Stimulation of ouabain binding to Na,K-ATPase in 40% dimethyl sulfoxide bya factor from Na,K-ATPase preparations

In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K -ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215-225, 1995). Furthermore, i n Me2SO the EO complex reacts very slowly with P-i and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzy me formation. Here we found that, unexpectedly, the ouabain binding decreas ed with the enzyme concentration in the Me2SO assay medium. We extracted th e enzyme preparation with Me2SO or chloroform/methanol and demonstrated tha t the extracted (depleted) enzyme bound ouabain poorly. Addition of such ex tracts to assays with low enzyme concentration or depleted enzyme fully res tored the high-affinity ouabain binding. Dialysis experiments indicated tha t the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g ., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney gam ma-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the gamma-proteolipid in comparison to the native untreated enzyme, as demo nstrated by immunoreaction with the antibody. (C) 1999 Academic Press.