Characterization and localization of crayfish Ca pump and exchanger

WHEATLY, M.G.*; ZHANG, Z.; STINER, L.T.M.: Characterization and localization of crayfish Ca pump and exchanger.

The crayfish molting cycle is used to study gene expression for membrane Ca translocating proteins specifically Plasma Membrane Ca ATPase (PMCA) and Na/Ca exchanger (NCX). Amino acid sequences deduced from cDNA sequences were used to design oligo-peptide antigens (15 mers from hydrophilic loop regions). Peptides were conjugated to cationized BSA and used to generate polyclonal antibodies in rabbits. Antiserum against PMCA has been shown to bind to a single strong band at 150-160 kDa in Western analysis of crayfish antennal gland; a weaker signal is detected in crayfish hepatopancreas. No signal is detected with preimmune serum. Antiserum against NCX binds with a strong band at 124 kDa in crayfish cardiac muscle. Immunohistochemistry reveals hybridization of antiserum against PMCA to basolateral membranes of antennal gland. Antiserum against NCX shows specific binding in heart muscle, axial abdominal muscle, antennal gland and hepatopancreas. Westerns are being used to quantify relative expression during different stages of the molting cycle. (NSF grant IBN 9603723).

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