Accessibility of tryptophan residues in Na,K-ATPase.
The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.
D020106:Acrylamide; D000178:Acrylamides; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002918:Chymotrypsin / Q000494:pharmacology; D004285:Dogs; D066298:In Vitro Techniques; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000178:Acrylamides; D020106:Acrylamide; D010042:Ouabain; D000244:Adenosine Diphosphate; D014364:Tryptophan; D012964:Sodium; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium