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A plasma membrane‐associated AAA‐ATPase from Glycine max
Author(s) -
HicksBerger Carrie,
Sokolchik Irina,
Kim Chinpal,
Morré D. James
Publication year - 2006
Publication title -
biofactors
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.204
H-Index - 94
eISSN - 1872-8081
pISSN - 0951-6433
DOI - 10.1002/biof.5520280207
Subject(s) - glycine , atpase , chemistry , biophysics , biochemistry , biology , enzyme , amino acid
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.