The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase

Pig kidney Na+,K+‐ATPase

“…The 368-375 region is assumed to be a common structural element for the catalytic site of all known transport ATPases (including FoFiATPases) [3 11. Another region (496-506) of the asubunit, apparently a part of the enzyme catalytic site, contains lysine residue (501) which can be modified with an irreversible inhibitor of the enzyme -fluorescein isothiocyanate [32,33]. The cytoplasmic region comprising the major part of the protein molecule includes the catalytic site [2,3,34], where the N-terminus is localized [34,35].…”

Family of human Na+,K+‐ATPase genes Structure of the gene for the catalytic subunit (αIII‐form) and its relationship with structural features of the protein

“…The total number of amino acid distinctions in these proteins is 139, of them 32 are concentrated in the N-terminus, the most variable part of E1E2 ion-transporting ATPases (residues 1-59), the sequence of the first 11 residues completely differs from the corresponding regions of other forms. The second cluster of variability (residues 478-487) directly precedes the lysine residue modified with fluorescein isothiocyanate [17,18] and probably pertains to the ATP-hydrolysing site. The homology degree of the remaining regions of the compared polypeptides is -90%.…”

Inactivation of the Na,K‐ATPase by modification of Lys‐501 with 4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulfonic acid (SITS)

“…It is not yet clear why SITS apparently covalently modifies Lys-501, while preliminary data suggests that H:DIDS is attached to a lysine residue closer to the carboxyl-terminus [10]. Lys-501 has been shown to be labeled by FITC (fluorescein isothiocyanate) which also inactivates the Na°K-ATPase [11,12] and also by NIPI (N-(2-nitro-4-isothiocyanophenyl)-imidazole) [13,14]. SITS, FITC, and NIPI are all aryl isothioeyanates.…”

“…The 300 A pore size CI8 (218-TP-5415) and C4 {214-TP-5415~ reverie-phase columns ~'~ere from the Sep~trations Group (Vyduc). A LDC-Milton Roy HPLC system was used as described previously [12]. Peptides were sequenc~xl on a Porton 2090E gas phase sequenator.…”