Cyclic AMP-plus ATP-dependent modulation of the NADH oxidase activity of porcine liver plasma membranes.
Plasma membranes of porcine liver, highly purified by aqueous two-phase partition, oxidized NADH in the absence of added external acceptors. The oxidation was resistant to cyanide and responded to nanomolar concentrations of ATP alone or ATP in the presence of cyclic AMP. Both the Km for NADH and the long-term activity of the oxidase were affected. Upon incubation at 37 degrees C with cyclic AMP (0.1-10 nM) and ATP (1-100 nM), the NADH oxidase activity was inhibited. The inhibition was complex and due to an approx. 5-fold increase in the Km for NADH compared to the NADH oxidase of membranes incubated in the absence of cyclic AMP + ATP. The response to cAMP + ATP was rapid and occurred within seconds of ATP addition. The response was inhibited by the selective inhibitor of cyclic AMP-dependent protein kinase, H-89. Neither cyclic AMP alone nor ATP alone at nanomolar concentrations elicited a rapid response. However, 10 nM ATP alone did result in similar alteration of Km and Vmax as did ATP + 0.1 nM cyclic AMP. The response to ATP alone or in preparations depleted of cyclic AMP required higher ATP concentrations than with cAMP present or occurred more slowly with a lag of 1-2 min. The NADH oxidase activity of porcine plasma membranes after cyclic AMP + ATP treatment retained high activity with storage at 4 degrees C, whereas that of unincubated or sham-incubated plasma membranes was reduced with time of storage at 4 degrees C. In some but not all instances, NADH oxidase activity inactivated by incubation with NADH at 37 degrees C or after storage at 4 degrees C could be reactivated by incubation with cyclic AMP plus ATP. As with the alteration in Km, cyclic AMP alone was without effect and ATP alone was much less effective than the combination. The results demonstrate ATP-dependent modulation of the NADH oxidase activity of isolated plasma membranes at physiological concentrations of ATP. This modulation may have functional significance in mediating the hormone and growth factor responsiveness of the plasma membrane NADH oxidase activity.
Morré
D J
DJ
Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907.
Navas
P
P
Rodriguez-Aguilera
J C
JC
Morré
D M
DM
Villalba
J M
JM
de Cabo
R
R
Lawrence
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015415:Biomarkers; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D000242:Cyclic AMP / Q000378:metabolism*; D004795:Enzyme Stability; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D013552:Swine
D015415:Biomarkers; D007546:Isoquinolines; D009097:Multienzyme Complexes; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide