Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs.
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 degrees 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, beta-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-alpha- and methyl-beta-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k(ass)) and dissociation rate constant (k(diss)) were determined for the lectin to be 4.3 x 10(5) M(-1) x sec(-1) and 2.2 x 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.
Kawsar
S M A
SM
Laboratory of Marine Biochemistry, Department of Environmental Biosciences, International Graduate School of Arts and Sciences, Yokohama City University, Yokohama, 236-0027, Japan.
Matsumoto
R
R
Fujii
Y
Y
Yasumitsu
H
H
Dogasaki
C
C
Hosono
M
M
Nitta
K
K
Hamako
J
J
Matsui
T
T
Kojima
N
N
Ozeki
Y
Y
D000818:Animals; D001048:Aplysia / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D037161:Galectins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits
D037161:Galectins