PAULSEN, R.S.*; WEIHRAUCH, D.; LIGNOT, J.-H.; TOWLE, D.W.: Na,K-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of ALPHA subunit mRNA and protein.
Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na,K-ATPase activity in posterior gills, but it is not clear whether the response is due to activation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3′ untranslated region of the mRNA coding for the ALPHA subunit of Na,K-ATPase were amplified and sequenced by RT-PCR. The resulting 3,433-nucleotide cDNA encodes a putative 1,011-amino acid protein with a predicted molecular mass of 112.5 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The ALPHA subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83% identity to other arthropods and 74-77% identity to vertebrate and non-arthropod invertebrate species. Semi-quantitative PCR showed high levels of ALPHA subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na,K-ATPase ALPHA subunit protein band of the expected size. Posterior gills contained a much greater level of ALPHA subunit protein compared with anterior gills, in agreement with historical measurements of enzyme activity. Transfer of blue crabs from 35 to 5 ppt salinity was accompanied by modest increases in ALPHA subunit mRNA and protein in posterior gills, providing a molecular explanation for the enhanced Na,K-ATPase enzyme activity that accompanies the hyperosmoregulatory response. Supported by NSF (IBN-9807539).