A 19-kDa C-terminal tryptic fragment of the alpha chain of Na/K-ATPase is essential for occlusion and transport of cations.

Abstract

Tryptic digestion of pig renal Na/K-ATPase in the presence ofRb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the et chain, a largely intact ( chain, and essentially normal Rb-and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-ValAsn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. We conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding site, located on the cytoplasmic domain of the a chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect-mediated, presumably, by conformational changes of the protein.The Na/K-ATPase or Na/K-pump has been purified, cloned, and sequenced (1,2). Much is known about transport modes, ion occlusion, and conformational changes (3). Nevertheless, the central question-how the free energy of hydrolysis of ATP is transduced into active pumping of cations-remains unanswered. ATP-binding residues are located on the central cytoplasmic loop of the a chain (1, 2), but little is known about the cation binding sites or pathway for cation movement (for a review, see ref. 4). Information on the cation binding sites would undoubtedly clarify the mechanism of coupling. In particular, by knowing whether the cation and ATP sites were adjacent or quite separate, one could distinguish between more or less direct mechanisms.One approach to identifying cation-binding residues is chemical labeling. Carboxyl residues are good candidates, and hence reagents such as N,N'-dicyclohexylcarbodiimide (DCCD) have been used extensively (5-8). We (8, 9) have studied inactivation by DCCD of Rb and Na occlusion and concluded that carboxyl residues are involved and Rb and Na bind to the same residues. Incorporation of about 2 mol of DCCD per mol of a chain accompanies full inactivation of Rb occlusion (10). Subsequent attempts to locate the label in the primary sequence could exploit selective proteolysis (11). In control experiments designed to produce extensive digestion, we were surprised to find a condition in which occlusion was retained while the a chain was largely but incompletely fragmented. In this condition a stable 19-kDa membraneemb...

Transmembrane Carboxyl Residues Are Essential for Cation-dependent Function in the Gastric H,K-ATPase

“…While this site is conserved within the Na,K-ATPase, it does not appear to be the major site of DCCD modification, despite structural similarities revealed by tryptic cleavage patterns (39,45). In each pump, tryptic cleavage in the presence of K ϩ produces a C-terminal peptide of similar size that is resistant to further cleavage.…”

Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

“…The detection of Asn 831 on the intracellular side of the plasma membrane initiated far reaching conclusions about the folding of the ␣-subunit. As the final result of this investigation, a model with 10 transmembrane spans was preferred over one with 8, although the latter could not be excluded (12,18).…”

“…In several studies, the detection of single amino acids on either the extracellular or intracellular surface of the plasma membrane was used together with the hydropathy data as a basis for the analysis of the transmembrane topology. Asn 831 is the amino-terminal amino acid of a 19-kDa hydrophobic fragment derived from the ␣-subunit that is involved in Rb ϩ occlusion (18,19). This fragment can be generated when Na ϩ ,K ϩ -ATPase in inside-out vesicles is extensively digested by trypsin.…”

Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

“…The noncompetitive binding disappears in the experiments with 19kDa membranes produced by extensive tryptic digestion of renal Na,KATPase. This preparation consists of membraneembedded fragments corresponding to transmembrane segments and intact short hairpin loops on the extracellular side whereas most of the cytoplasmic parts of the protein, including the ATP-binding site, have been digested away [13,48,78]. The competitive binding can be explained by a reaction scheme in which Mg 2+ (or any other cation) is allowed to bind to the first site whereas, to the second site near the membrane surface, only monovalent cations are able to bind (Fig.…”