| Abstract
| - Nephrocalcin (NC), a calcium-binding glycoprotein of 14 000molecular weight as a monomer,is known to inhibit the growth of calcium oxalate monohydrate (COM)crystals in renal tubules. Wehave isolated NC from bovine kidney tissue and purified into fourisoforms, fractions A−D. NC-A andNC-B strongly inhibit the growth of COM crystals, and NC-C and NC-Dinhibit crystal growth weakly.The strong inhibitor proteins are abundant in normal subjects,whereas stone formers excrete less of NC-Aand NC-B and more of NC-C and NC-D. NC-C was characterized withrespect to its metal binding sitesby using vanadyl ion (VO2+) as a paramagnetic probe inelectron paramagnetic resonance (EPR) andelectron nuclear double resonance (ENDOR) spectroscopic studies.We demonstrated that VO2+ bindsto NC-C with a stoichiometry of metal:protein binding of 4:1 and thatVO2+ competes with Ca2+ inbinding to NC-C. In NC-C, the metal ion is exposed to solventwater molecules and two water moleculesare detected in the inner coordination sphere of the metal ion byENDOR. In the metal binding environmentof NC-A, as reported previously (Mustafi, D., & Nakagawa, Y. (1994)Proc. Natl. Acad. Sci. U.S.A.91,11323−11327), inner sphere coordinated water is completely excluded.Based on the results of the metalbinding properties in both strong and weak inhibitor proteins, aprobable mechanism of inhibition ofCOM crystal growth by NC has been outlined.
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