Affiliations: Department of Medicinal Chemistry and Molecular
Pharmacology, Purdue University, West Lafayette, IN 47907, USA
Note: [] Address for correspondence: D.J. Morré, Department of
Medicinal Chemistry and Molecular Pharmacology, Purdue University, 136 Hansen
Life Sciences Research Building, 201 S. University Street, West Lafayette, IN
47907-2065, USA. Fax: +1 765 494 4007; E-mail: [email protected]
Abstract: 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.
Northern blot analysis detected one transcript of ∼2700
bp. The full-length cDNA sequence was that previously obtained (GenBank
Database; U20213) encoding a protein with two copies of the conserved
AAA-ATP-binding motif and regions of sequence homology with other AAA-ATPases.
Electron microscopic preparations of the recombinant SBPM AAA-ATPase revealed
hexamers typically formed by these proteins. The cloned and expressed protein
was identical to the protein isolated from the soybean plasma membrane as
confirmed using antisera raised to a non-conserved region of the derived
protein sequence and by N-terminal sequencing of peptides derived from the
isolated protein.
