CHARACTERIZATION OF CA2-BINDING SITES IN THE KIDNEY-STONE INHIBITOR GLYCOPROTEIN NEPHROCALCIN USING VANADYL IONS - DIFFERENT METAL-BINDING PROPERTIES IN STRONG AND WEAK INHIBITOR PROTEINS REVEALED BY EPR AND ENDOR()

Nephrocalcin (NC), a calcium-binding glycoprotein of 14 000 molecular weight as a monomer, is known to inhibit the growth of calcium oxalate monohydrate (COM) crystals in renal tubules. We have isolated NC from bovine kidney tissue and purified into four isoforms, fractions A-D, NC-A and NC-B strongly inhibit the growth of COM crystals, and NC-C an d NC-D inhibit crystal growth weakly. The strong inhibitor proteins ar e abundant in normal subjects, whereas stone formers excrete less of N C-A and NC-B and more of NC-C and NC-D. NC-C was characterized with re spect to its metal binding sites by using vanadyl ion (VO2+) as a para magnetic probe in electron paramagnetic resonance (EPR) and electron n uclear double resonance (ENDOR) spectroscopic studies. We demonstrated that VO2+ binds to NC-C with a stoichiometry of metal:protein binding of 4:1 and that VO2+ competes with Ca2+ in binding to NC-C. In NC-C, the metal ion is exposed to solvent water molecules and two water mole cules are detected in the inner coordination sphere of the metal ion b y ENDOR. In the metal binding environment of NC-A, as reported previou sly (Mustafi, D., & Nakagawa, Y. (1994) Proc. Natl. Acad Sci. U.S.A. 9 1, 11323-11327), inner sphere coordinated water is completely excluded . Based on the results of the metal binding properties in both strong and weak inhibitor proteins, a probable mechanism of inhibition of COM crystal growth by NC has been outlined.