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Isolation and identification of a putative NTPase from the cytoskeleton from pea stem tissue

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Program Information

Session
Number
Session Name
32 Cell Walls and Cytoskeleton

Presented by: , Eric_Davies@ncsu.edu

Authors:

Shibata, Koichi Laboratory of Molecular Cell Biology, College of Agriculture, Ehime University
Abe, Shunnosuke Laboratory of Molecular Cell Biology, College of Agriculture, Ehime University
Davies, Eric Botany Department, North Carolina State University
The cytoskeleton fractions from plant cells contain characteristic proteins of wide-ranging molecular weights and they likely play an important role in cell structure and function. However, isolation of these proteins was difficult because they aggregate when conventional chromatography protocols are used and because of the presence of massive amount of ribosomal proteins. In this study, we have isolated the cytoskeleton fraction from the first internode of Alaska peas grown in the dark for 5 days. The cytoskeleton pellet was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes, and the post-ribosomal supernatant, applied to a heparin column (which prevents protein aggregation). Proteins bound to the column were then eluted with a gradient of 0.15 to 1M KOAc followed by heparin. Among proteins separated was a 49-kDa protein (which we call B3) eluting at 0.7-0.87M KOAc and is composed of at least 8 isoforms as seen in IEF electrophoresis. These B3 isoforms were digested by V8 protease and the partial amino acid sequences of these fragments obtained. We cloned several cDNAs which encoded B3 isoproteins. Based upon both nucleotide and the encoded amino acid sequences, these appear to correspond to a calmodulin dependent nucleoside triphosphatase (NTPase). We also compared the amounts of B3 between purified nuclei and the cytoskeletal fractions, and found that about 10 times as much was present in the cytoskeletal fraction as in the nuclear fraction. We discuss this protein in its putative role in shuttling RNA between the nucleus and cytoplasm.

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