The long range goal of this research is to contribute significant insight into the biochemical aspects of regulation of biocalcification by organic matrix substances.
The specific aims are to (1) partially characterize biocalcification inhibitory substances present in crab nascent premolt cuticle tissue and (2) purify these substances to homogeneity prior to their extensive physicochemical characterization. The organism used in this research is the blue crab, Callinectes sapidus, an accessible invertebrate animal model for vertebrate biocalcification. Water soluble biomolecules which inhibit calcium carbonate precipitation in vitro, are present in the crab's nascent premolt cuticle. These substances are not present in the postmolt cuticle. Three proteins, present in the premolt cuticle, are absent in the postmolt cuticle. The involvement of protein in the inhibition of biocalcification by the crab's nascent premolt tissue will be indicated if the following results are obtained for the corresponding planned experiments. (1) Inhibition of in vitro calcium carbonate precipitation: (a) Dose-response: Inhibition is saturable at high tissue concentrations. (b) Heat inactivation: Inhibition is mitigated by pretreatment of the tissue with high temperatures. (2) Equilibrium dialysis against a calcium ion solution: Tissue binds calcium. Polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) will be conducted on cuticle tissue from all crab molt stages. The ability of specific proteins to bind calcium will be monitored by calcium-45 autoradiography of these gels. The ability of specific proteins to inhibit in vitro calcium carbonate precipitation will be determined by treatment of the gels with saturating levels of calcium and carbonate and location of clear areas which lack precipitate. The purification procedure for calcification inhibitory substances will adhere to the following general scheme: (1) initial staging of animals, removal of cuticle tissue, removal of hypodermis, homogenization, centrifugal removal of insoluble precipitates, (2) column chromatography with gel filtration, anion exchange, and/or hydroxyapatite matrices, (3) high performance liquid chromatography (HPLC) with reverse phase and/or anion exchange columns.
