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. Northern blot analysis detected one transcript of approximately 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.
Hicks-Berger
Carrie
C
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Sokolchik
Irina
I
Kim
Chinpal
C
Morré
D James
DJ
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D015152:Blotting, Northern; D002462:Cell Membrane / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D030561:Databases, Nucleic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D008666:Metalloendopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D013025:Glycine max / Q000201:enzymology*
D000906:Antibodies; D018076:DNA, Complementary; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C114583:m-AAA proteases