Colchicine-binding properties of hepatic tubulin. The role of "time-decay".
Determination of total and polymerized tubulin in the liver by the [3H]colchicine-binding assay is hampered by rapid loss of binding sites. One solution, which has been proposed to overcome this problem, is the use of a "time-decay" corrected assay that extrapolates to an initial binding capacity based on the results of testing multiple aliquots of liver supernatants over time. If such an approach is valid, it follows that the extrapolated initial value should be independent of the rate of time-decay. To test this relationship, we modulated the half-life (T1/2) for time-decay of a fixed amount of hepatic microtubule-derived tubulin using various concentrations of organic acids, sodium glutamate and glucose-1-phosphate. We found that the initial binding capacity based on simple first order decay did vary with the T1/2. Similar results were also obtained with bovine brain tubulin added to hepatic supernatants. These results indicate that the quantification of hepatic tubulin derived by extrapolated time-decay values is inaccurate and may overestimate the actual amount of tubulin present, especially when time-decay is rapid.
D000818:Animals; D001665:Binding Sites; D003078:Colchicine / Q000378:metabolism*; D006207:Half-Life; D008099:Liver / Q000032:analysis; D008297:Male; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D014404:Tubulin; D003078:Colchicine