Membrane ATPase

Ion-motive ATPases membrane-bound enzymes which, as part of their catalytic cycle, couple the transport of one or more ionic species across the membrane in which they are located, to either the hydrolysis of ATP to ADP and P or to the synthesis of ATP from ADP and Pj. They comprise 3 major classes P-type ATPases (see), F-type ATPases (see) and V-type ATPases (see). 

Wolosin JM, Forte JG. Stimulation of oxyntic cell triggers K and CL conductances in apical H -K -ATPase membrane. Am J Physiol 1984 246 C537-C545. 

Frank, J. Zouni, A. van Hoek, A. Visser, A. J. W. G. Clarke, R. J. Interaction of the fluorescent probe RH421 with ribulose-l,5-bisphosphate carboxylase/ oxygenase and with Na , K" -ATPase membrane fragments. Biochim. Biophys. Acta, Biomembr. 1996, 1280, 51-64. 

A) cytochrome oxidase-dimyristoyl phosphatidylcholine complexes of the indicated lipid/protein mole ratios, at T = 32°C B) (Na , K )-ATPase membranes at T = 8°C ... 

The oligomycin-sensitive ATPase complex, a major enzyme of the mitochondrial inner membrane, can serve to illustrate some of these principles. The ATPase complex is a water-insoluble enzyme which spontaneously associates into vesicular membranes in the presence of phospholipids. The ultrastructural appearance of the reconstituted ATPase membranes is similar to that of the native inner mitochondrial membrane. The complex consists of at least 10 different subunit polypeptides which have been resolved into 3 components, each with a measurable function (1) Five of the polypeptides are part of a catalytic unit called Fi, a water-soluble ATPase which neither requires phospholipids for activity nor has any detectable capacity for binding phospholipids. (2) Four other polypeptides form a unit which has no presently known enzymatic function, but when combined with Fi modifies its physical and catalytic properties. The proteins of this unit are extremely insoluble in water and can combine with phospholipids to form membranes. One of the proteins of the membrane unit is characterized by an unusually large proportion of nonpolar amino acids and a high affinity for phospholipids. (3) The third component (OSCP) is a single polypeptide whose function is to link F to the hydrophobic membrane unit. In the purified state, this protein is completely water soluble. ... 

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

Active Transport. Maintenance of the appropriate concentrations of K" and Na" in the intra- and extracellular fluids involves active transport, ie, a process requiring energy (53). Sodium ion in the extracellular fluid (0.136—0.145 AfNa" ) diffuses passively and continuously into the intracellular fluid (<0.01 M Na" ) and must be removed. This sodium ion is pumped from the intracellular to the extracellular fluid, while K" is pumped from the extracellular (ca 0.004 M K" ) to the intracellular fluid (ca 0.14 M K" ) (53—55). The energy for these processes is provided by hydrolysis of adenosine triphosphate (ATP) and requires the enzyme Na" -K" ATPase, a membrane-bound enzyme which is widely distributed in the body. In some cells, eg, brain and kidney, 60—70 wt % of the ATP is used to maintain the required Na" -K" distribution. 

FIGURE 10.8 A schematic diagram of the Na, K -ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ions are transported out of the cell, and ions are transported in. The transport stoichiometry is 3 Na out and 2 in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na, K -ATPase by binding on the extracellular surface of the pump protein. 

Na, K -ATPase, a K ATPase Ca-+-ATPase, SR Ca" -ATPase, plasma membrane H -ATPase, yeast H" -ATPase, Neurospora... 

FIGURE 10.13 Some of the sequence homologies in the nucleotide binding and phosphorylation domains of Na, K -ATPase, Ca -ATPase, and gastric H, K -ATPase. (Adapted from j0rgensm, P. L., and Andersen, J. R, 1988. Structnral basis for Ei - E2 confoyinational transitions in Na, K -pnmp and Cc -pnmp proteins. Journal of Membrane Biology 103 95-120)... 

M FIGURE 10.14 The arrangement of Ca -ATPase in the sarcoplasmic reticulum membrane. Ten transmembrane segments are postulated on the basis of hydropathy analysis. 

The trigger for all musele eontraetion is an increase in Ca eoneentration in the vicinity of the muscle fibers of skeletal muscle or the myocytes of cardiac and smooth muscle. In all these cases, this increase in Ca is due to the flow of Ca through calcium channels (Figure 17.24). A muscle contraction ends when the Ca concentration is reduced by specific calcium pumps (such as the SR Ca -ATPase, Chapter 10). The sarcoplasmic reticulum, t-tubule, and sarcolemmal membranes all contain Ca channels. As we shall see, the Ca channels of the SR function together with the t-tubules in a remarkable coupled process. 

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes FjFo-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis. 

It is possible that the stationary-state situations leading to an active ion transport occur only in localized regions of the membrane, i.e., at ATPase molecule units with diameters of about 50 A and a length of 80 A. The vectorial ion currents at locations with a mixed potential and special equipotential lines would appear phenomenologically like ionic channels. If the membrane area where the passive diffusion occurs is large, it may determine the rest potential of the whole cell. 

Additional cellular events linked to the activity of blood pressure regulating substances involve membrane sodium transport mechanisms Na+/K.+ ATPase Na+fLi countertransport Na+ -H exchange Na+-Ca2+ exchange Na+-K+ 2C1 transport passive Na+ transport potassium channels cell volume and intracellular pH changes and calcium channels. 

Cardiac glycosides (CG) are potent and highly specific inhibitors of the intrinsic plasma membrane Na+/K+-ATPase, also known as the sodium pump. They modulate electrophysiological properties of the heart and its contractile functions. 

The pharmacological receptor of cardiac glycosides is the sarcolemmal Na+/K+-ATPase expressed on most eucaryotic membranes. It was characterised biochemically in 1957 by J. Skou, who was awarded with the Nobel Prize in chemistry in 1997. The sodium... 

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