Barry J.R. Pitts

Inhibition of bovine heart Na+, K+-ATPase by palmitylcarnitine and palmityl-CoA.

Abstract The activity of a partially purified bovine heart Na+,K+-ATPase is inhibited by DL- and L- palmitylcarnitine (I50=44–48μM). Palmitylcarnitine with a I50 of 25μM also markedly inhibits K+-phosphatase activity. Palmityl-CoA decreases Na+,K+-ATPase activity, but to a lesser extent (I50=80μM). Both palmitic acid and hexanoic acid produce 10 to 15% inhibition of activity at concentrations of 70μM and 3–5mM, respectively. These free fatty acids protect the enzyme against inhibition by 40μM palmitylcarnitine. However, at 50μM palmitylcarnitine, the protective effect by hexanoic acid is no longer apparent. Addition of 40μM palmitylcarnitine to the Na+,K+-ATPase in the presence of varying concentrations of palmityl-CoA produces an additive inhibition of enzyme activity, suggesting two different sites on the enzyme susceptible to inhibition by the two ester forms of the fatty acid.

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: A possible role in myocardial ischemia

Abstract Palmitylcarnitine is a time-dependent inhibitor of the Ca 2 + -ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.

Improved purification and partial characterization of (Na+, K+)-ATPase from cardiac muscle.

A method is described for purification of (Na+, K+)-ATPase which yielded approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 mumol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600-700 pmol of [3H]ouabain/mg protein when incubated in the presence of either Mg2+ plus Pi, or Mg2+ plus ATP plus Na+, and incorporated more than 600 pmol 32P/mg protein when incubated with gamma-32P-labelled ATP in the presence of Mg2+ and Na+. The preparation is approximately 35% pure.

A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney.

Abstract The rates of association of [3H]ouabain to Na+,K+-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na+,K+-ATPase being the fastest. Although the major determinant of the affinity of the Na+,K+-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na+,K+-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I50 (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na+,K+-ATPase activity, approached the value calculated from [3H]ouabain binding. The ratio of the I50 value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na+,K+-ATPase is not the reason for the faster dissociation rate of this enzyme.

Nature of the transport ATPase-digitalis complex: VI. purification of and ouabain binding to a highly active cardiac Na+,K+-ATPase

Abstract A Na+,K+-ATPase has been isolated from canine heart with a specific activity as high as 200 μmoles of inorganic phosphate/mg protein/hour. Activity is not due to simple detergent activation since specific ouabain binding (i.e., [Mg++,Na+,ATP] or [Mg++,Pi]-ligand dependent) ranged from 200–450 pmoles/mg protein. Specific ouabain binding activities are up to ten times greater than heretofore reported.

Cardenolide analogs. An explanation for the unusual properties of AY 22241

AY 22241 (AY) was compared with digitoxigenin with respect to effects on myocardial contractility and inhibition of Na+, K+-ATPase. Ay was less potent than digitoxigenin but otherwise showed the same type of rapid onset and rapid reversal of activity usually associated with genins. Since AY is a glycoside, its behaviour was regarded as anomalous since glycosides usually show both slow onset and slow reversal of activity. Attempts were made to account for the genin-like properties of AY in terms of the model for the digitalis receptor proposed by Thomas et al. (1974a,b). It is suggested that the low potency and genin-like properties of AY may be due to the fact that the steroid is attached to the lactone through the carbon atom that is alpha to the carbonyl group. This feature could result in non-alignment of the sugar residue with the sugar binding site and might also reduce the effectiveness of the interaction of the steroid moiety with its binding site. A notable feature of AY toxicity was its extremely rapid progression to irreversible fibrillation.

Effect of palmitylcarnitine on ouabain binding to Na, K-ATPase.

Abstract Palmitylcarnitine, an endogenous long-chain fatty acyl ester, inhibited cardiac Na, K-ATPase activity and binding of [ 3 H]ouabain to the enzyme. The inhibitory effects on enzyme hydrolytic activity and drug binding were time and concentration dependent, but also dependent upon the ratio of palmitylcarnitine to protein. Palmitylcarnitine inhibitory effects were irreversible, but could be prevented by bovine serum albumin. In the presence of Mg 2+ + ATP or Mg 2+ + Pi, [ 3 H]ouabain binding was fully inhibited by 100 μ m palmitylcarnitine. The addition of sodium, or sodium plus potassium to the drug-binding medium reduced the inhibitory effect. The protective action of Na + was concentration dependent and was optimal at 75 μ m palmitylcarnitine. Equimolar amounts of choline chloride did not have the same protective effect as sodium chloride. Binding of ouabain to the enzyme in the absence of palmitylcarnitine prevented the protective effect of Na + . Inhibition of Na, K-ATPase functional properties occurred at a concentration range of palmitylcarnitine reported to occur in the cytosol of ischemic cells during episodes of experimental myocardial ischemia. It is suggested that elevated levels of palmitylcarnitine in ischemic myocardium may play a role in altering cellular function as well as the inotropic response of ischemic cardiac muscle to digitalis glycosides.

On the lack of inotropy of cardiac glycosides on skeletal muscle: A comparison of Na+,K+-ATPases from skeletal and cardiac muscle☆

Abstract Comparison of Na,K-ATPase from skeletal and cardiac muscle revealed that, although the skeletal muscle enzyme was only slightly less sensitive to inhibition by ouabain, the rates of [ 3 H]ouabain binding to, and dissociation from, the skeletal enzyme were much faster than the corresponding rates for the cardiac enzyme. The skeletal muscle enzyme required higher concentrations of potassium to stabilize the ouabainenzyme complex and to stimulate the K + -phosphatase activity. The K + -phosphatase activity was only 8% of the Na,K-ATPase activity of the skeletal muscle enzyme, compared to 22% for the cardiac preparation. The glycoprotein subunit found in Na,K-ATPases from cardiac and many other tissues appeared to be absent in the enzyme from skeletal muscle. The differences in binding and dissociation rates for ouabain suggest that there may be significant differences in the structure of the digitalis receptor in the two enzymes. The I 50 for ouabain inhibition of the skeletal muscle Na,K-ATPase was, however, only slightly higher than for the cardiac enzyme, suggesting that the lack of an inotropic effect of cardiac glycosides on skeletal muscle could not be due to failure of the digitalis drugs to bind to and inhibit the membrane-linked sodium pump.

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases: Effect on calcium binding

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.