Directional immobilization of sodium- and potassium-activated ATPase to expose its cytoplasmic part to the liquid phase on microtiter plates by wheat germ agglutinin.
A convenient method for highly efficient and directional immobilization of intact sodium- and potassium-activated ATPase (Na,K-ATPase) using wheat germ agglutinin linked on microtiter plates was developed. Wheat germ agglutinin, which bound tightly to the beta-subunit of Na,K-ATPase and had no effect on the Na,K-ATPase activity, the potassium-activated p-nitrophenylphosphatase activity, or the inhibitory action of ouabain, was covalently linked to microtiter plates and used as an immobilizer of the enzyme. The amount of Na,K-ATPase coupled to microtiter plates in this immobilizing system was more than 10-fold greater than that used in the direct immobilizing system (O. Urayama, M. Nakao, H. Nagamune, and H. Sugiyama, (1984) Anal. Biochem. 141, 194-198). Also in this system, the cytoplasmic domain of Na,K-ATPase was exposed to the liquid phase. This technique was useful for investigating the reactivities of monoclonal antibody specific for the cytoplasmic domain of the enzyme. Moreover, because this technique was used successfully in the immobilization of periodic acid--Schiff positive staining glycoprotein 1 prepared from human erythrocytes and human alpha 2-macroglobulin, the technique should also be useful for other membrane or secreted proteins that possess N-linked sugar chains containing bisecting N-acetylglucosamine or a high amount of sialic acid.
Nagamune
H
H
Department of Oral Microbiology and Immunology, School of Dentistry, University of Tokushima, Japan.
Urayama
O
O
Hara
Y
Y
Ota
F
F
Hirota
K
K
Satomi
Y
Y
Seo
K
K
Fukui
K
K
Nakao
M
M
D000818:Animals; D004800:Enzymes, Immobilized / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis; D006041:Goats; D006801:Humans; D007124:Immunoenzyme Techniques; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D014909:Wheat Germ Agglutinins / Q000494:pharmacology
D004800:Enzymes, Immobilized; D006023:Glycoproteins; D014909:Wheat Germ Agglutinins; D000254:Sodium-Potassium-Exchanging ATPase