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

Abstract

The sodium pump or Na,K-ATPase, maintains the Na ÷ and K" gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothioeyanatostilbene.2,2'-disulfoni¢ acid (SITS) inhibited tile ATPase activity. Both the labeling of the protein and the loss of ATPase acti~,ity were prevented by co-incubation with ADP (acting as an ATP analog) or KCI, Only the a-subunit was labeled by SITS, The ~t.subunit fi'om ll~e inhibited ellzymc was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino ~tcid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated tlmt SITS modifies Lys-501 (X) on the ct-subunit of Na,K-ATPase.

DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism

“…In regard to Na-KATPase inhibition, DIDS and SITS were considered to be ineffective when applied to the exterior of intact cells because they do not readily penetrate the plasma membrane. This reasoning was based on the notion that their principle mechanism of action was a direct inhibitory effect caused by interaction with a Lys residue on the cytoplasmic portion of P-type ATPases (27,38). In the present study we show evidence that DIDS is able to cause Na-K-ATPase inhibition in ocular nonpigmented ciliary epithelium (NPE) by a mechanism that is associated with SFK activation.…”

Ligand‐Induced Conformational Changes in the Na,K‐ATPase α Subunita

“…Formation of a cross‐link by H 2 DIDS (and presumably DIDS) had been the major source of the difficulty in determining its site of modification in earlier experiments. Previous work from our laboratory, 14 demonstrated that the single isothiocyano‐substituted stilbene, SITS, was an irreversible inhibitor of the Na,K‐ATPase. Like DIDS, SITS inactivation was completely preventable by the presence of either ATP or low K + .…”

“…The segment of this loop located between D369 and G502 is particularly interesting. It has been observed that a variety of aryl isothiocyanates, including NIPI, 13 SITS, 14 and FITC, 17,18 covalently label K501 in the absence of ATP but not in its presence, consistent with K501 residing in or close to the ATP site. Also, the photoaffinity ATP analogs, 2‐azido‐ATP and 8‐azido‐ATP, have been shown to modify K480 and G502, respectively; these modifications are eliminated by the simultaneous presence of ATP 18 .…”

Cys577 Is a Conformationally Mobile Residue in the ATP-binding Domain of the Na,K-ATPase α-Subunit

“…The MIANS-labeled proteins were visualized by illumination with a long wave UV lamp. Only the ␣-subunit was labeled in all cases where MIANS was present (lanes 2-6) hydro-4,4Ј-DIDS (5, 24), 4-acetamido-4Ј-isothiocyanatostilbene-2,2Ј-disulfonate (25), and fluorescein isothiocyanate (6), compounds that also inactivate Na,K-ATPase in an ATP-protectable manner and whose sites of action have been localized to the ATP-binding loop (i.e. K501).…”