The article explores the mechanism of action of colchicine, focusing on its binding to cellular proteins. It demonstrates that colchicine-³H binds to a noncovalent complex with a soluble macromolecule in tissue culture cells. This binding is consistent in both in vivo and in vitro conditions, with the same equilibrium and kinetic constants. The bound radioactivity is shown to be part of a molecule with the same chromatographic behavior and specific antimototic activity as colchicine. The binding activity correlates with the presence of microtubules in various cell types, including dividing cells, mitotic apparatus, cilia, sperm tails, and brain tissue. However, binding to extracts of slime mold or purified muscle proteins is very low or undetectable. The binding site has a sedimentation constant of 6S and is suggested to be a subunit of microtubules. The study also examines the binding of colchicine to various cell types and model systems, showing that the binding activity is not restricted to dividing cells, as evidenced by the high activity in brain tissue. The binding activity was found to correlate with the presence of microtubules rather than mitotic activity or motility. Microtubules are present in the mitotic spindle, cilia, sperm tails, and neuronal processes, and colchicine can cause the disassembly of microtubules in the axopods of Heliozoa. These findings suggest that the binding sites are the subunit proteins of microtubules. The article also discusses the chemical nature of the bound colchicine, showing that it is not chemically modified. Indirect experiments, such as chromatography and hydrolysis, support the conclusion that the bound radioactivity is due to chemically unchanged colchicine. The study concludes that colchicine binds to a macromolecule in intact cells, and the binding is reversible, dependent on the macromolecule being in its native state, and does not involve chemical modification of colchicine. The kinetics of the reaction can be described as the simple formation of a noncovalent complex with a single class of binding sites. The binding of colchicine to a protein present in neurons and axoplasm is of particular interest, as the acute physiological disturbances caused by colchicine poisoning are primarily due to its toxic action on the central nervous system. The complete absence of binding to muscle proteins is consistent with the general results that muscle proteins are not related to the mitotic apparatus. The study provides a comprehensive understanding of colchicine's mechanism of action, highlighting its interaction with microtubules and its diverse biological effects.The article explores the mechanism of action of colchicine, focusing on its binding to cellular proteins. It demonstrates that colchicine-³H binds to a noncovalent complex with a soluble macromolecule in tissue culture cells. This binding is consistent in both in vivo and in vitro conditions, with the same equilibrium and kinetic constants. The bound radioactivity is shown to be part of a molecule with the same chromatographic behavior and specific antimototic activity as colchicine. The binding activity correlates with the presence of microtubules in various cell types, including dividing cells, mitotic apparatus, cilia, sperm tails, and brain tissue. However, binding to extracts of slime mold or purified muscle proteins is very low or undetectable. The binding site has a sedimentation constant of 6S and is suggested to be a subunit of microtubules. The study also examines the binding of colchicine to various cell types and model systems, showing that the binding activity is not restricted to dividing cells, as evidenced by the high activity in brain tissue. The binding activity was found to correlate with the presence of microtubules rather than mitotic activity or motility. Microtubules are present in the mitotic spindle, cilia, sperm tails, and neuronal processes, and colchicine can cause the disassembly of microtubules in the axopods of Heliozoa. These findings suggest that the binding sites are the subunit proteins of microtubules. The article also discusses the chemical nature of the bound colchicine, showing that it is not chemically modified. Indirect experiments, such as chromatography and hydrolysis, support the conclusion that the bound radioactivity is due to chemically unchanged colchicine. The study concludes that colchicine binds to a macromolecule in intact cells, and the binding is reversible, dependent on the macromolecule being in its native state, and does not involve chemical modification of colchicine. The kinetics of the reaction can be described as the simple formation of a noncovalent complex with a single class of binding sites. The binding of colchicine to a protein present in neurons and axoplasm is of particular interest, as the acute physiological disturbances caused by colchicine poisoning are primarily due to its toxic action on the central nervous system. The complete absence of binding to muscle proteins is consistent with the general results that muscle proteins are not related to the mitotic apparatus. The study provides a comprehensive understanding of colchicine's mechanism of action, highlighting its interaction with microtubules and its diverse biological effects.