Transmembrane modulation of the concanavalin A inhibition of 5'-nucleotidase is not due to a direct association of the enzyme with the cytoskeleton.
The inhibition of the cell surface enzyme 5'-nucleotidase by concanavalin A is being studied as a model for understanding transmembrane modulation of cell surface functions. Nucleotidase of 13762 MAT-C1 ascites rat mammary adenocarcinoma cells is inhibited by concanavalin A in a noncooperative process. When cells are treated with the cytoplasmic effectors cytochalasins, colchicine, energy poisons, calcium plus ionophore or hypotonic buffers, the concanavalin A inhibition of the enzyme becomes cooperative. 5'-Nucleotidase of isolated MAT-C1 microvilli is also inhibited by concanavalin A in a noncooperative process; however, treatment of the microvilli with the same cytoplasmic effectors does not induce cooperativity. Since previous studies in several systems have suggested an association of nucleotidase with actin-containing microfilaments or the cell cytoskeleton, one explanation for the cooperativity changes is that they result from a change in the association of the enzyme with the cytoskeleton. However, Triton X-100 extractability of nucleotidase is the same for MAT-C1 cells exhibiting cooperative or noncooperative concanavalin A inhibition. Moreover, enzyme from cells exhibiting cooperative inhibition can be extracted into the zwitterionic detergent Zwittergent in a cooperative form, while enzyme exhibiting noncooperative behavior can be extracted into Zwittergent in a noncooperative form. Gel filtration and rate-zonal sucrose density gradient centrifugation showed little discernible size or sedimentation difference between enzyme samples exhibiting noncooperative and cooperative inhibition. These results indicate that changes in the cooperativity of the concanavalin A inhibition of nucleotidase are not a result of changes in the association of the enzyme with the cytoskeleton. These studies emphasize the caution which must be exercised in interpreting the effects of cytoskeletal perturbants on cell surface functions.
Craik
J D
JD
Carraway
C A
CA
Carraway
K L
KL
D015720:5'-Nucleotidase; D000230:Adenocarcinoma / Q000201:enzymology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003208:Concanavalin A / Q000494:pharmacology*; D003599:Cytoskeleton / Q000201:enzymology*; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology; D008871:Microvilli / Q000201:enzymology; D009708:Nucleotidases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D051381:Rats
D003208:Concanavalin A; D009708:Nucleotidases; D015720:5'-Nucleotidase