isoform-like proteins in marine euryhaline milkfish (Chanos

528C.-H. TANG ET AL.Fig. 6. The summary of Na 1 /K 1 -ATPase (NKA) a3-isoformin gills of milkfish sampled at a series of different timesafter transfer from SW to HSW or FW. (A) Representativeimmunoblots of NKA a3-isoform probed with a polyclonalantibody revealed single immunoreactive bands at approximately105 kDa. (B) The relative protein abundance followingtransfer from SW to HSW indicated that NKA a3-isoformdecreased significantly after 12 hr post-transfer. On the otherhand, following transfer from SW to FW, changes in therelative protein abundance of NKA a3-isoform levels declinedgradually during 48 hr post-transfer and significantly decreasedat 96 and 168 hr post-transfer. Values were mean7SEM (n 5 6). The asterisks indicated significant differences(Po0.05) compared with the 0 hr using Dunnett’s testfollowing a one-way ANOVA. M, marker; SW, seawater;HSW, hypersaline water.SW-acclimated milkfish (Lin and Lee, 2005).Therefore, alteration of NKA activity in gills offish following salinity changes may result fromdifferent NKA isoforms.There is accumulating evidence to presume thatNKA a-isoforms may change in teleosts to copewith the environmental fluctuations, e.g., salinityor temperature. The mRNA expression patterns ofgill NKA a1a and a1b examined in varioussalmonid species were found to express differentiallyin FW- and SW-acclimated individuals(Richards et al., 2003; Bystriansky et al., 2006,2007a,b; Nilsen et al., 2007). Changes in mRNAexpression are often assumed to parallel changes inprotein abundance. The lack of correlationbetween mRNA and protein of branchial NKAa-isoforms was reported (Morrison et al., 2006). Thislack of correlation between mRNA and proteinsuggested the correlation with posttranscriptionalregulation (Scott et al., 2004). This study,therefore, investigates the effect of environmentalsalinity on the protein expression of branchialNKA a-isoforms-like proteins in milkfish.The amino acid identities of individual isoformsacross species are higher than the identities ofdifferent isoforms within one species (Blanco andMercer, 1998). Therefore, the heterologous antibodiesto NKA a-isoforms (polyclonal antibodiesdirected against the rat a-specific isoforms) wereused in Atlantic salmon (Salmo salar) (D’Cottaet al., 2000) as well as in this study. Among theisoform-specific heterologous antibodies used inmilkfish, mouse monoclonal antibody a6F to theavian NKA a1-isoform and goat polyclonal antibodysc-16052 to human NKA a3-isoform had beenapplied in teleosts previously (tilapia, Oreochromismossambicus; Lee et al., 1998; Antarctic fish,Trematomus bernacchii; Brauer et al., 2005). Thisstudy identified three NKA a-isoform-like proteinsin milkfish gills with these isoform-specific antibodiesby immunoblotting analysis (Figs. 1A, 2A,and 3A). Three NKA a-isoform-like proteins werealso found in the gills of Antarctic fish (Guynnet al., 2002), while only a1 anda3 werereportedintilapia gills (Lee et al., 1998; Feng et al., 2002). Theimmunoblots of milkfish gill lysates probed witheach isoform-specific antibody revealed single immunoreactivebands at approximately 105–115 kDa(Figs. 1A, 2A, and 3A), similar to those recognizedin tilapia (Lee et al., 1998) and Antarctic fish(Guynn et al., 2002; Brauer et al., 2005).This study revealed that the SW-acclimatedgroup was significantly higher than the FW- orHSW-acclimated groups in the levels of NKAa1- and a3-like proteins (Figs. 1 and 3). Expressionpattern of NKA a2-like protein in the gills were notsalinity-dependent (Fig. 2B). It was suggested thata1- and a3-like proteins might play the role forosmo- or ionoregulation but a2-like protein probablyperformed the other physiological function.The affinity of gill NKA for sodium increasedsignificantly in warm-acclimated Antarctic teleost(T. bernacchii; Morrison et al., 2006) that isopposite to milkfish, which has lower sodiumaffinity in SW than FW (Lin and Lee, 2005).Upregulation of gill a1- and a2-like proteinsinfluenced sodium affinities and fulfilled some ofthe requirements for the subtly moderated enzymebehaviors (e.g., elevated NKA activity) in warmacclimatedT. bernacchii (Morrison et al., 2006),while the changes in gill NKA a1- and a3-likeproteins may play the potential role for reductionof sodium affinities to decrease NKA activity inSW-exposed milkfish. In addition, the role of NKAJ. Exp. Zool.