Shoji Okamura

Lack of inhibition of carrot colchicine binding activity by podophyllotoxin

The binding of [3H]colchicine to carrot cell extract was not inhibited by an excess amount of podophyllotoxin. Under the same experimental condition, porcine brain tubulin almost completely lost its [3H]colchicine binding activity. The components in the carrot cell extract did not affect the interaction of brain tubulin and podophyllotoxin.

Isolation of Plasma Membrane and Analysis of Membrane Glycoproteins from the Slime Mould Physarum polycephalum

SUMMARY: Plasma membranes were isolated from the acellular slime mould Physarum polycephalum by differential centrifugation and an aqueous two-phase polymer method. ATPase activity in the membrane fraction was optimal at pH 6.5 and was severely inhibited by vanadate but resistant to oligomycin. The protein components of the plasma membrane were analysed by polyacrylamide gel electrophoresis. Glycoproteins were located on Western blots by incubation of the sheets with lectin-peroxidase reagents. The results indicated that most of the membrane proteins were glycosylated. Pulse-chase experiments with [3H]glucosamine showed that when the plasmodia were induced to differentiate to macrocysts, turnover of the membrane glycoproteins occurred more rapidly than in growing plasmodia.

β-Tubulin isotypes in the tobacco BY2 cell cycle

A well known feature of the tubulin isotypes is that the difference in the primary structure is concentrated at the C-terminal variable acidic region (Cva). The topological and electrostatic feature of Cva suggest special roles in interacting with other molecules such as MAPs, motor and even other proteins. In spite of the apparent importance of Cva, it is still obscure whether any isotype has a special role in the cell cycle. In general, more dynamic changes in microtubule distribution occur in plant than in animal cell cycle, namely, the preprophase band, spindle, phragmoplast and cortical microtubule arrays. Tobacco BY2 cell is advantageous for various reasons. It is a fast-growing higher plant cell culture, and the population is quite homogeneous and free of aggregates. Above all, fairly good synchronous culture can be induced by aphidicolin treatment (Nagata and Kumagai, 1999). The 3#-ends including about 50 C-terminal amino acid-coding region of the -tubulin cDNA were amplified by 3#RACE. According to the difference in the predicted amino acid sequences of the C-terminal, they were grouped into five different isotypes, NTB1–5 * Corresponding author. Tel.: +81-76-434-7547; fax: +81-76-434-4656. E-mail address: okamura@toyama-mpu.ac.jp (S. Okamura). Fig. 1. Predicted C-terminal amino acid sequences of tobacco -tubulin isotypes.

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/105 cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.

Identification of a carbohydrate‐binding site in physarum haemagglutinin I

Carbohydrate‐binding peptides from trypsin‐digests of Physarum lectins (haemagglutinins I and II) were isolated by affinity column chromatography. The amino acid sequence of the peptlde fragment from haemagglutinin I was determined to be 48TVHQSWY54. A similar amino acid sequence was found in the peptide fragment from haemagglutinin II, in which alignment of valine, histidine, tryptophan and tyrosine was identical. Deletion of the heptapeptide sequence (TVHQSWY) by site‐directed mutation abolished the haemagglutinating activity. The replacement of Trp53 by alanine resulted in a complete loss of the haemagglutinating activity, suggesting that the tryptophan residue in the heptapeptide sequence is essential for carbohydrate binding.