Proc. Natl. Acad. Sci. USA Vol. 91, pp. 11323-11327, November 1994 Biochemistry Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: Electron paramagnetic resonance and electron nuclear double resonance spectroscopy DEVKUMAR MUSTAFI* AND YASUSHI NAKAGAWA Department of Biochemistry and Molecular Biology, University of Chicago, Cummings Life Science Center, 920 East 58th Street, Chicago, IL 60637 Communicated by Clyde A. Hutchison, Jr., July 25, 1994 (received for review April 26, 1994) ABSTRACT Nephrocalin (NC) is a calcium-binding gly- coprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystalsin renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography Into four isoforms, deignated s fractious A-D. They differ pri- marily according to the content of phosphate and 'carboxy- glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crysal, whereas frac- tions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca2+-binding affnit, was characterized with respect to its metal-bding sites by using the vanadyl ion (VO+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (EN- DOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichi- ometry of metabl:protein binding of 4:1. Also, the binding of V02+ to NC was shown to be competitive with Ca2+. Only protein residues were deteted by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO2+- reconstituted NC, differs sigfcantly from the binding sites in other Ca2+-binding proteins. Nephrocalcin (NC), a calcium-binding glycoprotein of 14,000 molecular weight, is known to inhibit growth of calcium oxalate monohydrate (COM) crystals in renal tubules (1). This inhibitor protein has been isolated in four isoforms from urine of normal subjects (1) and kidney stone patients (2), from surgically removed calcium oxalate kidney stones (3), and from kidney tissue of nine species of vertebrates (4). Among these nine species, the dissociation constant of these inhibitor proteins for binding COM crystals varies from 6 x 10-6 to 4 x 10-8 M. Purification of NC from bovine kidney tissue by DEAE-cellulose chromatography showed that it also consists of four isoforms, which have been designated as fractions A-D (4-6). Fractions A-D of NC were also ob- tained similarly from other species, as described earlier (1-4). Fractions A and B, the "strong" inhibitors, are abundant in normal subjects (1), whereas stone formers excrete less of fractions A and B and more of C and D, which are "weak" inhibitors (2). NC is an acidic glycoprotein containing 33% acidic amino acid residues (Asp and Glu) and about 5% aromatic and basic amino acid residues (5). Fractions A-D differ according to the content of carbohydrate and phos- phate. Also, the strong inhibitors contain three or four y-tcarboxyglutamic acid residues per molecule, which are not present in NC of stone-forming patients (5). We have previously shown that, on the basis of 31P NMR studies of Ca2+-NC complexes and by equilibrium dialysis with 45Ca, a total of 4 mol of Ca2+ are bound per mol of NC (6). In this study we characterized the metal-binding prop- erties of normal NCt (fraction A) by EPR and electron nuclear double resonance (ENDOR) using the vanadyl ion (VO2+) as a paramagnetic probe. The VO2+ ion has been shown to be an effective paramagnetic substitute for many divalent metal ions in metalloproteins and metalloenzymes (7-10) and is, in particular, an effective substitute for Ca2+ and Mg2+ (11, 12). VO2+ is an ideal substitute for Ca2+ because VO2+, like Ca2+, has a high affinity for binding to oxygen-donor ligands (7). By EPR spectrometric titration, we have shown that VO2+ binds to NC-A with a stoichiometry of 4 mol/mol of protein. Furthermore, we have also demon- strated that VO2+ competes with Ca2+ in binding to NC. An unusual result of this preliminary study of VO2+-NC com- plexes obtained by ENDOR spectroscopy showed that the coordinating ligands to the metal ions are from protein residues with complete exclusion of solvent water from the inner coordination sphere. EXPERIMENTAL PROCEDURES Materials. NC was isolated from fresh bovine kidney tissue and separated into four fractions by DEAE-cellulose chro- matography (Whatman DE-52), as described (1). These frac- tions were eluted by varying the ionic strength: NC-A at 10-15 inS, NC-B at 16-23 mS, NC-C at 24-27 mS, and NC-D at 32-38 inS, using a linear gradient of NaCl from 0.1 M to 0.5 M in 0.05 M Tris HCl at pH 7.3. Fraction A (NC-At), which exhibits the highest affinity for Ca2+ binding, was used in this study. This fraction of protein has been found in normal (non-kidney stone) subjects and can be used as a model system for a strong inhibitor protein. Fraction A of NC was first eluted at a conductivity between 10 and 12 mS and then further purified by Sephacryl S-200 gel filtration chromatog- raphy (Pharmacia LKB, 2 x 100 cm) with 0.05 M Tris'HCl buffer at pH 7.3 containing 0.2 M NaCl and 0.02% sodium azide. Chromatographically purified NC-A was dialyzed and lyophilized and then dissolved in 0.03 M Pipes at pH 5.8 to a concentration of 12-14 mg/ml for spectroscopic studies. This solution was then treated with Chelex-100 by dialysis to ensure removal of metal ions. Metal ion removal (mostly Ca2+) was confirmed by atomic absorption (1). Abbreviations: COM, calcium oxalate monohydrate; ENDOR, elec- tron nuclear double resonance; hf, hyperfine; NC, nephrocalcin; NC-A, fraction A of NC. *To whom reprint requests should be addressed. tSince this study is primarily with use of normal NC, the abbreviation NC is often used for normal NC (fraction A). The three other fractions B-D, if used, are always abbreviated as NC-B, NC-C, and NC-D, respectively. 11323 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.