Erythrosin 5′‐isothiocyanate labels Cys⁵⁴⁹ as part of the low‐affinity ATP binding site of Na⁺/K⁺‐ATPase¹

Abstract

The high-affinity E I ATP site of Na + /K + -ATPase labeled with fluorescein 5P-isothiocyanate and its E 2 ATP site labeled with erythrosin 5P-isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998)

Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities

“…At high ATP concentrations, RB and EB inhibit the activity arisen from the low-affinity site non-competitively. Some fluorescein-related pseudo ATP analogs have been shown to be useful probes for biochemical study of the two ATP binding sites of P-type ATPase [36,37,38]. RB and EB inhibit the two ATP binding sites differently; therefore, this result suggests that these fluorescein analogs may also be a useful tool for unraveling the functional significance of the two nucleotide-binding sites of SecA.…”

The phosphatase activity of the isolated H4‐H5 loop of Na+/K+ ATPase resides outside its ATP binding site

“…It remains, however, unclear whether F548 directly interacts with ligands or whether its importance lies in the formation of the structural backbone of the ATP binding pocket. The adjacent amino acid residue C549, was shown to be labeled by erythrosin isothiocyanate in the membrane‐embedded Na + /K + ATPase after blocking of the E 1 ATP binding site with FITC [16]. Modification of this site with the sulfhydryl‐reactive 8‐thiocyano‐ATP forming a mixed disulfide bridge may inactivate Na + /K + ATPase [63].…”

“…The observation of high and low affinity ATP sites with approximate K d values of 1 µ m (E 1 ATP site) and 200 µ m (E 2 ATP site) in the membrane‐embedded Na + /K + ATPase, and the finding that reaction inert MgATP complex analogues such as Cr(H 2 O) 4 ATP and Co(NH 3 ) 4 ATP may react specifically with these ATP sites [6] as well as the complex kinetics with the fluorescent 2′(3′)‐ O ‐(6‐ N ′, N ′‐dimethylaminonaphthalenesulfonyl)ATP (DANSyl‐ATP) [7,8], lead to the suggestion that high and low affinity ATP sites coexist and that they interact during catalysis. Consistent with this conclusion is the finding that the activity of a K + activated phosphatase, which represents a partial function of the ATP site [9–12], was almost unaffected by the blockade of the ATP site due to modification of K501 with fluorescein isothiocyanate (FITC) [13], but was lost when the enzyme additionally reacted with Co(NH 3 ) 4 ATP [14,15] or erythrosin isothiocyanate [16]. The latter binds to C549 within the nucleotide (N)‐domain of Na + /K + ATPase [16].…”

“…Consistent with this conclusion is the finding that the activity of a K + activated phosphatase, which represents a partial function of the ATP site [9–12], was almost unaffected by the blockade of the ATP site due to modification of K501 with fluorescein isothiocyanate (FITC) [13], but was lost when the enzyme additionally reacted with Co(NH 3 ) 4 ATP [14,15] or erythrosin isothiocyanate [16]. The latter binds to C549 within the nucleotide (N)‐domain of Na + /K + ATPase [16]. Molecular distance measurements after specific labeling of the high and low affinity ATP sites with these fluorescent probes gave evidence for the existence of an (αβ) 2 dimeric structure [17].…”

Structure-function relationship in P-type ATPases—a biophysical approach

“…Further fluorescent labels applied to monitor conformational changes are eosin Esmann 1981, 1983;Lin et al 1997) and erythrosin 5 0 -isothiocyanate (Linnertz et al 1998a(Linnertz et al , 1998b. Various fluorescent labels which can be bound simultaneously at different locations of the pump protein can also be used to determine distances between selected domains by the Förster resonance energy transfer mechanism (Linnertz et al 1998b).…”