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32Pi- and45Ca-metabolism by matrix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage by isosmotic percoll density-gradient fractionation

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Summary

Matrix vesicle-enriched fractions were isolated from different zones of epiphyseal cartilage by nonenzymatic methods involving tissue homogenization, differential centrifugation, and isosmotic Percoll gradient fractionation. Uptakes of both32Pi and45Ca were studied concomitantly over periods from 20 min to 24 h. Percoll density gradients separated epiphyseal microsomes into two alkaline phosphatase-rich fractions: a low-density noncalcifiable fraction (P-I), and a higher-density fraction (P-II) which readily mineralized. The P-II fraction was found only in calcifying regions of the growth plate. Based on chemical and physical properties and enzyme activities, both fractions were similar except that P-II contained significantly higher levels of mineral ions than did P-I, and had lower levels of alkaline phosphatase. The mineral appeared to be primarily in a noncrystalline form. Metabolism of32Pi and45Ca by P-II followed a complex kinetic pattern in which accumulation of large amounts of both ions was preceded by an initial limited burst of uptake and a lag-phase of variable duration. During mineral ion loading, the density of the P-II fraction progressively increased as evidenced by co-migration of45Ca,32Pi, and alkaline phosphatase to increasingly higher densities. During the period of early mineral deposition (1–5 h), Ca/P uptake ratios were very low (1.0–1.2) and X-ray diffraction patterns showed a predominantly amorphous pattern. This suggests that the mineral accumulated in matrix vesicles is initially some form of noncrystalline calcium monohydrogenphosphate. L-tetramisole, a potent inhibitor of alkaline phosphatase, inhibited accumulation of both45Ca and32Piin the absence of organic P substrates,32Pi being preferentially inhibited over45Ca. This finding, coupled with recent studies on the behavior of alkaline phosphatase at physiological pH, suggests that the protein is not acting as a phosphohydrolase, but rather as a Pi-binding or transport agent in vesicle-mediated calcification.

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Authors and Affiliations

  1. Department of Chemistry, School of Medicine, University of South Carolina, 29208, Columbia, South Carolina, USA

    Gregory P. Warner, H. Lee Hubbard, Georgia C. Lloyd &  Roy E. Wuthier

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  1. Gregory P. Warner
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  2. H. Lee Hubbard
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  3. Georgia C. Lloyd
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  4. Roy E. Wuthier
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Warner, G.P., Lee Hubbard, H., Lloyd, G.C. et al. 32Pi- and45Ca-metabolism by matrix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage by isosmotic percoll density-gradient fractionation. Calcif Tissue Int 35, 327–338 (1983). https://doi.org/10.1007/BF02405054

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