Comparison of binding sites for wheat germ agglutinin on Raji lymphoblastoid cells and their isolated nuclei and plasma membranes

Abstract

Raji lymphoblastoid cells and the cell nuclei and plasma membranes isolated by the glycerol-lysis technique [Jett, M., Seed, T., & Jamieson, G. A. (1977) J. Biol. Chem. 252, 2134-2142] have been examined for their ability to bind wheat germ agglutinin. Intact cells and isolated nuclei showed similarities (i) in the total number of binding sites (3.38 X 10(6) and 4.06 X 10(8), respectively), indicating at least a 2-fold higher receptor density on the nuclei, (ii) in the ratios of the number of high-affinity sites and low-affinity sites (1.05 and 1.07), and (iii) in the apparent association constants at the high-affinity sites (28 nM and 48 nM) and at the low-affinity site (116 nM and 370 nM). Isolated plasma membranes had a similar number of total binding sites calculated on an equivalent cell basis (2.01 X 10(6)) but showed differences in the ratio of high- to low-affinity sites (1.5) and in their apparent association constants (3 nM and 22 nM). These results suggest similarities in the lectin receptors on the outer surface of lymphoblastoid cells and the cell nuclei. The differences obtained with isolated membranes may be due to inversion of the membrane vesicles or to their decreased rigidity as compared with the intact cell.

Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

“…AlexaFluor-488-conjugated wheat germ agglutinin, a lectin that binds to N-acetylglucosamine and sialic acid residues, 29, 30 was used to label the plasma membrane. The software ImageJ 18 was used to calculate Manders coefficients for polyplexes formed with Cy5-labeled pDNA and the wheat germ agglutinin on the plasma membrane as a measure of colocalization.…”

“…To further study how polymer length and structure influence the interaction of polyplexes with the plasma membrane and the mechanism of cellular uptake, confocal microscopy was used to monitor colocalization of polyplexes and the plasma membrane 30 min after transfection. AlexaFluor-488-conjugated wheat germ agglutinin, a lectin that binds to N -acetylglucosamine and sialic acid residues, , was used to label the plasma membrane. The software ImageJ was used to calculate Manders coefficients for polyplexes formed with Cy5-labeled pDNA and the wheat germ agglutinin on the plasma membrane as a measure of colocalization. , …”

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells

“…While the bound lectin subsequently appears to be internalized into the cell, and localizes to the nucleus within as little as 30 min, the resulting biochemical events remain elusive. Interestingly, however, WGA reactivity with components of nuclear membrane has been studied extensively (Kramer and Canellakis, 1979;Jett and Jamieson, 1981;Yoneda et al, 1987;Burrus et al, 1988;Meikrantz et al, 1991;Vannier-Santos et al, 1991;Moore and Blobel, 1992;Sterne-Marr et al, 1992;Kita et al, 1993;Maison Radu et al, 1993;Miller and Hanover, 1994;Beltinger et al, 1995;Dargemont et al, 1995). Accordingly, several glycoprotein components of nuclear pore complexes show WGA binding reactivity, and active transport events at the nuclear pore complex are interrupted after WGA binding (Moore and Blobel, 1992;Sterne-Marr et al, 1992;Kita et al, 1993;Michaud and Goldfarb, 1993;Radu et al, 1993;Adam and Adam, 1994;Bustamante et al, 1994;Miller and Hanover, 1994; Heese-Peck al, 1995).…”