Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

Abstract

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms, It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.The basic idea in this paper is a change in the shape of the sodium and potassium pump during its reaction cycle. This change is not necessarily that assumed for translocation (1), but rather a change in the conformation of an active center. The evidence is indirect. It is assumed that the reactivity of a phosphoryl group attached covalently to an active site in an active center depends on the conformation of the active center surrounding the site. Thus a change in reactivity implies a change in conformation. It should be enough to show that this group at a single active site changes its reactivity in the course of the normal reaction sequence. The phosphoryl group is attached to sodiumpotassium-dependent adenosine triphosphatase, an aspect of the pump.

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

“…During the recent years the transport cycle [1,3,27] has been broken down into a series of defined reaction steps and their kinetic properties were investigated and characterized [5,10,11,15,17,40]. In the case of the sodium transport branch the reaction sequence, 3 Na + cyt + E 1 → Na 3 E 1 → ͑Na 3 ͒E 1 -P → P-E 2 ͑Na 3 ͒ → P-E 2 ͑Na 2 ͒ + Na + → P-E 2 + Na + ext , is able to explain all experimental data available.…”

Outside of the box: recent news about phospholipid translocation by P4 ATPases

“…Loss of ions into the luminal space from that conformation is the key to dephosphorylation to the E2 form. The latter is also conformationally unstable, and the enzyme thus converts back into the E1 conformation [1,22].There is a subtlety about this sequence of events which should be mentioned here, particularly as it applies to the E2P conformation. This latter designation must define two conformations; indeed, both have been crystallized in the case of the Ca-ATPase [15,16,27].…”

“…The conformational changes link a binding pocket for the transported ion to either the cytoplasm (the E1 conformation) or the extracytoplasmic, or lumenal space (the E2 conformation). In the classic Post-Albers model [1,22], binding of ions in the E1 conformation enables phosphorylation to the E1P form; this form is conformationally unstable, and converts to the E2P conformation. Loss of ions into the luminal space from that conformation is the key to dephosphorylation to the E2 form.…”

“…Loss of ions into the luminal space from that conformation is the key to dephosphorylation to the E2 form. The latter is also conformationally unstable, and the enzyme thus converts back into the E1 conformation [1,22].…”

Kinetic characterization of Na,K-ATPase inhibition by Eosin

“…Indeed, the homeostatic role of the Na pump is so critical that Na,K-ATPase activity accounts for approximately 23% of ATP hydrolysis in humans during rest [5] and is the major user of ATP in red blood cells from most species. The Na pump reaction cycle is conveniently described by a model proposed independently by Albers and Post [6,7], which describes two major conformations: the first has cation accessibility from the cytoplasm (E in ) and the second has cation access from the extracellular space (E out ). Briefly, the binding of 3 intracellular Na + ions shifts the pump to a conformation where it binds one molecule of ATP to a high affinity nucleotide binding site.…”