Carbohydrate-binding properties of the hemolytic lectin CEL-III from the holothuroidea Cucumaria echinata as analyzed using carbohydrate-coated microplate.
The carbohydrate-binding properties of the hemolytic lectin CEL-III from the Holothuroidea Cucumaria echinata were studied using the microplate assay system which we have recently developed [Hatakeyama et al. (1996) Anal. Biochem. 237, 188-192]. When the binding of CEL-III to lactose covalently immobilized on a microplate was examined using colloidal gold solution, the binding was detected with as little as 1 microgram/ml protein. Affinity of several carbohydrates to CEL-III was assessed by means of an inhibition experiment using the lactose-coated plate and it was found that N-acetylgalactosamine has the highest affinity for CEL-III, followed by lactose and lactulose. Examination of the binding of CEL-III to the lactose-coated plate at various pH values and temperatures revealed that the affinity is higher in the acidic pH region and at lower temperatures. From the Ca(2+)-dependence profile for the binding of CEL-III to the lactose-coated plate, the apparent dissociation constant for Ca2+ was estimated to be 2.3 mM. These results suggested that the carbohydrate-binding properties of CEL-III are closely related to its hemolytic activity, although an additional interaction between the protein and the lipid bilayer, which is enhanced in the alkaline pH region, also seems to be necessary for its hemolytic action.
Hatakeyama
T
T
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka. thata@net.nagasaki.u.ac.jp
Miyamoto
Y
Y
Nagatomo
H
H
Sallay
I
I
Yamasaki
N
N
D000818:Animals; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D017979:Gold Colloid; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D012616:Sea Cucumbers / Q000737:chemistry*; D013696:Temperature
D002241:Carbohydrates; D017979:Gold Colloid; D037102:Lectins; D007785:Lactose; D002118:Calcium