Affiliations: Department of Medicinal Chemistry and Molecular
Pharmacology, HANS Life Sciences Research Building, Purdue University, 201
South University Street, West Lafayette, IN 47907-2064, USA | 1945 Ridgetop Drive, Charlottesville, VA, USA
Note: [] Address for correspondence: D.J. Morré, Department of
Medicinal Chemistry and Molecular Pharmacology, HANS Life Sciences Research
Building, Purdue University, 201 South University Street, West Lafayette, IN
47907-2064, USA. Fax: +1 765 494 4007; E-mail: [email protected]
Abstract: A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane
associated AAA-ATPase (ATPase Associated with
Different Cellular Activities) were combined in stoichometric
proportions into liposomes together with albumin as a source of protein thiols.
Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and
dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine
was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl
phosphatidylcholines. The lipids were dried to a film and reconstituted into
vesicles by resuspension in buffer containing the recombinant proteins in
equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these
vesicles enlarged in an ATP-dependent manner based on light-scattering
measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized,
the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor
specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX,
the recombinant AAA-ATPase, a source of protein thiols and ATP all were
required. In control experiments, no ATP-dependent vesicle enlargement was
observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of
ATP was without any effect when only the single proteins were incorporated into
the lipid vesicles. A model has been developed whereby the plasma membrane
AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX
protein, to membrane structural proteins. Binding of ATP and subsequent
hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby
both thinning the membrane and increasing the vesicle surface.
Journal: BioFactors, vol. 28, no. 2, pp. 105-117, 2006
