|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
Na++K+-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of 
-subunit mRNA and protein
1 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA and Lake Forest College, Lake Forest, IL 60045, USA,
2 Duke University, Durham, NC 27706, USA and
3 Université Montpellier II, 34095 Montpellier Cedex 5, France
*e-mail: dtowle{at}mdibl.org
Accepted September 3, 2001
Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na++K+-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the 
-subunit of Na++K+-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The -subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81–83 % identity to those of other arthropods and 74–77 % identity to those of vertebrate species. Quantitative RT-PCR analysis showed high levels of -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 -subunit protein band of the expected size. The posterior gills contained a much higher level of -subunit protein than the anterior gills, in agreement with previous measurements of enzyme activity. Immunocytochemical analysis showed that the Na++K+-ATPase -subunit protein detected by 5 antibody is localized to the basolateral membrane region of gill epithelial cells. Transfer of blue crabs from 35 to 5 
salinity was not accompanied by notable differences in the relative proportions of -subunit mRNA and protein in the posterior gills, suggesting that the enhanced Na++K+-ATPase enzyme activity that accompanies the hyperosmoregulatory response may result from post-translational regulatory processes.
Key words: Na++K+-ATPase, gills, blue crab, Callinectes sapidus, cDNA sequencing, salinity, osmoregulation, quantitative polymerase chain reaction.
This article has been cited by other articles:
|
|
 |
|
  H. H. Taylor and P. Greenaway Osmoregulation in the terrestrial Christmas Island red crab Gecarcoidea natalis (Brachyura: Gecarcinidae): modulation of branchial chloride uptake from the urine J. Exp. Biol., October 15, 2002; 205(20): 3251 - 3260. [Abstract] [Full Text] [PDF]
|
|
