellulose phosphate-bound enzyme.
Rabbit plasma HPRG contains 53 histidine residues, of which 34 are
located in the histidine-proline-rich domain containing 15 repeats of
the sequence (H/P)(H/P)PHG that has been proposed to mediate
interactions with transition metals, although no evidence of a specific
binding has been given (4). The HPRG component of rabbit skeletal
muscle AMP deaminase contains 10 mol of histidine residues/10,000 g of
protein (3). The abundance of such potential metal ligands suggests
that HPRG has the ability and perhaps the function to bind several
metal ions, and it has been established (5, 6) that HPRG from rabbit
serum binds Hg2+, Cu2+, Zn2+,
Ni2+, Cd2+, and Co2+ in descending
order of binding affinity. However, no attempts to characterize the
structure of the metal-binding site(s) of HPRG have been performed.
This prompted us to an investigation by x-ray absorption spectroscopy
of the zinc-binding sites of the HPRG variant that we have isolated
from rabbit skeletal muscle as a first step toward determining the
physicochemical properties of this novel protein.
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EXPERIMENTAL PROCEDURES |
Reagents--
Chelating Fast Flow Sepharose was from Amersham
Biosciences. Phosphocellulose resin (P-11) was supplied by Whatman
International Ltd., Maidstone, UK). All of the chemicals and other
reagents used were of analytical grade.
AMP Deaminase and Its HPRG Component--
AMP deaminase was
prepared as described previously (7) from fresh muscle dissected from
the back and hind leg of rabbits. Because rabbit skeletal muscle AMP
deaminase undergoes progressive fragmentation with storage,
homogenization of the muscle and phosphocellulose purification of the
enzyme was carried out at each step using a buffer system containing 5 mM NaN3 to reduce the rate of proteolytic processes. In the presence of azide, the 85-70-kDa band transition of
the purified enzyme occurred with a half-time of one month, significantly slower than that previously described (half-time of 2 weeks) (8). Following the identification (described in the present
paper) of the peptide that elutes at 0.6 M KCl from cellulose phosphate-bound AMP deaminase with the HPRG component of the
enzyme, the fractions that formed the peak eluted with 5 mM
NaN3, 0.6 M KCl, pH 7.0, were concentrated by
ultrafiltration with an Amicon Microcon YM-30 centrifugal filter device
(Millipore) and stored in aliquots at
20 °C. An 1 mM
HPRG sample was prepared for XAS by further concentration of the pooled
fractions. The protein concentrations of the various enzyme fractions
were determined spectrophotometrically by using
A2801%,1 cm values of 9.1 and 8.2, respectively, for AMP deaminase and its isolated HPRG component, which
were calculated on the basis of protein determinations by the method
described in Ref. 9 using bovine serum albumin as standard.
The isolated rabbit skeletal muscle variant of HPRG was assumed to have
the same molecular mass of 58 kDa calculated for the rabbit plasma
protein (4). The AMP deaminase activity was determined spectrophotometrically as previously described using a Shimadzu UV-260
spectrophotometer (10).
SDS/PAGE--
Electrophoresis in the presence of
0.1% (w/v) SDS was carried out under reducing conditions on 10% (w/v)
polyacrylamide slab gels in 0.1 M Tris, 0.1 M
Bicine, pH 8.3. Protein standards (Sigma) were used to determine
molecular weights.
Electroblotting and Sequence Analysis--
Electroblotting was
performed by the method of LeGendre and Matsudaira (11) using 10 mM CAPS, pH 11, containing 10% (v/v) methanol. Transfers
were performed for 60-90 min at 400 mA. N-terminal sequencing was
performed using an Applied Biosystems model 476A protein sequencer.
UV Spectra--
The interaction of rabbit muscle HPRG with
metals was assessed by changes in absorption. Absorption spectra were
recorded by using a Shimadzu UV-260 spectrophotometer at room
temperature. Spectral measurements were made within 1 min of mixing
protein with metal, and no absorbance changes after this time were noted.
Equal concentrations of metal in the reference cuvette served as
reference solutions. The fractional saturation of HPRG,
, is defined
as the ratio of the observed increase in absorbance (A) at
275 nm to the maximum
A at saturation of the protein.
XAS Sample Preparation--
Two equivalents of Zn2+
from a ZnSO4 solution in ultrapure water were added to 1.0 mM rabbit muscle HPRG solution (prepared as described
above) to obtain 50 µl of Zn-HPRG 2:1 complex. 45 µl of the above
solution were filled into a plastic cell covered with Kapton windows.
Both the cells and the Kapton foils used for the windows were
thoroughly washed with ultrapure water and absolute ethanol and then
dried before use. The sample cell was mounted in a two-stage Displex
cryostat (modified Oxford instruments) and kept at 20 K during the
data collection.
XAS Data Collection and Analysis--
The XAS data were
collected at Deutsches Elektronen Synchrotron (DESY) (Hamburg,
Germany) at the EMBL bending magnet beam line D2 using Si (111) double
monochromator for the measurement at the zinc edge. During the
measurements the DESY storage ring was operating under normal
conditions (4.5 GeV, 90-140 mA). Ionization chambers in front and
behind the sample were used to monitor the beam intensity. The XAS data
have been recorded by measuring the zinc-K
fluorescence using
a Canberra 13-element solid state detector over the energy range
between 9324 and 10624 eV using variable energy step widths. In the
x-ray absorption near-edge structure and the extended x-ray absorption
fine structure (EXAFS) regions, steps of 0.3 and 0.5-1.2 eV were used,
respectively. An absolute energy calibration of the spectra was
obtained by recording known Bragg reflections of a Si (220) crystal in
back reflection geometry following a reported procedure (12)
(E0,Zn = 9663.5 eV). 25 scans for a total of
more than 1.5 million counts/experimental point were averaged to obtain
good signal/noise statistics. The pre-edge background removal has been
performed by a linear fit, whereas the removal of the atomic background
above the edge and the EXAFS extraction has been performed by fitting a
cubic spline using the EXPROG program package (13).
The full k3 weighted EXAFS spectrum (23-750 eV
above E0) and its Fourier transform (FT)
calculated over the range 3.0-14.0 Å
1 have been
compared with theoretical simulations obtained by the set of programs
EXCURVE9.20 (14). The edge energy E0 was
adjusted at the beginning of the refinement to bring the experiments
and the simulations on the same scale and left unchanged during the fitting. A fixed amplitude factor of 0.95 was used to compensate for
amplitude reduction of the signal caused by multiple excitations. The
k3 weighted full spectrum was simulated by
varying the atom types and the coordination numbers (as integers) and
iteratively refining the distance (r) and the Debye-Waller
factor (2
2) for each atomic shell. Multiple scattering
contributions were included for histidine imidazole ligands. A
zinc-bound imidazole ring was generated by molecular modeling with a
zinc-N(His) distance obtained from the EXAFS first shell
analysis. The coordinates from the model were input to EXCURVE9.20 to
provide a single unit with the correct geometry. A single distance fit
was able to reproduce the closer atomic shell, allowing us to minimize
the number of parameters in the refinement procedure by simulating the
zinc histidine ligands by a single zinc-bound imidazole ring with a fixed coordination number. In this way the imidazole outer shell distances were defined by the zinc-N(His) distance, and the imidazole outer atoms were constrained to vary within 0.1 Å from the idealized positions. All of the histidine numbers between 1 and 4 were tried with
3, giving the correct fit. The imidazole ring plane was kept coincident
with the zinc-nitrogen bond throughout the refinement.
The quality of the fit obtained was assessed by the following
goodness-of-fit function,
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(Eq. 1)
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where Nind is the number of independent
data points (Nind = (2
k
r)/
),
p is the number of parameters, N is the number of data points, and w is the weight of the spectrum. The
quality of the fit obtained was also assessed by the R-factor as
defined within EXCURVE9.20 as follows.
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(Eq. 2)
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RESULTS AND DISCUSSION |
Selective Purification of the AMP Deaminase-associated HPRG by
Cellulose Phosphate Ion Exchange Chromatography--
A rapid method
for the preparation of AMP deaminase from frozen rabbit skeletal muscle
was introduced on the basis of the observation that the enzyme remained
bound to cellulose phosphate under conditions (0.45 M KCl,
pH 7.0) at which apparently no other proteins are bound (15). Thus,
elution with 1.0 M KCl, pH 7.0, yielded a homogeneous
preparation of the enzyme at a high degree of purity. However, by
eluting cellulose phosphate column with a linear gradient from 0.45 to
1.0 M KCl, sometimes a small protein peak that was not
examined was found by the same authors to precede the main activity
peak (16). The constant presence of presumably the same additional
peptide was observed in the enzyme prepared in our laboratory from
fresh rabbit muscle. Therefore, we introduced a modification in the
cellulose phosphate chromatography (i.e. the enzyme was
eluted with 1.0 M KCl after the column had been washed with
0.6 M KCl) that effectively separated the contaminant peptide from the purified enzyme (7). We have now found that the HPRG
component of rabbit skeletal muscle AMP deaminase is dissociated from
the cellulose phosphate-bound enzyme at a high degree of purity by
elution with 0.6 M KCl, indicating its correspondence to
the peptide previously discarded as a contaminant of the enzyme preparation.
Fig. 1 shows the elution profile of
cellulose phosphate-bound AMP deaminase obtained by two successive
washing steps with 0.6 and 1.0 M KCl, after having washed
the column with 0.45 M KCl. In five separate enzyme
preparations the yield of the protein eluted with 0.6 M KCl
(peak 1) and with 1.0 M KCl (peak 2) was 13 ± 4 and 73 ± 11 mg/kg of rabbit skeletal muscle, respectively. The protein in peak 1 was not completely devoid of AMP deaminase activity, but its specific
activity (0.5 units/mg of protein) (1 unit = 1 µmol of AMP
deaminated per min when assayed in 50 mM imidazole HCl, pH
6.5, 100 mM KCl, 0.1 mM AMP at 20 °C) was negligible in comparison with that of the protein in peak 2 (210 units/mg of protein). Analysis by SDS/PAGE of the two freshly pooled
peaks (Fig. 2, A and
B, lane 1) showed that both gave rise to a main
band of approximately 85 kDa, but an additional faint 95-kDa band that
was almost completely proteolyzed in the first week of storage at
4 °C was present in the electrophoretogram of pool 2 (Fig.
2B, lanes 1 and 2).

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Fig. 1.
Fractionation of AMP deaminase components by
stepwise elution of the cellulose phosphate-bound enzyme. AMP
deaminase was prepared by following the protocol described in Ref. 15
up to the washing of cellulose phosphate-bound AMP deaminase with 0.45 M KCl adjusted to pH 7.0 with 1 M
K2HPO4. When no more protein was detectable in
the wash, the cellulose phosphate was transferred to a column (2 × 25 cm) and allowed to settle in 0.45 M KCl, pH 7.0. The
enzyme was eluted by two successive steps with 100 ml of 0.6 M KCl, pH 7.0, and 100 ml of 1.0 M KCl, pH 7.0. Fractions of 5 ml were collected, and A280
(solid line) and A260 (dashed
line) were measured. The horizontal bars indicate the
fractions that were pooled.
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Fig. 2.
Alteration of the SDS/PAGE behavior with time
of storage at 4 °C of whole rabbit skeletal muscle AMP deaminase and
its HPRG component separated by ion exchange chromatography.
A, 3-µg samples of pool 1 (Fig. 1) stored at 4 °C in 5 mM NaN3, 0.6 M KCl, pH 7.0. B,
4-µg samples of pool 2 (Fig. 1) stored at 4 °C in 5 mM
NaN3, 1.0 M KCl, pH 7.0. C, 4-µg samples of
AMP deaminase prepared as described in Ref. 15 and stored at 4 °C in
5 mM NaN3, 1.0 M KCl, pH 7.0. Samples of
freshly prepared proteins (lanes 1), proteins stored for 5 days (lanes 2), 12 days (lanes 3), 21 days
(lanes 4), or 27 days (lanes 5), or freshly
prepared protein incubated with 0.1% -mercaptoethanol for 1 h
at room temperature (A, lane 6) were denatured
with 0.1% SDS and run on 10% (w/v) polyacrylamide slab gel.
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With aging of pool 2, a 85-70-kDa band transition also occurred with a
half-time of 1 month (Fig. 2B, lanes 2-5),
confirming our previous demonstration that limited proteolysis of
rabbit skeletal muscle AMP deaminase, removing the 95-residue-long N terminus of the enzyme, converts the native 85-kDa subunit to an
approximately 70-kDa core that is resistant to further proteolysis (17). In contrast, the 85-kDa band of pool 1 was almost completely transformed in the first week of storage, giving rise to a 95-kDa band
that was resistant to proteolysis, even if the enzyme was stored at
4 °C for several months (Fig. 2A, lanes 2-5).
By electroblotting and sequencing analysis, the 85-kDa original band of
each pool yielded no N-terminal sequences, confirming the previous
suggestion that the N terminus of rabbit skeletal muscle AMP deaminase
is modified (3). In contrast, the 95-kDa bands revealed the single sequence LTPTDXKTTKPL corresponding to the N-terminal
sequence of rabbit plasma HPRG. The yield of the sequence increased
after storage for few days at 4 °C, indicating that the native
protein has a blocked N terminus and that it undergoes a proteolytic
process starting with its isolation. It should be noted that rabbit
plasma HPRG migrates in SDS/PAGE with an apparent molecular mass of 90 or 94 kDa (4, 18, 19) higher than that deduced from its sequence (58 kDa) or that calculated taking account of its 17.5% carbohydrate
content (70 kDa) (4). Interestingly, a 1-h incubation of a sample of
freshly prepared pool 1 with 0.1%
-mercaptoethanol at room
temperature before analysis by SDS/PAGE caused the same 85-95 kDa
transition that was observed on storage (Fig. 2A, lane 6). It seems likely, on the basis of this observation, that the shift in the migration on SDS/PAGE is due to a change in conformation consequent to the reduction of a disulfide bond present in freshly isolated HPRG. The presence in the HPRG component of AMP deaminase of a
disulfide bridge homologous to that supposed to connect Cys-6 and
Cys-497 in rabbit plasma HPRG (4) was inferred on the basis of
electroblotting and sequencing analysis of the SDS/PAGE bands corresponding to presumably disulfide-linked fragments liberated by
trypsin cleavage (3). It was also observed that the reduction of that
disulfide bridge in rabbit plasma HPRG required prior denaturation of
the protein and that even after reduction, the separation of the N- and
C-terminal domains of the protein by ion exchange chromatography was
difficult, because of the hydrophobicity of the contact area (4).
To ascertain whether the catalytic subunit of AMP deaminase was present
only in traces in peak 1 in Fig. 1, as indicated by the
determination of the enzyme activity, we applied a sample of pool 1, denatured by overnight dialysis against 0.5 M NaCl, 20 mM sodium phosphate buffer, pH 7.0, containing 0.1%
-mercaptoethanol and 3 M urea, to a metal affinity
column (Chelating Fast Flow Sepharose; Amersham Biosciences) charged
with Zn2+ and equilibrated with the dialysis buffer
(results not shown). We have previously used zinc affinity
chromatography to separate the 95-kDa HPRG component from the 85- and
70-kDa components present in whole AMP deaminase because the 85- and
70-kDa species are not retained by this resin and eluted in the void
volume (20). In contrast, all of the protein present in the
urea-denatured sample of pool 1 was retained by the resin and was
eluted only when the resin was washed with the EDTA containing buffer,
which strips the metal ions from the gel. Analysis by SDS/PAGE of the eluted fractions revealed identity of migration with the 95-kDa peptide
used as starting material. Electroblotting and sequencing of the
EDTA-eluted peptide revealed the sequence
LTPTDXKTTKPLAEKALDLI, corresponding to the rabbit plasma
HPRG N-terminal sequence (4).
The presence of HPRG as an apparently single component in peak 1 (Fig.
1) indicates that 0.6 M KCl selectively elutes the HPRG
component from the AMP deaminase complex adsorbed to cellulose phosphate. However, a significant amount of HPRG is still present as
the 85-kDa component in the 1 M KCl-eluted enzyme because
the same N-terminal sequence (LTPTDXK) shown by the 95-kDa
band was obtained by electroblotting and sequencing of the 70-kDa band; furthermore its yield increased with time of storage of the enzyme at
4 °C. A plausible interpretation of this data is that the 70-kDa band contains a C-terminally truncated version of HPRG. This is in
agreement with the analysis of the peptides liberated by limited proteolysis of plasma HPRG and of the HPRG component of AMP deaminase showing that both proteins behave as approximately 70-kDa fragments when they are split inside the disulfide bridge connecting the N-terminal domain to the C-terminal domain of the molecule (3, 4). On
this basis, we may reasonably assume that on SDS/PAGE of the 1 M KCl-eluted enzyme under reducing conditions, the HPRG component of AMP deaminase and the catalytic subunit both migrate as
85-kDa species, this observation being probably because of an
interaction between the two proteins that further reduces the approachability of
-mercaptoethanol to that disulfide bridge.
To establish the extent of the diminution of HPRG content occurring in
AMP deaminase as a consequence of the introduction of the washing step
with 0.6 M KCl in the phosphocellulose chromatography before the elution of the enzyme with 1.0 M KCl, we
compared the yield of the protein obtained from the skeletal muscle of
the same rabbit by following either that protocol or the direct elution with 1.0 M KCl after the washing step with 0.45 M KCl. The total protein yield in terms of mass was about
the same with the two methods, because the protein eluted successively
with 0.6 and 1.0 M KCl accounted for about 15 and 85%,
respectively, of the total protein eluted with 1.0 M KCl as
a single step (Table I). Taking into
account the 85-kDa molecular mass of the catalytic subunit of AMP
deaminase and assuming for the AMP deaminase associated HPRG the same
molecular weight of 58-kDa calculated for the rabbit plasma protein
(4), these data are consistent with an approximate 30% diminution of
the HPRG content of the phosphocellulose-bound enzyme as consequence of
the washing step with 0.6 M KCl.
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Table I
Purification of rabbit skeletal muscle AMP deaminase: elution of the
enzyme held by cellulose phosphate after the washing step with 0.45 M KCl
The results are the average values of the determinations carried out on
two different AMP deaminase preparations. In each, the three protein
fractions were obtained by following the two different elution
procedures of the cellulose phosphate-adsorbed enzyme prepared from the
muscle of the same rabbit.
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The enzyme obtained with the one-step elution with 1.0 M
KCl showed a 20% lower specific activity and a lower
A280:A260 ratio, indicating a possible contamination by nucleic acids or nucleotides. By
following the procedure described under "Experimental Procedures" for the determination of the
A2801%,1 cm, the high value of
12.6 was calculated for this enzyme, in comparison with that of 9.1 obtained for the enzyme purified adopting the 0.6 M KCl
washing step. Altogether, these data indicate that elution with 0.6 M KCl of the AMP deaminase adsorbed to cellulose phosphate probably removes from the resin a protein-protein complex with an
extremely high HPRG/AMP deaminase molar ratio, thereby
increasing the specific activity of the enzyme isolated in the
successive elution of the column with 1.0 M KCl. It should
be noted that AMP deaminase prepared as described in Ref. 15 from
frozen rabbit skeletal muscle showed an
A280:A260 ratio of 1.8 and an A2801%,1 cm value of 9.1 (21). Analysis by sedimentation-equilibrium techniques revealed that
that enzyme had a molecular weight of 278,000 (21), somewhat lower than
that calculated for a molecular aggregate of four identical 85-kDa
subunits. This observation was previously explained with the finding
that freezing of the muscle causes the same 85-70 kDa transition
observed with aging of the purified enzyme (8). In contrast, our
determination by sedimentation-equilibrium analysis of the molecular
mass of freshly prepared rabbit skeletal muscle AMP deaminase in 1 M KCl, pH 7.0, indicated the presence of two species of 173 and 309 kDa, which were interpreted as being consistent with the
existence of a dimer-tetramer equilibrium (22). In the light of the
data of the present paper, the heterogeneity observed in
sedimentation-equilibrium centrifugation of the native enzyme should be
interpreted as being due to the presence of HPRG/AMP deaminase
protein-protein complexes with different molar ratio, the observed
309-kDa molecular mass determined for the heavier component being in
agreement with a model for AMP deaminase quaternary structure in which
two 85-kDa catalytic subunits assemble with two approximately 70-kDa
HPRG subunits (assuming a carbohydrate content similar to that of the
plasma protein).
As far as the effect of the diminution of the HPRG content in the
preparation of AMP deaminase on the properties of the enzyme is
concerned, comparison of the results obtained with the enzymes prepared
by following the two different protocols has not given any evidence of
clear differences in the kinetics (results not shown) or in the
behavior on SDS/PAGE. However, the HPRG-enriched enzyme showed an
apparent reduction in the rate of the proteolytic phenomena with
storage; it is evident from Fig. 2C (lanes 1-5) that both the disappearance of the 95-kDa band and the 85-70-kDa band
transition are significantly slower for the HPRG-enriched enzyme. Our
sequence data show that the HPRG variant present in the AMP deaminase
preparation shares with the plasma protein an almost totally conserved
cystatin domain at the N terminus (23), diverging only at one amino
acid residue among the 46 sequenced up to
now2; therefore, we suggest a
protective role for the HPRG component against the protease-induced
fragmentation of AMP deaminase in addition to its recently suggested
action in assuring the molecular integrity of the enzyme (20).
It has been established (5, 6) that HPRG from rabbit serum binds
Hg2+, Cu2+, Zn2+, Ni2+,
Cd2+, and Co2+ in descending order of binding
affinity. To obtain a preliminary indication of specific
versus nonspecific binding of zinc to the muscle specific
form of HPRG, the interaction of the protein with metals was assessed
directly by monitoring changes in UV absorption. When compared with
HPRG alone, the protein in the presence of increasing equivalents of
Zn2+, Cu2+, or Ni2+ showed an
increase in absorption around 275 nm (Fig.
3), a feature consistent with the
interaction altering the environment of tyrosine and phenylalanine
residues. The change in absorbance at 275 nm produced by each of the
three metals showed a maximum when the HPRG samples contained near 40 equivalents of metal ions. Further additions of each metal up to 50 metal ions/HPRG molecule caused only minor changes in the spectra of
HPRG that were almost superimposable to those obtained at 40 metal
ions/protein. At metal/HPRG ratios higher than 50, different
perturbation of the ultraviolet absorption spectrum of HPRG was
observed with a decrease rather than an increase of the absorbance at
275 nm. These data may be interpreted as being due to the existence of
competing effects (high affinity binding causing an increase in
absorbance and low affinity binding causing a decrease in absorbance).
However, because an almost constant value of the maximum
A275 was determined in the range 40-50 metal
ions/protein before the second perturbation became evident, we adopted
that value as representing the apparent saturation of the sites with
higher affinity.

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Fig. 3.
Perturbations produced by Zn2+ in
the ultraviolet absorption spectrum of the HPRG component of rabbit
skeletal muscle AMP deaminase and titrations of metal-HPRG
interactions. Shown are the spectra of HPRG alone (dashed
line), or in the presence of increasing (15-80 µM)
concentrations of ZnCl2 (solid lines), or in the
presence of 100 µM ZnCl2 (dotted
line). The inset shows the fractional saturation ( )
of HPRG with increasing amounts of ZnCl2 ( ),
NiCl2 ( ), or CuCl2 ( ). In each case, the
concentration of HPRG in 0.2 M KCl, 5 mM
Na2HPO4, pH 8.0, was 1.6 µM, and
the increase in absorbance at 275 nm was used to determine .
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Titration of the interaction of HPRG with Zn2+ or
Ni2+ shows a sigmoidal relationship (Hill coefficient,
h = 4.0 and 3.5, respectively), indicating that these
metals are bound in a cooperative manner with interactions among
various sites (Fig. 3). Cu2+ binds to HPRG with the same
apparent stoichiometry observed with Zn2+ and
Ni2+ but shows a minor positive cooperativity of binding
(h = 1.5).
Our results are in partial agreement with the previously reported
effects of metals on the fluorescence of rabbit plasma HPRG (5),
showing that HPRG interacts with various divalent metal ions with an
apparent stoichiometry of 10 (Cu2+, Co2+,
Ni2+, and Hg2+) to 20 (Zn2+ and
Cd2+) metal ions bound per HPRG molecule. The existence of
some nonoverlapping sites was suggested by the sigmoidal binding of
Zn2+ and Cd2+ (h = 4.4 and 2.8, respectively), whereas the other metals were bound in a noncooperative
manner. Moreover, only Zn2+ and Cd2+ caused an
enhancement rather than a quenching of fluorescence (5). Altogether,
these results were interpreted as an indication that all of the metal
ions probably share the same set of binding sites, although the
interaction of each metal with the protein may vary because of the
involvement of a different group of protein ligands in a different way.
This interpretation, which can be extended to the data of protein-metal
interactions we have obtained with the muscle-specific form of HPRG,
permits one to conclude that most of such a large number of
metal-binding sites are nonspecific and that most of the zinc is simply
adventitiously bound. Therefore, to minimize the effect of a
distribution of zinc-binding sites and to enhance the chance of
characterizing the structure of a specific binding site, our
investigation by XAS of the zinc binding behavior of HPRG was carried
out with a sample obtained by adding only 2 equivalents of zinc to the
protein. The usefulness of this approach was confirmed by the
inspection of XAS data obtained with HPRG samples containing 8 or 15 equivalents of zinc (results not shown). In these spectra, different
EXAFS patterns were superimposed on the spectrum reported in the
present paper, preventing a thorough analysis.
X-ray Absorption Spectroscopy--
The full-length cDNA
sequence of plasma human and murine HPRG and a partial cDNA
sequence of the rabbit protein have been reported (2, 4, 24). Alignment
of the predicted amino acid sequences indicates that all HPRG species
share an overall domain structure comprising an N-terminal domain
containing two cystatin-like repeats, a histidine-proline-rich domain,
and a C-terminal domain. Alignment of our partial amino acid sequence data of the rabbit skeletal muscle AMP deaminase-associated variant of
HPRG with the predicted sequence of rabbit plasma HPRG indicates that
the rabbit muscle and plasma forms of HPRG share high identity, diverging only at 5 amino acid residues among the 78 sequenced (93.6%
identity) that span all the three proposed overall domains of
HPRG.2
The 34 histidine residues located in the histidine-proline-rich domain
of rabbit plasma HPRG have been shown to mediate interactions with
transition metals, although no evidence has been given of the existence
of any specific metal-binding site (4). Because the HPRG component of
rabbit skeletal muscle AMP deaminase contains 10 mol of histidine
residues/10,000 g of protein (3), we exploited metal affinity
chromatography in an attempt to separate the novel component of the
enzyme from the catalytic subunit. The isolation of the HPRG
component from the purified enzyme under denaturing conditions was achieved by zinc affinity chromatography (20). The
observation of the present paper that under nondenaturing conditions
the HPRG component can be partially dissociated at a high degree of
purity from cellulose phosphate-bound AMP deaminase by an one-step
elution with 0.6 M KCl prompted us to utilize this procedure to obtain an HPRG sample suitable for investigation by XAS of
the zinc-binding behavior of the protein.
The zinc-K-edge region of the sample is reported in Fig.
4, whereas the EXAFS spectrum and the FT
are reported in Fig. 5 (panels a and b, respectively). The presence of the
characteristic camelback features in the EXAFS spectrum denotes
histidine binding to zinc. In addition, the EXAFS shows beat nodes at
about 9.0 and 10.5 Å
1 that evidence destructive
interference of the signal caused by the existence of different
backscatterer distances in the zinc coordination. These features render
the simulation of the spectrum quite difficult, and several different
models of the zinc coordination in HPRG were attempted before a
satisfactory fit was obtained. Table II
reports the structural parameters relative to the zinc coordination in
the HPRG protein obtained from the best fit to the spectrum.

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Fig. 5.
a, EXAFS spectrum (solid
line) of the zinc-HPRG 2:1 complex; b, Fourier
transform (solid line) superimposed to the best fit
(open circles) obtained with the parameters reported in
Table II. The phase of the FT is calculated from the first shell atom
backscattering factors.
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Table II
Zinc-HPRG 2:1 complex and fitting results of the whole k3
weighted EXAFS spectrum
2 (k3 weight) was 0.27. The R-factor was
35.4 (see "Experimental Procedures"). The bond valence sum was
2.23-2.52 (calculated as in Ref. 31 without and with
the O atom contribution, respectively).
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The presence of different bond lengths in the zinc coordination
polyhedron is evident from the EXAFS Fourier transform (Fig. 5b) where the main feature shows two closely spaced maxima
corresponding to zinc ligand distances of about 2.0 and 2.3 Å.
Although the 2.0 Å distance is typical of oxygen/nitrogen donors, the
2.3 Å distance suggests the presence of heavier donor atoms in the
zinc first coordination shell. All possible different combinations of
oxygen/nitrogen-His donors summing up to 4, 5, and 6 were tested to
model the 2.0 Å shell, whereas sulfur, chlorine, and zinc
backscatterers were tried to model the 2.3 Å shell. It was immediately
evident that zinc backscattering could not be the cause of the outer
distance shell that was, on the contrary, easily modeled by one sulfur ligand. An identical fit was obtained by replacing sulfur with a
chlorine atom at a slightly shorter distance (Table II). Introduction of multiple scattering effects from histidine imidazole rings was able
to fully reproduce the spectrum. The best EXAFS and FT fit results in
an average zinc coordination polyhedron composed of 3 nitrogen atoms
from histidine residues at 1.99 Å and 1 sulfur ligand at 2.28 Å (Cl
at 2.25 Å). Besides the peaks at about 3.0 and 4.2 Å, caused by
multiple scattering from the histidine second and third shell atoms,
the spectrum FT shows a peak at about 3.6 Å (Fig. 5b). The
position of this peak does not change if the FT is performed over a
different k range, and it is present also in the FT of the
EXAFS spectrum of a HPRG-zinc complex collected at a lower HPRG
concentration (100 µM) and a higher zinc-HPRG ratio (8:1
and 15:1; data not shown), suggesting that this peak is not an artifact
but a real component of the HPRG spectrum. The 3.6 Å FT peak could
easily be reproduced in the fit by assuming the presence of a second
zinc ion at this distance. On the contrary, this feature could not be
reproduced by fitting a shell of four or five carbon atoms between 3.5 and 3.7 Å. Although perfectly reproducing the FT of the spectrum, the
inclusion of the zinc shell at 3.68 Å only slightly improves the fit
R-factor (2%). The unambiguous detection of metal-metal
scattering at distances larger than 3.0 Å in protein EXAFS is always
difficult (25), because many factors like multiple scattering from
other ligands, metal carbon scattering, etc., usually interfere with
the metal-metal scattering (25). However, the example of the EXAFS
spectrum of urease from jack bean, Klebsiella
aerogenes and Bacillus pasteurii (26, 27) are
particularly instructive because the spectra FTs of both native and
-mercaptoethanol-inhibited enzymes always show an outer shell
pattern identical to the HPRG-Zn2 complex with a peak at
about 3.5 Å in between the histidine outer shell peaks. A dinuclear
Ni2 cluster is present in urease with nickel-nickel distances ranging between 3.1 and 3.5 Å (26-28). In summary, although not conclusive, the evidence from our EXAFS data suggests that the
zinc-binding site in HPRG may host two metal ions at a distance of
about 3.7 Å.
On average the zinc ion in HPRG appears to be 4-coordinated. However,
it is known that coordination numbers obtained from EXAFS analysis
alone are affected by large errors, especially when in the presence of
a mixture of nitrogen/oxygen- and sulfur-type ligands (29, 30). Indeed,
a significant improvement of the fit index and the R-factor
(6%) was obtained by adding one further oxygen backscatterer at 2.43 Å. For this reason the presence of five or of a mixture of 4- and
5-coordinated zinc sites cannot be excluded on the basis of our data,
although bond valence sum analysis (30) of the observed distances
support the presence of 4-coordinated zinc sites in HPRG (Table
II).
The question arises whether one of the zinc ligands is a sulfur atom
from a Cys thiolate group or whether it is a chloride anion from the
solution where the Cl
concentration was about 0.6 M. The EXAFS data alone cannot provide a definitive answer
to this question because the two different zinc ligands provide
identical fits. However, at physiological pH, chloride is a weaker zinc
ligand than water and much weaker than hydroxide and thiolate sulfur,
and few examples are known where chloride binding to a protein metal
cofactor has been demonstrated (32, 33). Furthermore, many zinc
proteins have been crystallized in the presence of Cl
concentrations ranging between 0.2 to 1.2 M, but only in a
few cases has chloride coordination to zinc been observed by x-ray crystallography (e.g. Protein Data Bank entries 1BEN and
1D0G). Our data do not allow us to definitely ascertain the nature of the ligand at 2.23-2.25 Å, but a thiolate sulfur seems the most probable candidate.
The EXAFS analysis of the 2:1 zinc-HPRG complex shows that the protein
is able to bind zinc, possibly in a dinuclear metal-binding site where
two Zn2+ ions are bound to histidine residues. In
principle, the EXAFS analysis gives only the average zinc coordination
in the sample and does not establish whether one zinc ion is in a
histidine only environment (i.e. zinc-N(His)4)
and the other in a nitrogen/sulfur coordination (i.e.
zinc-N(His)2,S2) or whether the zinc-binding site is a more symmetric one with common bridging ligands. However, the
zinc-zinc distance of 3.68 Å in HPRG is typical of dinuclear first
transition row metal sites with bridging ligands like carboxylates or
cysteine thiolate sulfur. Examples of dinuclear zinc sites can be seen
in dihydroorotase (34), where two Zn2+ ions, bridged by the
carboxylate group of a carbamylated Lys residue, are at
3.48 Å distance. This also occurs in phosphotriesterase where two
Zn2+ ions are at 3.45 Å (35). In
-mercaptoethanol
inhibited urease (Protein Data Bank entry 1UBP), the two
Ni2+ ions in the active site are bridged by the inhibitor
thiolate sulfur bringing them as close as 3.1 Å (28). Possible models of the zinc site found in HPRG that are consistent with the structural data obtained from EXAFS are reported in Fig.
6.

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Fig. 6.
Possible models of the zinc-binding site in
HPRG. a, mononuclear tetrahedral site where one of the
ligands might be a thiolate from a Cys residue (chloride from
solution). b, dinuclear site where the bridge is provided by
a thiolate from a Cys residue (or chloride). In the case of a
five-coordinate zinc (not shown) two water molecules or a carboxylate
group might be bound to the dinuclear site.
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The finding that the XAS data support the presence of a dinuclear
cysteine-bridged zinc-binding site in the HPRG molecule dissociated
from rabbit skeletal muscle AMP deaminase deserves consideration in
light of the determination of the disulfide bridge arrangement of
bovine plasma HPRG that has shown that all the 12 half-cysteine
residues found in the protein are involved in the formation of six
disulfide bridges (36). Alignment of the partial amino acid sequence
data of bovine plasma HPRG with the available predicted amino acid
sequences of human, mouse, rat, and rabbit HPRG indicates that 11 of
the 12 cysteine residues are totally conserved in all species,
suggesting that five disulfide bridges are likely to be essential for
the proper folding of the protein of each species. The two cysteine
residues that in bovine HPRG form the disulfide bond within one of the
two proline-rich regions that flank the histidine-rich region are
conserved in rabbit HPRG (residues 264 and 294) but not in the human
protein (Cys-264 of the rabbit HPRG sequence is replaced by proline)
and in mouse and rat HPRG, where Cys-294 is absent. It should be noted that a number of other cysteine residues are present at different positions in the histidine-proline-rich domain of human, mouse, and rat
HPRG. This analysis suggests that the cysteine residue that is not
involved in disulfide bond formation and that is therefore the
candidate for zinc ligation in the muscle variant of HPRG as suggested
by EXAFS belongs to this region of the protein. The same conclusion is
likely to be true for the six histidine residues used by the protein as
zinc ligands, because it was shown that one isolated 30-kDa
His-Pro-Gly-rich peptide retains the ability of rabbit plasma HPRG to
bind metals (18).
The characterization of skeletal muscle AMP deaminase as a zinc
metalloenzyme was reported for the rat enzyme (37) and for the rabbit
enzyme (21) on the basis of its interaction with chelating agents and
metal ions. Overall, the results of the present study clearly indicate
the presence in HPRG of a zinc-binding site and permit the assertion
that the two components of AMP deaminase manifest as a common property
the ability to interact with zinc ions. The stoichiometry of zinc
binding to rabbit skeletal muscle AMP deaminase as found in
vivo has yet to be absolutely established (21), because the native
enzyme as isolated, which our results indicate to be made up of the
catalytic subunit and the HPRG component, contains 2.6 g atoms of
zinc/mol (molecular mass, 278 kDa), whereas the apoenzyme binds 4 g atoms of zinc/mol. However, the increase of
Vmax caused by the addition of the fourth zinc
atom is only 28% of that expected. This suggests that the fourth zinc
atom is not directly associated with activity (21).
Alignment of the amino acid sequence for yeast AMP deaminase with that
for mouse adenosine deaminase demonstrates conservation of the four
amino acids (three His and one Asp) known from the x-ray crystal
structure of adenosine deaminase to bind zinc in contact with the
attacking water nucleophile (38). On the basis of these similarities,
the same model of a pentacoordinated zinc bound at the catalytic site
that was described for adenosine deaminase, a 352-amino acid protein,
has also been proposed for the 810-amino acid monomer of yeast AMP
deaminase (39). However, we may point out that alignment of the amino
acid residues supposed to be in contact with zinc in yeast AMP
deaminase with the deduced amino acid sequence for the skeletal muscle
enzyme demonstrates conservation of only three amino acids (His-363,
His-572, and Asp-649, corresponding respectively to His-422, His-630,
and Asp-707 in yeast AMP deaminase), whereas residue 424 of the yeast
enzyme sequence (His) is replaced by Gly-365 in both rat and human
skeletal muscle enzymes. Altogether, these observations confirm that
zinc is a firmly bound component of skeletal muscle AMP deaminase
essential for the enzyme activity but also suggest that the model of
zinc-binding site proposed for yeast AMP deaminase cannot be
unambiguously extended to the skeletal muscle enzyme.
The separation of HPRG using zinc affinity chromatography under
denaturing conditions induced a marked reduction in the solubility of
the catalytic subunit of skeletal muscle AMP deaminase, strongly suggesting an additional role for HPRG in the maintenance of the native
quaternary structure of the enzyme that could be envisaged from the
formation of a 1:1 HPRG-AMP deaminase molecular adduct (20). The
observation of the present paper that with the HPRG-enriched enzyme the
rate of the fragmentation of AMP deaminase with storage is reduced
(Fig. 2) suggests another possible role of the HPRG component in
preserving the molecular integrity of the enzyme. It seems to be
premature to assign a physiological role to the participation of HPRG
in the structure of skeletal muscle AMP deaminase. However, the
addition of HPRG into the family of metallochaperones (40) could be
envisaged by coupling the previous suggestion of the presence in the
whole enzyme of additional zinc not required for activity (21) with the
finding of the present study that rabbit muscle HPRG is able to bind
zinc, possibly in a dinuclear metal-binding site, and by considering
the absence of significant differences in the kinetics of the enzymes
with different HPRG content. In this view, HPRG may enhance the
stability of AMP deaminase in vivo through insertion of zinc
or by modulating the intracellular zinc availability.