8.2.3 Enzyme Targets: Adenosine TriphosphataseAdenosine triphosphatase (ATPase, E.C. 3.6.1.3) comprises a group of extremely wide-
spread membrane-bound enzymes. All cell membranes contain the Na+–K+-dependent
enzyme, which, among its other roles, is extremely important in the establishment of the
mitochondrial proton gradient and in bacterial permease systems that transport amino
acids. As a result of the proton gradient in mitochondria, or a very steep Na+–K+gradient,
ATP synthesis rather than ATP hydrolysis can take place, according to the Mitchell
chemiosmotic hypothesis. This model suggests that Na+–K+-activated ATPase located in
neural membranes is responsible for maintaining transmembrane ionic asymmetry, or ion
gradients, across these membranes. Adenosine triphosphatase is phosphorylated by ATP
and then dephosphorylated by K+. The free energy of ATP hydrolysis is used to transport
sodium and potassium against their respective gradients in a 3 Na+:2 K+: 1 ATP ratio, but
Na+must be inside and K+outside the cell in order to activate the enzyme. A rich source
for ATPase isolation is the electroplax of Torpedo orElectrophorus.
8.2.3.1 Physicochemical Properties of ATPase
The Na+–K+–ATPase has been purified and sequenced. It is a dimeric protein with an α
subunit of 100 kD and a βsubunit of 55 kD. The αsubunit contains the ATP hydroly-
sis subsite in which an aspartate accepts the γphosphate of ATP. The function of the
glycoproteinβsubunit is not clear. ATPases from different sources (electroplax, sheep
kidney, rat, bacterial K+pump) have been shown, through cDNA cloning, to have a high
degree of homology, indicating their ancient common evolutionary history.
Determination of the primary sequence, proteolytic digestion, and labeling experiments
have enabled investigators to elucidate the folding of the protein within the cell mem-
brane. There are eight transmembrane domains and a large cytosolic portion. ATP binds
to Lys-501, and Asp-369 is phosphorylated. The cardiac glycoside binding site, which
is partially located on the outside of the αsubunit, inhibits the ATP-driven ion transport
and the Na+-dependent conformational change. Investigators also discovered that the
mammalian brain and heart contain distinct isozymes with different αsubunits show-
ing different steroid binding capabilities. The sarcoplasmic reticulum of muscle cells
contains a Ca^2 +-dependent ATPase, responsible for Ca^2 +concentration changes in the
muscle that are intimately connected with the contraction process.
8.2.3.2 Cardiac Steroid Glycosides and Na+-K+/ATPase
Myocardial cell membrane ATPase, the enzyme present in heart muscle, is the site of
action of the cardiac steroid glycosides, which have a specific action on the heart muscle.
These drugs increase the force of contraction of the muscle (positive inotropic effect) as
well as its conductivity and automaticity. They are also valuable in treating congestive
heart failure, in which the circulatory needs of organs are no longer satisfied, and heart
arrhythmias, in which the rhythm of the cardiac contractions is upset. The effect of the
drug is that the force of contraction increases and the heart rate is slowed (chronotropic
effect). Consequently, the cardiac output is elevated while the size of the heart decreases.
The exterior of the myocardial enzyme, situated in the plasmalemma, is considered
to be the specific binding site of cardiac steroid glycosides. It is believed that the
positive inotropic effect is due to the inhibition of enzyme dephosphorylation and
492 MEDICINAL CHEMISTRY