Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Biochemical characterization of tomato annexin p35
T2 - independence of calcium-binding and phosphatase activities
AU - Lim, Eng-Kiat
AU - Roberts, Michael
AU - Bowles, D. J.
PY - 1999/12/25
Y1 - 1999/12/25
N2 - 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 mutantforms 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.
AB - 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 mutantforms 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.
KW - annexin
KW - calcium
KW - phosphatase
U2 - 10.1074/jbc.273.52.34920
DO - 10.1074/jbc.273.52.34920
M3 - Journal article
VL - 273
SP - 34920
EP - 34925
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 1083-351X
IS - 52
ER -