A plasma membrane-associated AAA-ATPase from Glycine max.

An AAA-ATPase (ATPases Associated with a Variety of Cellular Activities) localized to the plasma membrane of soybean (Glycine max) was isolated, partially sequenced and cloned (SBPM AAA-ATPase). The protein with an apparent monomer molecular mass of about 97 kDa was isolated using a combination of anion exchange, preparative SDS-PAGE, reverse phase HPLC, and ATP affinity chromatography. The cDNA for the full-length SBPM AAA-ATPase was cloned by screening an expression library using an antibody against the highly conserved Walker B AAA-ATP-binding motif.

ATP-dependent and drug-inhibited vesicle enlargement reconstituted using synthetic lipids and recombinant proteins.

A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichiometric 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.

Inside-out but not right side-out plasma membrane vesicles from soybean enlarge when treated with ATP + 2,4-D as determined by electron microscopy and light scattering: evidence for involvement of a plasma membrane AAA-ATPase.

The concept that the location of an AAA-ATPase associated with the plant plasma membrane may be indicative of a functional relationship to growth or cell enlargement by analogy with roles in physical membrane displacements as proposed for AAA-ATPases associated with internal membranes was tested. A plant growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans was utilized in a completely cell-free system. The rate of enlargement was accelerated by the synthetic plant growth factor 2,4-dichlorophenoxyacetic acid (2,4-D) in a log dose-dependent manner and was increased approximately 2-fold with the addition of 1 microM 2,4-D plus 100 microM ATP compared to 100 microM ATP alone, 1 microM 2,4-D alone or no additions.

Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases.

The salivary apyrases of blood-feeding arthropods are nucleotide-hydrolyzing enzymes implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular adenosine diphosphate. A human cDNA homologous to the apyrase cDNA of the blood-feeding bed bug was identified, revealing an open reading frame encoding a 371-amino acid protein. A cleavable signal peptide generates a secreted protein of 333 residues with a predicted core molecular mass of 37,193 Da.