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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Isolation of a Crystal Matrix Protein Associated with Calcium Oxalate Precipitation in Vacuoles of Specialized Cells¹

Department of Genetics and Cell Biology (X.L.), School of Biological Sciences (D.Z., V.J.L.-H., V.R.F.), and Institute of Biological Chemistry (T.W.O.), Washington State University, Pullman, Washington 99164–4236

The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to ³H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. ⁴⁵Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.

¹ This work was supported in part by the National Science Foundation (grant nos. MCB 9632027 and MCB 9904562 to V.R.F.) and by a Loyal Davis Fellowship (to X.L.).

² These authors contributed equally to the paper.

^* Corresponding author; e-mail vfrances{at}mail.wsu.edu; fax 509–335–3184.

Received March 13, 2003; returned for revision May 12, 2003; accepted June 2, 2003.

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