ABSTRACT
The literature suggests that when Na(+)-K(+)-ATPase has reduced access to its glycosphingolipid cofactor sulfogalactosyl ceramide (SGC), it is converted to a Na(+) uniporter. We recently showed that such segregation can occur within a single membrane when Na(+)-K(+)-ATPase is excluded from membrane microdomains or 'lipid rafts' enriched in SGC (D. Lingwood, G. Harauz, J.S. Ballantyne, J. Biol. Chem. 280, 36545-36550). Specifically we demonstrated that Na(+)-K(+)-ATPase localizes to SGC-enriched rafts in the gill basolateral membrane (BLM) of rainbow trout exposed to seawater (SW) but not freshwater (FW). We therefore proposed that since the freshwater gill Na(+)-K(+)-ATPase was separated from BLM SGC it should also transport Na(+) only, suggesting a new role for the pump in this epithelium. In this paper we discuss the biochemical evidence for SGC-based modulation of transport stoichiometry and highlight how a unique asparagine-lysine substitution in the FW pump isoform and FW gill transport energetics gear the Na(+)-K(+)-ATPase to perform Na(+) uniport.
Subject(s)
Fishes/metabolism , Galactosylceramides/metabolism , Gills/enzymology , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Sequence , Animals , Fresh Water , Molecular Sequence DataABSTRACT
At present, alkaline phosphatase (AP) conjugates are major workhorses of immunological detection. However, APs are membrane bound enzymes, and therefore have the potential to interact with lipids. Using TLC overlay, we screened AP-conjugated immunoglobulins (IgGs), and AP-conjugated streptavidin, for their ability to bind sphingolipids and phospholipids non-specifically. Horseradish peroxidase (HRP)-conjugated IgG was tested as a negative control. AP-conjugates bound to all sphingolipids and phospholipids assayed, whereas no HRP-IgG binding was observed. AP conjugate-lipid binding could be reduced by pretreatment of chromatograms with polyisobutylmethacrylate. Addition of Tween 20 also abolished AP-lipid binding, except to lactosyl ceramide, suggesting a degree of specificity. This study serves to prevent spurious interpretation of AP-conjugate based binding assays, be they against purified lipids/lipid mixtures or tissue samples from which lipids have not been removed.
Subject(s)
Alkaline Phosphatase/metabolism , Immunoconjugates/metabolism , Immunoenzyme Techniques/methods , Immunoglobulins/metabolism , Phospholipids/metabolism , Sphingolipids/metabolism , Alkaline Phosphatase/chemistry , Antibody Specificity , Chromatography, Thin Layer , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Immunoconjugates/chemistry , Immunoglobulins/chemistry , Polysorbates/pharmacologyABSTRACT
We investigated the effect of salinity on the relationship between Na(+)-K(+)-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na(+)-K(+)-ATPase activity, especially in Na(+)-K(+)-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na(+)-K(+)-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na(+)-K(+)-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na(+)-K(+)-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na(+)-K(+)-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.
