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• W2024706102 abstract "The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn2+ ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase. The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn2+ ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase. histidine-proline-rich glycoprotein x-ray absorption spectroscopy N,N-bis(2-hydroxyethyl)glycine 3-(cyclohexylamino)propanesulfonic acid extended x-ray absorption fine structure Fourier transform Deutsches Elektronen Synchrotron 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 (1Peterson C.B. Morgan W.T. Blackburn M.N. J. Biol. Chem. 1987; 262: 7567-7574Google Scholar). 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 (2Hulett M.D. Parish C.R. Immunol. Cell Biol. 2000; 78: 280-287Google Scholar). 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 (3Ranieri-Raggi M. Montali U. Ronca F. Sabbatini A. Brown P.E. Moir A.J.G. Raggi A. Biochem. J. 1997; 326: 641-648Google Scholar). 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar). The HPRG component of rabbit skeletal muscle AMP deaminase contains 10 mol of histidine residues/10,000 g of protein (3Ranieri-Raggi M. Montali U. Ronca F. Sabbatini A. Brown P.E. Moir A.J.G. Raggi A. Biochem. J. 1997; 326: 641-648Google Scholar). 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 (5Morgan W.T. Biochemistry. 1981; 20: 1054-1061Google Scholar, 6Guthans S.L. Morgan W.T. Arch. Biochem. Biophys. 1982; 218: 320-328Google Scholar) 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. Chelating Fast Flow Sepharose was from AmershamBiosciences. 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 was prepared as described previously (7Ranieri-Raggi M. Raggi A. FEBS Lett. 1979; 102: 59-63Google Scholar) 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) (8Ranieri-Raggi M. Raggi A. Biochem. J. 1980; 189: 367-368Google Scholar). 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 mmNaN3, 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 mmHPRG sample was prepared for XAS by further concentration of the pooled fractions. The protein concentrations of the various enzyme fractions were determined spectrophotometrically by usingA 2801%,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. 9Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Google Scholar 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar). The AMP deaminase activity was determined spectrophotometrically as previously described using a Shimadzu UV-260 spectrophotometer (10Martini D. Ranieri-Raggi M. Sabbatini A.R.M. Raggi A. Biochim. Biophys. Acta. 2001; 1544: 123-132Google Scholar). 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 mBicine, pH 8.3. Protein standards (Sigma) were used to determine molecular weights. Electroblotting was performed by the method of LeGendre and Matsudaira (11LeGendre N. Matsudaira P. Matsudaira P. A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, New York1989: 49-69Google Scholar) 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. 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, α, is defined as the ratio of the observed increase in absorbance (A) at 275 nm to the maximum ΔA at saturation of the protein. 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. 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-Kα 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 (12Pettifer R.F. Hermes C. J. Appl. Crystallogr. 1985; 18: 404-412Google Scholar) (E 0,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 (13Nolting H.F. Hermes C. EXPROG: EMBL EXAFS data analysis and evaluation program package for PC/AT. European Molecular Biology Laboratory, DESY, Hamburg, Germany1993Google Scholar). The full k 3 weighted EXAFS spectrum (23–750 eV above E 0) 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 (14Binsted N. Hasnain S.S. J. Synchrotron Rad. 1996; 3: 185-196Google Scholar). The edge energy E 0 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. Thek3 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 (2ς2) 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,ε2=1/(Nind−p)(Nind/N)∑iNwi(χiexp(k)−χith(k))2Equation 1 where N ind is the number of independent data points (N ind = (2ΔkΔr)/π),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.Re xafs=∑iN1/ς(‖χiexp(k)−χith(k)‖)·100Equation 2 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 (15Smiley K.L. Berry A.J. Suelter C.H. J. Biol. Chem. 1967; 242: 2502-2506Google Scholar). 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 (16Zielke C.L. Suelter C.H. J. Biol. Chem. 1971; 246: 1313-1317Google Scholar). 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 (7Ranieri-Raggi M. Raggi A. FEBS Lett. 1979; 102: 59-63Google Scholar). 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 andB, 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. 2 B, lanes 1 and 2).Figure 2Alteration 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 mmNaN3, 1.0 M KCl, pH 7.0. C, 4-μg samples of AMP deaminase prepared as described in Ref. 15Smiley K.L. Berry A.J. Suelter C.H. J. Biol. Chem. 1967; 242: 2502-2506Google Scholar 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% β-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.View Large Image Figure ViewerDownload (PPT) With aging of pool 2, a 85–70-kDa band transition also occurred with a half-time of 1 month (Fig. 2 B, 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 (17Ronca F. Ranieri-Raggi M. Brown P.E. Moir A.J.G. Raggi A. Biochim. Biophys. Acta. 1994; 1209: 123-129Google Scholar). 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. 2 A, 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 (3Ranieri-Raggi M. Montali U. Ronca F. Sabbatini A. Brown P.E. Moir A.J.G. Raggi A. Biochem. J. 1997; 326: 641-648Google Scholar). 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar, 18Morgan W.T. Biochemistry. 1985; 24: 1496-1501Google Scholar, 19Mori S. Nishibori M. Yamaoka K. Okamoto M. Arch. Biochem. Biophys. 2000; 383: 191-196Google Scholar) higher than that deduced from its sequence (58 kDa) or that calculated taking account of its 17.5% carbohydrate content (70 kDa) (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar). Interestingly, a 1-h incubation of a sample of freshly prepared pool 1 with 0.1% β-mercaptoethanol at room temperature before analysis by SDS/PAGE caused the same 85–95 kDa transition that was observed on storage (Fig. 2 A, 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar) 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 (3Ranieri-Raggi M. Montali U. Ronca F. Sabbatini A. Brown P.E. Moir A.J.G. Raggi A. Biochem. J. 1997; 326: 641-648Google Scholar). 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar). 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% β-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 (20Ranieri-Raggi M. Martini D. Sabbatini A.R.M. Moir A.J.G. Raggi A. Biochim. Biophys. Acta. 2003; 1645: 81-88Google Scholar). 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar). 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 (3Ranieri-Raggi M. Montali U. Ronca F. Sabbatini A. Brown P.E. Moir A.J.G. Raggi A. Biochem. J. 1997; 326: 641-648Google Scholar, 4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar). On this basis, we may reasonably assume that on SDS/PAGE of the 1m 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 β-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 0.45m 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 (4Borza D.B. Tatum F.M. Morgan W.T. Biochemistry. 1996; 35: 1925-1934Google Scholar), 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.Table IPurification of rabbit skeletal muscle AMP deaminase: elution of the enzyme held by cellulose phosphate after the washing step with 0.45m KClOne-step elution with 1.0 mKClTwo-step elutionWith 0.6 mKClWith 1.0 m KClTotal protein (mg/100 g of muscle)8.5 ± 1.61.0 ± 0.17.0 ± 1.4Activity (units/mg)1700.5210A 280/A 2601.61.21.8The 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. Open table in a new tab 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. The enzyme obtained with the one-step elution with 1.0 mKCl showed a 20% lower specific activity and a lowerA 280:A 260 ratio, indicating a possible contamination by nucleic acids or nucleotides. By following the procedure described under “Experimental Procedures” for the determination of theA 2801%,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 0.6m 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. 15Smiley K.L. Berry A.J. Suelter C.H. J. Biol. Chem. 1967; 242: 2502-2506Google Scholar from frozen rabbit skeletal muscle showed anA 280:A 260 ratio of 1.8 and an A 2801%,1 cm value of 9.1 (21Zielke C.L. Suelter C.H. J. Biol. Chem. 1971; 246: 2179-2186Google Scholar). Analysis by sedimentation-equilibrium techniques revealed that that enzyme had a molecular weight of 278,000 (21Zielke C.L. Suelter C.H. J. Biol. Chem. 1971; 246: 2179-2186Google Scholar), 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 (8Ranieri-Raggi M. Raggi A. Biochem. J. 1980; 189: 367-368Google Scholar). In contrast, our determination by sedimentation-equilibrium analysis of the molecular mass of freshly prepared rabbit skeletal muscle AMP deaminase in 1m 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 (22Ranieri-Raggi M. Raggi A. Biochem. J. 1990; 272: 755-759Google Scholar). 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. 2 C (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 (23Ko" @default.
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• W2024706102 date "2003-01-01" @default.
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• W2024706102 title "Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase" @default.
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