Later, a phosphorylated intermediate associated with the ouabaininsensitive Na ATPase was identified by De Souza et al. in microsomal fractions of cultured MDCK I cells and by Ventrella et al.0 in homogenate fractions of rat kidney and microsomal fractions of rainbow trout gills. Both articles have several discrepancies, but the most important is that furosemide totally inhibits the Na stimulated phosphorylation in MDCK cells but enhances phosphorylation in rat kidney and trout gills. The data emerging from these studies, which used homogenates or microsomal fractions in which different ATPase and phosphatase activities coexist, are very difficult to interpret. However, the results obtained with the purified Na ATPase demonstrated that furosemide stabilizes the phosphorylated intermediate in an E1.P.Na form, sensitive to ADP, increasing the observed phosphorylation . Cloning of the ouabain insensitive Na ATPase The atna complementary DNA that codes for the ouabain insensitive, K independent, Na ATPase was recently cloned from guinea pig intestinal epithelial cells . It was amplified by two strategies based on degenerate PCR.
The first approach was based on the use of degenerate primers designed from consensus sequences for the two bestconserved P type ATPase structural motifs , since the ouabaininsensitive mTOR inhibitor selleck chemicals Na ATPase has features of this protein family. This strategy allowed seven P type ATPase cDNAs to be cloned, which belonged to subtypes P2A , P2B , and P2C . They included a new ATPase cDNA fragment of 902 bp, strongly related to atp1a1, which was named atna. The second strategy was based on successive reverse transcription PCR and hemi nested PCR, which employed primers targeted to the three peptides identified by tandem mass spectrometry of the purified ouabain insensitive Na ATPase . Interestingly, these three peptides are shared by the ? subunit of the Na and Na K ATPases. As expected, when this strategy was applied, two different cDNA fragments were cloned: one fragment corresponded to the ?1 isoform of Na K ATPase and the other matched with the atna fragment, cloned in the first strategy. The sequence of guinea pig atna cDNAwas completed by RLM RACE for 5 and 3 ends.
It has 2,787 nucleotides that include the following: the 5 untranslated region of 163 residues that begins with adenosine; an open reading frame of 2,436 bases that encodes a protein with 811 amino acids; and a 3 untranslated region 188 bases long in which the polyA signal and polyAsite, necessary for messenger RNA maturation, were identified . celestone It was demonstrated that this cDNA codes for the ouabain insensitive Na ATPase through silencing experiments in MDCK cells, a dog kidney cellular lineage that express a K independent, ouabain insensitive Na ATPase . The atna cDNA was cloned from MDCK cells, employing the second strategy applied in guinea pig.