A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes.
A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.
D001395:Azotobacter / Q000378:metabolism*; D002250:Carbon Radioisotopes; D008744:Methylamines / Q000378:metabolism*; D008851:Micropore Filters; D012996:Solutions; D013012:Sorbitol / Q000378:metabolism; D013997:Time Factors
D002250:Carbon Radioisotopes; D008744:Methylamines; D012996:Solutions; D013012:Sorbitol; C027451:methylamine