Abstract. To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,KATPase, equilibrium Na + -titration experiments were performed using two fluorescent dyes, RH421 1 and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na + in the presence of various Mg 2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na + ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na + ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na + ions and followed by the conformational transition E 1 /E 2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na + ions are bound.

Abstract.In the E 1 state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di-and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br 2 -Titu 3+ is bound with the highest affinity (< M), among the tested divalent cations Ca 2+ binds the strongest, and Na + binds with about the same equilibrium dissociation constant as Mg 2+ (∼0.8 mM). For alkali ions it exhibits binding affinities following the order of Rb + Ӎ K + > Na + > Cs + > Li + . The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na + ions the equilibrium dissociation constant is < 0.01 mM. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na + , no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mM.

Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the ␣ subunit of Na ؉ ,K ؉ -ATPase acts as an entrance port for Na ؉ and K ؉ ions. 1) In defined conditions chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa membranes, produced by extensive proteolysis of Na ؉ ,K ؉ -ATPase, and in parallel inactivates Rb ؉ occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca 2؉ , Mg 2؉ , La 3؉ , p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo-2,4,6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br 2 -TITU 3؉ ). 3) Ca 2؉ ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the ␣ subunit. It appears that negatively charged residues in L6/7 recognize either Na ؉ or K ؉ ions or the competitive cation antagonists. Na ؉ and K ؉ ions are then occluded within trans-membrane segments and can be transported, whereas the cation antagonists are not occluded and block transport at the entrance port. The cytoplasmic segment of the ␤ subunit appears to be close to or contributes to the entrance port, as inferred from the following observations. 1) Specific chymotryptic cleavage of the 16-kDa fragment of the ␤ subunit to 15-kDa at 20°C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309 -10316) markedly reduces affinity for Br 2 -TITU 3؉ and for Na ؉ ions, detected by Na ؉ occlusion assays or electrogenic Na ؉ binding, whereas Rb ؉ occlusion is unchanged. 2) Na ؉ ions specifically protect the 16-kDa fragment against this chymotryptic cleavage.An understanding of the working of P-type active cation pumps such as Na ϩ ,K ϩ -, H ϩ ,K ϩ -, H ϩ -, and Ca 2ϩ -ATPase will require knowledge of high resolution molecular structure. The most detailed structures available are those of Ca 2ϩ -ATPase and H ϩ -ATPase at 8-Å resolution, based on cryoelectron microscopy of two-dimensional crystals. These studies reveal the overall shape of these proteins and presence of 10 trans-membrane ␣-helical rods most of which are tilted at an angle to the membrane (1, 2). These structural studies fit well with the trans-membrane topology of ␣ subunits of P2-type pumps determined with a variety of biochemical techniques (3). Attempts are being made to infer the packing arrangement of the trans-membrane segments (1, 4). Biochemical and molecular studies are providing much information on functional sites for ATP and cations. The cation occlusion sites are located within trans-membrane segments as indicated by proteolysis experiments (5, 6) and site-directed mutagenesis (7,8), and the latter approach suggests that carboxyl and other oxygen-containing side chains of residues within trans-membrane segments M4, M5, and M6, and probably M8, ligate the occluded cations (7-10). Thus, the trans-membrane helices are packed so as to create the cation occlusion "cage...

Eubacterium acidaminophilum combines the oxidation of amino acids such as alanine or valine with the reduction of glycine to acetate in a two-substrate fermentation (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited production of hydrogen and acetate. Experiments with 14 C-labeled carbonate revealed that acetate was formed under these conditions by net reduction of CO 2 /HCO 3 3 ; 14 C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO 2 ; dense cell suspensions under H 2 /CO 2 produced only very small amounts ( 6 0.5 mM) of acetate. There was no activity of carbon monoxide dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochemical and energetic considerations. ß