Characterization of Ca(2+)-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 and NC-D inhibit crystal growth weakly. The strongly inhibitor proteins are abundant in normal subjects, whereas stone formers excrete less of NC-A and NC-B and more of NC-C and NC-D. NC-C was characterized with respect to its metal binding sites by using vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear 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 molecules are detected in the inner coordination sphere of the metal ion by ENDOR. In the metal binding environment of 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 metal binding properties in both strong and weak inhibitor proteins, a probable mechanism of inhibition of COM crystal growth by NC has been outlined.
Mustafi
D
D
Department of Biochemistry and Molecular Biology, University of Chicago, Cummings Life Science Center, Illinois 60637, USA. Mustafi@biovax.uchicago.edu
Nakagawa
Y
Y
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002129:Calcium Oxalate / Q000037:antagonists & inhibitors*; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D007477:Ions; D007669:Kidney Calculi / Q000737:chemistry; D008670:Metals / Q000378:metabolism; D011487:Protein Conformation; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism*
D006023:Glycoproteins; D007477:Ions; D008670:Metals; C029490:nephrocalcin; D002129:Calcium Oxalate; D014638:Vanadates; D002118:Calcium