Biochemical characterization of tomato annexin p35. Independence of calcium binding and phosphatase activities.
Tomato annexin p35 has been cloned and used in a site-directed mutagenesis study to explore the phospholipid binding and catalytic properties of the protein in detail. Analysis of the cDNA sequence of p35 reveals that the annexin has only two typical endonexin folds, corresponding to repeats I and IV. Expression of recombinant p35 in Escherichia coli confirmed both phospholipid binding and a nucleotide phosphatase activity that could be inhibited on interaction of the recombinant annexin with phospholipids. Site-directed mutagenesis in which the acidic residues Glu-68 (repeat I), and Asp-297 (repeat IV) were changed to Asn, generated two mutant forms, E68N and D297N, respectively. Both mutant forms of the annexin continued to express catalytic activity. Changing repeat I had little effect on phospholipid binding, whereas the change to repeat IV abolished this property. These data show that, in this plant annexin, repeat IV plays a more critical role in calcium-dependent phospholipid binding than repeat I, and that the catalytic and phospholipid binding activity of the protein can be separated experimentally.
Lim
E K
EK
Plant Laboratory, Department of Biology, University of York, P. O. Box 373, York YO10 5YW, United Kingdom.
Roberts
M R
MR
Bowles
D J
DJ
D000595:Amino Acid Sequence; D017302:Annexins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D017302:Annexins; D009711:Nucleotides; D010743:Phospholipids; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010744:Phosphoric Monoester Hydrolases