Lead (Pb2+) produces toxic effects in bone of humans and experimental animals. The exact mechanism of action is unknown. At the molecular level lead has been shown to bind with high affinity to osteocalcin, an abundant noncollagenous bone protein. Since osteocalcin has been shown to play a significant role in bone formation and remodeling, we hypothesize that it is a likely target for lead. We have shown that Pb2+ alters the structure of osteocalcin at a stoichiometry of only 1:1 where as a stoichiometry of 3:1 is needed for Ca2+. In addition, low Pb2+ levels in addition to physiological Ca2+ levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca2+ alone. We propose that the molecular mechanism of lead toxicity is an increase in mineral bound osteocalcin. This increase in mineral bound osteocalcin might play a role in perturbed bone remodeling and abnormal bone crystal size, perfection and biomechanical properties. The following specific aims have been designed to test this hypothesis: 1) Determine the high resolution structure of the 3 g-carboxyglutamic acid (Gla) form Pb2+- osteocalcin using X-ray crystallography and that of Ca2+- and Mg2+-osteocalcin for comparison. A mechanism for mineral binding will be proposed. 2) Measure serum and mineral bound osteocalcin and investigate detailed mineral properties (crystal size and perfection) using Fourier Transform Infrared Microspectroscopy (FTIRM), in bones from mice with various levels of Pb2+ exposure. 3) Measure osteocalcin levels and collect FTIRM mineral data in mice on lower Mg2+ diets for comparison to Pb2+ data and 4) Investigate functional effects of these treatments on the bone mineral by measuring biomechanical properties (density, strength, and fragility) in the bones from these mice. Comparisons of all mineral data will help determine the toxic effect of lead at the molecular level, increase our understanding of the physiology of osteocalcin and aid in developing effective pharmacotherapeutic strategies for lead exposed children and the elderly.
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