CHYMOTRYPTIC DIGESTION OF THE CYTOPLASMIC DOMAIN OF THE BETA-SUBUNIT OF NA K-ATPASE ALTERS KINETICS OF OCCLUSION OF RB+ IONS/

This paper demonstrates that specific chymotryptic digestion of the cy toplasmic domain of the beta subunit of Na/K-ATPase leads to changes i n the kinetics of occlusion of Rb+ ions. The experiments utilize exten sively trypsinized Na/K-ATPase, ''19-kDa membranes,'' which lack cytop lasmic loops of the alpha subunit, whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three fragments of 8.1-11.7 kDa) containi ng transmembrane segments and extracellular loops are intact. The beta subunit is partially split into NH2- and COOH-terminal fragments of 1 6 and approximate to 50 kDa, respectively. Cation occlusion and ouabai n binding are preserved. The 19-kDa membranes were incubated, at 37 de grees C, with a selection of proteases, in the presence of Rb+ ions. I n these conditions, only alpha-chymotrypsin destroyed the ability to o cclude Rb+ ions. This process was associated with truncation of the 16 -kDa fragment of the beta subunit in two stages. In the first stage, c hymotrypsin removed 10 residues from the 16-kDa fragment to form a 15- kDa fragment (NH2-terminal Ile(15)) and 4 or 6 residues from the NH2 t erminus of the alpha subunit fragment beginning at Asp(68). In these m embranes Rb+ occlusion was still intact at 37 degrees C. Strikingly, h owever, deocclusion of two Rb+ ions, which is characteristically bipha sic in 10-kDa membranes, displayed deocclusion kinetic with mainly one fast phase. These membranes also showed a much lower affinity for Rb ions compared with 19-kDa membranes; and, consistent with the lower R b+ affinity, Rb+ ions, at nonsaturating concentrations, protected less well against thermal inactivation of Rb+ occlusion. In the second sta ge, the 15-kDa fragment was truncated further to a 14-kDa fragment (NH 2-terminal Leu(24)), followed by thermal destabilization of Rb+ occlus ion even at high concentrations of Rb+ ions. Eventually, the thermally inactivated complex of fragments of alpha and beta subunits was diges ted to the limit peptides. The results suggest that the cytoplasmic do main of the beta subunit interacts with that of the alpha subunit, pos sibly with residues leading into the first transmembrane segment, and controls access of Rb+ ions into or out of the occlusion sites.