J. Biochem, 1988, Vol. 104, No. 3 329-332
© 1988 Japanese Biochemical Society
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Preparation of Tubulin from Caulerpa, a Marine Green Alga, Using Casein as a Protective Agent against Proteolytic Degradation1
*Laboratory of Neurochemistry, National Institute for Physiological Sciences Okazaki, Aichi 444
**Laboratory of Cell Biology, National Institute for Basic Biology Okazaki, Aichi 444
***Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka 560
Bidirectional organelle movements taking place in the cytoplasm of the rhizomes of Caulerpa, a coenocytic marine green alga, have been indicated to be dependent on microtubules (Kuroda, K. & Manabe, E. (1983) Proc. Jpn. Acad. 59B, 131–134; Manabe, E. & Kuroda, K. (1984) Proc. Jpn. Acad. 60B, 118–121). However, when a crude extract of Caulerpa rhizomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with monoclonal anti-tubulin antibody, no reacting band could be detected. This apparent absence of tubulin in the extract was found to be a result of the complete degradation of tubulin by potent intrinsic proteolytic activity. All of the commercially available protease inhibitors so far tested (p-chloromercuriphenylsulfonic acid, phenyl methylsulfonyl fluoride, 1-chloro-4-phenyl-3-tosylamido-2-butanone, 7-amino-1-chloro-3-tosylamido-2-heptanone, p-tosyl-L-arginine methyl ester, soybean trypsin inhibitor, antipain, chymostatin, leupeptin, and pepstatin) failed to inhibit the activity completely. But addition of casein at the concentra tion of 1% (weight per volume) to the solutions used for preparation was effective in protecting tubulin from proteolytic degradation, thus making it possible to prepare tubulin from the crude extract of Caulerpa. On SDS-PAGE, the Caulerpa 
-tubulin thus prepared was a little smaller in molecular weight than that of rabbit brain.
1This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Y. Ishizaki was the recipient of a Fellowship of the Japan Society for the Promotion of Science for Japanese Junior Scientists.
2Present address: Section of Physiological Chemistry, Faculty of Dentistry, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113.
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