Immunochemical and spectroscopic characterization of two fluorescein 5'-isothiocyanate labeling sites on sodium-potassium ATPase

Abstract

Fluorescein 5'-isothiocyanate (FITC) covalently modifies the Lys-501 residue of the catalytic (alpha) subunit of Na+,K(+)-ATPase and resides at a conformation-sensitive site in or near the ATP binding site. In these studies, FITC-directed antibodies which quench this hapten's fluorescence were used to infer the solvent accessibility of the enzyme-bound probe. These antibodies identified two FITC labeling populations. An antibody-accessible population, representing 20-50% of the bound FITC fluorescence, was essentially (95%) quenched by the antibody. The second population was irreversibly labeled, was inaccessible to antibody, and was the fraction of probe whose fluorescence intensity is sensitive to the enzyme's conformation. The anti-FITC antibodies therefore permitted the selective investigation of FITC at this active site. Distinct differences between the two labeling sites were then demonstrated. Shifts in the absorption spectrum suggested that the active-site-bound probe resides in a hydrophobic environment, while polarization values indicated a rigid, rotationally restricted location. These two properties were not altered by ligand additions. Iodide quenching studies, however, showed that in the E1Na+ conformation there was a 50% decrease in solvent access to the active-site-bound probe as compared to free probe while the E1Na(+)----E2K+ transition decreased this accessibility an additional 50%. Similarly, there was a significant decrease in the relative quantum yield of FITC linked at this site that was reduced further by the E1Na(+)----E2K+ transition. In contrast, frequency domain spectroscopy showed no significant differences in the lifetimes of fluorescence decay for the two different labeling populations nor for the high (E1Na+) and low (E2K+) fluorescence intensity conformations. We have found that static (lifetime independent) quenching rather than collisional processes or protonation changes accounts for the fluorescence intensity changes undergone by FITC bound at the ATP-protectable site.

Enzyme‐catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation

“…The spectrum of FITC bound at the uridine subsite is red-shifted about 5 nm relative to that for the free fluorescein. The environment at this subsite appears to be similar to that observed for fluorescein in ethanol [10] and for FITC bound at the conformation-sensitive site of Na+,K+-ATPase from lamb kidney [11]. These data agree with FITC residing in a hydrophobic pocket of the UPase active site.…”

Mechanism for reactivation of the ATP‐sensitive K+ channel by MgATP complexes in guinea‐pig ventricular myocytes.

“…FITC is known to react with the nucleotide-binding sites on various ATPases with a high specificity for ATP (Muallem & Karlish, 1983;Scott, 1985;Farley & Faller, 1985;Abbott, Amler & Ball, 1991). Since reactivation of the K+ATP channel also showed a high specificity to ATP (Fig.…”

“…FITC reacts with the lysine residue of the nucleotidebinding sites on various ATPases with high specificity for ATP over other NTPs, such as Ca2+-ATPases in the sarcoplasmic reticulum (Scott, 1985) or erythrocyte (Muallem & Karlish, 1983), Na+,K+-ATPases (Abbott et al 1991), and H+,K+-ATPase (Farley & Faller, 1985), inhibiting ATP binding to these sites and their enzyme activity. Since reactivation of the run-down KATP channels also showed a high specificity for ATP (Fig.…”

Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site

“…Consequently, the E 2 ATP site seems to be sterically more open as compared with the E X ATP site. The fact that the high-affinity binding site (EiATP site) is sterically poorly accessible was shown previously by FITC labeling [19].…”