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First published online July 26, 2004
Journal of Experimental Biology 207, 2991-3002 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01101
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Characterization and expression of plasma membrane Ca2+ ATPase (PMCA3) in the crayfish Procambarus clarkii antennal gland during molting

Yongping Gao and Michele G. Wheatly*

Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA




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  		Fig. 1. (A) Nucleotide sequence of open reading frame (ORF) and deduced amino acid sequence of crayfish antennal gland plasma membrane Ca2+-ATPase (PMCA3). Nucleotides and amino acids are numbered on the right. The putative start codon ATG and stop codon TAG are in bold. Sequences corresponding to the primers used in both initial PCR and 5'/3' RACE are underlined. (B) Comparison of the deduced amino acid sequence of crayfish antennal gland plasma membrane Ca2+-ATPase (PMCA3) with that of human PMCA3a (GenBank accession No. U57931), tilapia PMCA2 (AF23669) and bullfrog PMCA2a (AF337956). Stars below amino acids indicate identity between residues in PMCA from different species. Amino acids are numbered on the right. Transmembrane regions are underlined and labeled TM1-TM10. The phosphorylation site, fluorescein isothiocyanate (FITC) site (assumed to be part of the ATP-binding site), the 5'-p-fluorosulphonylbenzoyladenosine/r-(N-2-chloroethyl-N-methylamino) benzylamine ATP-binding (FSBA) site, calmodulin (CaM) binding site and PMCA3 Ab peptides location are underlined in both A and B. This sequence has been accepted by GenBank (accession number AY455931).

 


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  		Fig. 2. Hydropathy plot of crayfish antennal gland plasma membrane Ca2+-ATPase (PMCA3) in comparison with that of human PMCA3a (U57931), human PMCA3b (U60414), mouse PMCA3 (AKO32322) and rat PMCA3 (J05087). Hydrophobicity values were determined by the method of Kyte and Doolittle (1982Go) using a window of 19 residues (http://arbl.cvmbs.colostate.edu/molkit/hydropathy/index.html). Putative transmembrane segments are indicated by the numbers 1-10.

 


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  		Fig. 3. (Top) Northern blot analysis of plasma membrane Ca2+-ATPase (PMCA3) in various crayfish tissues in intermolt (A), premolt (B) and postmolt (C). Total RNA (20 µg) from gill, antennal gland, cardiac muscle and axial abdominal muscle was loaded in each lane. The membrane was hybridized to a 1164 bp crayfish PMCA3 probe and exposed to X-ray film for 24 h. (Bottom) To normalize the hybridization signal, 18s rRNA concentration was run on a corresponding formaldehyde-agarose gel and visualized by ethidium bromide staining under UV light before being transferred to the membrane. The band size is indicated to the left.

 


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  		Fig. 4. Quantification of PMCA3 mRNA (A) and PMCA3 protein (B) from gill, antennal gland, cardiac muscle and axial muscle during molt stages. Values indicate the percentage expression difference compared with the intermolt (control values). Values were obtained from three different scanned X-ray film images and analyzed using KODAK 1D image analysis software. Values are means ± S.E.M. (N=3). All values, except premolt antennal gland, were significantly different (P<0.001) from their intermolt values. Statistical comparison (t-test) between postmolt and premolt expression is indicated as either not significant (NS, P>0.05) or significant (*P<0.01; **P<0.001).

 


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  		Fig. 5. Western blot analysis of plasma membrane Ca2+-ATPase (PMCA3) in various crayfish tissues in intermolt (A), premolt (B) and postmolt (C). Total plasma membrane protein (30 µg) from gill, antennal gland, cardiac muscle and axial abdominal muscle was loaded in each lane. The membrane was hybridized to a polyclonal antibody (designed against amino acid residues 751-775 EGKEFNRRVRDESGGC of crayfish antennal gland PMCA3 deduced protein sequence; see Fig. 1A for location) and exposed to X-ray film for 2 min.

 


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  		Fig. 6. Phylogram comparison of crayfish PMCA3 amino acid sequence with PMCAs from a diversity of species that are available in GenBank (accession numbers are provided in parentheses). Phylogram values were determined by the Clustal method (DNASTAR, Madison, WI, USA).

 

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© The Company of Biologists Ltd 2004