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ry effect Decitabine was particular 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 on the enzyme.A minimum reaction cycle for the NaATPase was proposedin which the enzyme has an E1 form that could bephosphorylated from ATP in the presence of Mg2andNa, creating the E1.P.Na form, sensitive to ADP.Furosemide stabilizes the E1.P.Na form. The enzyme thenchanges to the E2.P.Na form, insensitive to ADP, which issusceptible to dephosphorylation. A conformational changeinduces Natranslocation through 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 a number of discrepancies, but the most important isthat furosemide completely inhibits the Nastimulated phosphorylationin MDCK cells but enhances phosphorylation in ratkidney and trout gills. The data emerging from these studies,which applied homogenates or microsomal fractions in whichdifferent ATPase and phosphatase activities coexist, are verydifficult to interpret. On the other hand, the results obtained with thepurified NaATPase demonstrated that furosemide stabilizesthe phosphorylated intermediate in an E1.P.Na form, sensitiveto ADP, increasing the observed phosphorylation.
Cloning on the ouabaininsensitive NaATPaseThe atna complementary DNAthat codes for theouabaininsensitive, Kindependent, Doxorubicin NaATPase wasrecently cloned from guinea pig intestinal epithelial cells. It was amplified bytwo methods according to degenerate PCR.The very first method was according to the use of degenerateprimers created from consensus sequences for the two bestconservedPtype ATPase structural motifs, considering that the ouabaininsensitiveNaATPase has functions of this protein loved ones.This method allowed seven Ptype ATPase cDNAs to becloned, which belonged to subtypes P2A, P2B, and P2C. They included a new ATPasecDNA fragment of 902 bp, strongly related to atp1a1, whichwas named atna.
The second method was according to successive reverse transcriptionPCRand heminested PCR, whichemployed primers targeted PARP to the three peptides identified bytandemmass spectrometry on the purified ouabaininsensitiveNaATPase. Interestingly, these three peptides are sharedby the αsubunit on the Naand NaKATPases. Asexpected, when this method was applied, two distinct cDNAfragments had been cloned: a single fragment corresponded to the α1isoform of NaKATPaseand the other matchedwith the atna fragment, cloned in the initial method.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 lengthy in which the polyAsignal and polyAsite,necessary for messenger RNAmaturation, wereidentified.
It was demonstrated that this cDNA codes forthe ouabaininsensitive NaATPase through 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 method applied in guinea pig. A specificsmallinterfering RNA was created from this cDNAsequence, and interference experiments had been performed inMDCK cells. The silencing on 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 on the ATNAprotein has all Ptype ATPases structural motifs described forthis protein loved ones, including the Ptype ATPasesignaturemotifDKTGTT,the dehalogenasemotifand the phosphatasemotif.The amino acid residues considered important for PtypeATPase functionseem to be present in ATNA.Sequence alignment Decitabine through 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. In reality, all important residues are identical inATNA and AT1A1 and differ in only a single position fromSERCA1.Even though it is reasonable to suppose that homologous residuesplay equivalent functions, this requires experimental demonstration.Nevertheless, homology analysis stronglysuggests that Doxorubicin ATNACAVPO has the amino acid residuesessential for ATP hydrolysis, includingthe phosphorylatable amino