TY - JOUR
T1 - Biochemical determination of tubulin-microtubule equilibrium in cultured cells
AU - Ostlund, Richard E.
AU - Leung, Joyce T.
AU - Hajek, Shirley Vaerewyck
N1 - Funding Information:
’ Supported by NIH Grant AM 20421 and a grant from the St. Louis Diabetic Children’s Welfare Fund.
PY - 1979/7/1
Y1 - 1979/7/1
N2 - The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH3 cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34-41% of tissue culture cell tubulin. Colchicine (10-6 to 10-5 m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.
AB - The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH3 cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34-41% of tissue culture cell tubulin. Colchicine (10-6 to 10-5 m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.
UR - http://www.scopus.com/inward/record.url?scp=0018362186&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(79)90568-2
DO - 10.1016/0003-2697(79)90568-2
M3 - Article
C2 - 495980
AN - SCOPUS:0018362186
SN - 0003-2697
VL - 96
SP - 155
EP - 164
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -