CHARACTERIZATION OF CALCIUM-BINDING SITES IN THE KIDNEY-STONE INHIBITOR GLYCOPROTEIN NEPHROCALCIN WITH VANADYL IONS - ELECTRON-PARAMAGNETIC-RESONANCE AND ELECTRON-NUCLEAR DOUBLE-RESONANCE SPECTROSCOPY

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecula r weight. It inhibits the growth of calcium oxalate monohydrate crysta ls in renal tubules. The NC used in this study was isolated from bovin e kidney tissue and purified with the use of DEAE-cellulose chromatogr aphy into four isoforms, designated as fractions A-D. They differ prim arily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calciu m oxalate monohydrate crystal, whereas fractions C and D inhibit cryst al growth weakly. Fraction A, with the highest Ca2+-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic r esonance (EPR) and electron nuclear double resonance (ENDOR) spectrosc opic studies. By EPR spectrometric titration, it was shown that fracti on A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the i nner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO2+-reconstituted NC, differs significan tly from the binding sites in other Ca2+-binding proteins.