2Department of Food and Hygiene, Faculty of Environmental Health Science, Azabu University, 1-17-71, Fuchinobe, Sagamihara City, Kanagawa 229-8501, Japan; E-mail: email@example.com
3Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsusima, Aoba-ku, Sendai 981-8558, Japan; E-mail: firstname.lastname@example.org
4Faculty of Medical Management and Information Science, School of Health Sciences, Fujita Health University, Toyoake, Aichi 470-1192, Japan; E-mail: email@example.com
5Department of Biology, School of Health Sciences, Fujita Health University, Toyoake, Aichi 470-1192, Japan; E-mail: firstname.lastname@example.org
6Laboratory of Environmental Biosciences, Y. S. F. H., 6 Ono, Tsurumi-ku, Yokohama 230-0046, Japan; E-mail: email@example.com
* To whom correspondence should be addressed.
Received September 19, 2008; Revision received February 18, 2009
A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80°C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, β-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-α- and methyl-β-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (kass) and dissociation rate constant (kdiss) were determined for the lectin to be 4.3·105 M–1·sec–1 and 2.2·10–3 sec–1, respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.
KEY WORDS: Aplysia kurodai, cell proliferating inhibition, D-galactose-binding lectin, galacturonic acid, sea hare, surface plasmon resonance