A comparison of calcium binding in Callinectes sapidus premolt and postmolt cuticle homogenates: implications for regulation of biomineralization.

Cuticle tissue homogenates (CTHs) from Callinectes sapidus premolt cuticle bound approximately 367% more Ca2+ ions than did those from the postmolt cuticle. The pH-stat assay which was used to compare in vitro CaCO3 nucleation times confirmed that the premolt CTHs had greater inhibitory activity than did the postmolt CTHs. This inhibitory activity was indicated by CaCO3 nucleation times in excess of control values. Premolt nucleation times exceeded those of postmolt samples by approximately 340%. A positive correlation was observed between Ca2+ binding and calcification inhibitory activity for both premolt and postmolt CTHs. Heat pretreatment of CTHs at 70 degrees C for a 24-hr period had no significant effect on their Ca2+ binding. However, this heat pretreatment decreased their calcification inhibitory activity. Pretreatment of CTHs with Ca2+ diminished their calcification inhibitory activity. These results are consistent with a mechanism for inhibition of biocalcification by these proteins which involves their initial reversible binding to nascent calcite nuclei growth steps and kinks, rather than their in vivo interaction with free Ca2+ ions in solution.

Thermal inactivation of butyrylcholinesterase and acetylcholinesterase.

Differences were observed in the extent of thermal inactivation of human butyrylcholinesterase (BuChE) and eel acetylcholinesterase (AChE). BuChE was more resistant to 57 degrees C inactivation than was AChE. Thermal inactivation of BuChE was reversible and followed first-order kinetics. AChE thermal inactivation was irreversible and did not follow first-order kinetics. AChE was marginally protected from thermal inactivation by the "nonspecific salts" ammonium sulfate and sodium chloride and to a greater extent by the "active site-specific salts" choline chloride, sodium acetate, and acetylcholine iodide. This protection was accompanied by a loss of absorbance at 280 nm. This data supports the hypothesis that thermal inactivation of AChE occurs by conformational scrambling and that aromatic amino acid residue(s) are involved in this process.

Novel protein inhibits in vitro precipitation of calcium carbonate.

Organic molecules both coexist and interact with inorganic crystal lattices in biomineralizing tissues. Mineral precipitation and crystal morphology are tightly regulated by the actions of these molecules. Polyacrylamide gel electrophoresis studies on water soluble extracts from the cuticle of Callinectes sapidus (Atlantic blue crab) reveal the presence, in unmineralized nascent premolt cuticle, of proteins which are absent in the mineralized postmolt cuticle. In the present studies, homogenates from both premolt and postmolt C. sapidus cuticles have been tested for their effect on the in vitro precipitation of calcium carbonate. The role of protein in this process was determined by heat pretreatment and trypsin pretreatment of the cuticle homogenates prior to the precipitation assay. The results from these experiments indicate that proteins, with molecular weights of approximately 75,000 and between 10,000 and 20,000, concentrated in the C. sapidus premolt cuticle, inhibit calcium carbonate precipitation in vitro. The inhibitory activity of these proteins appears to be a result of specific interactions since trypsin, myoglobin, and ovalbumin are not inhibitory. The presence of lower amounts of these inhibitory proteins in C. sapidus postmolt cuticle may be responsible for the subsequent mineralization of this tissue.