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ry effect Decitabine was distinct for Naand independent ofanions. Phosphorylation was insensitive to ouabain butstimulated by furosemide with an EC50 of 1.80.54 mM.Additionally, 0.5 mM ADP partiallyinhibited it.Phosphorylation was also sensitive to alkaline pH andhydroxylamine, suggesting an acylphosphate bond associatedwith the 100 kDa polypeptide in the enzyme.A minimum reaction cycle for the NaATPase was proposedin which the enzyme has an E1 type which will bephosphorylated from ATP in the presence of Mg2andNa, generating the E1.P.Na type, sensitive to ADP.Furosemide stabilizes the E1.P.Na type. The enzyme thenchanges to the E2.P.Na type, insensitive to ADP, which issusceptible to dephosphorylation. A conformational changeinduces Natranslocation by means of the membrane.
Later, aphosphorylated intermediate associated with the ouabaininsensitiveNaATPase was identified by De Souza et al.in microsomal fractions of cultured MDCK I cells andby Ventrella et al. 2010in Decitabine homogenate fractions of ratkidney and microsomal fractions of rainbow trout gills. Botharticles have various discrepancies, but the most important isthat furosemide totally inhibits the Nastimulated phosphorylationin MDCK cells but enhances phosphorylation in ratkidney and trout gills. The data emerging from these studies,which used homogenates or microsomal fractions in whichdifferent ATPase and phosphatase activities coexist, are verydifficult to interpret. However, the results obtained with thepurified NaATPase demonstrated that furosemide stabilizesthe phosphorylated intermediate in an E1.P.Na type, sensitiveto ADP, increasing the observed phosphorylation.
Cloning in the ouabaininsensitive NaATPaseThe atna complementary DNAthat codes for theouabaininsensitive, Kindependent, Doxorubicin NaATPase wasrecently cloned from guinea pig intestinal epithelial cells. It was amplified bytwo strategies based on degenerate PCR.The first method was based on the use of degenerateprimers developed from consensus sequences for the two bestconservedPtype ATPase structural motifs, since the ouabaininsensitiveNaATPase has characteristics of this protein family.This strategy allowed seven Ptype ATPase cDNAs to becloned, which belonged to subtypes P2A, P2B, and P2C. They integrated a new ATPasecDNA fragment of 902 bp, strongly related to atp1a1, whichwas named atna.
The second strategy was based on successive reverse transcriptionPCRand heminested PCR, whichemployed primers targeted PARP to the three peptides identified bytandemmass spectrometry in the purified ouabaininsensitiveNaATPase. Interestingly, these three peptides are sharedby the αsubunit in the Naand NaKATPases. Asexpected, when this strategy was applied, two distinct cDNAfragments had been cloned: 1 fragment corresponded to the α1isoform of NaKATPaseand the other matchedwith the atna fragment, cloned in the first strategy.The sequence of guinea pig atna cDNAwas completed byRLMRACE for 5and 3ends. It has 2,787 nucleotides thatinclude the following:the 5untranslated regionof 163 residues that begins with adenosine;an openreading frameof 2,436 bases that encodes a proteinwith 811 amino acids; anda 3untranslated region188 bases long in which the polyAsignal and polyAsite,important for messenger RNAmaturation, wereidentified.
It was demonstrated that this cDNA codes forthe ouabaininsensitive NaATPase by means of silencing experimentsin MDCK cells, a dog kidney cellular lineage thatexpress a Kindependent, ouabaininsensitive NaATPase. The atna Doxorubicin cDNA was cloned from MDCK cells,employing the second strategy applied in guinea pig. A specificsmallinterfering RNA was developed from this cDNAsequence, and interference experiments had been performed inMDCK cells. The silencing in the atna cDNA specificallyinhibited both the ouabaininsensitive NaATPase activityand the expression of its αsubunit.Structural analysis of ATNA proteinThe ATNAencoded protein has 811 amino acids with a probablemolecularweight of 88,940 Da and an estimated pI of 5.70.As shown in Fig.
5a, the amino acid sequence in the ATNAprotein has all Ptype ATPases structural motifs described forthis protein family, which includes the Ptype ATPasesignaturemotifDKTGTT,the dehalogenasemotifand the phosphatasemotif.The amino acid residues considered essential for PtypeATPase functionseem to be present in ATNA.Sequence alignment Decitabine by means of ClustalWandthreedimensional topology prediction by CPHmodels 3.0programallow the homologous residues atthe corresponding positions described for AT1A1PIG andSERCA1RABIT ATPases, whose crystalline structure waspreviously elucidated, to be identified inATNA. The homology comparison is summarized inTable 1. Actually, all essential residues are identical inATNA and AT1A1 and differ in only 1 position fromSERCA1.Despite the fact that it is reasonable to suppose that homologous residuesplay similar functions, this needs experimental demonstration.Nevertheless, homology analysis stronglysuggests that Doxorubicin ATNACAVPO has the amino acid residuesessential for ATP hydrolysis, includingthe phosphorylatable amino