Demonstration of the association of the cell-surface enzyme, 5'-nucleotidase, with microvillar microfilaments by phalloidin shift on velocity sedimentation gradients.
The cell-surface enzyme 5'-nucleotidase in microvilli from 13762 rat mammary adenocarcinoma cells remains largely associated with microfilament-containing high-speed pellets from Triton X-100 extracts of the microvilli. The fraction remaining with the insoluble portion is higher under ionic conditions which enhance microfilament stability. To minimize trapping and cosedimentation we have analyzed the distribution of microfilaments and 5'-nucleotidase activity on velocity sedimentation sucrose gradients of the microvillar extracts. A large fraction of the total enzyme activity is found in the filament fractions in the middle of the gradient. When phalloidin is included in the extraction buffer to stabilize the microfilaments, both the microfilaments and the bulk of the nucleotidase activity are shifted further into the gradients. Both the position of the filament fraction and the percentage of the total nucleotidase activity remaining with the filament fraction varies with extraction buffer composition and conditions. Nonetheless, under all conditions tested, a large percentage of the activity was shifted, along with the microfilaments, in the presence of phalloidin. These results are consistent with a specific association of 5'-nucleotidase with microfilaments in the ascites tumor cell microvilli.
D015720:5'-Nucleotidase; D008841:Actin Cytoskeleton / Q000378:metabolism*; D000230:Adenocarcinoma / Q000648:ultrastructure; D000818:Animals; D002451:Cell Compartmentation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003599:Cytoskeleton / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000648:ultrastructure; D008871:Microvilli / Q000201:enzymology* / Q000648:ultrastructure; D009708:Nucleotidases / Q000378:metabolism*; D010590:Phalloidine; D051381:Rats
D010590:Phalloidine; D009708:Nucleotidases; D015720:5'-Nucleotidase