Microtubules are involved in a variety of cellular functions which are thought to be performed by microtubules assembled from heterogeneous tubulin subunits. There is compelling evidence from electrophoresis, isoelectric focusing, protein and DNA sequencing that Alpha and Beta tubulin subunits are actually populations of heterogeneous proteins. There is little or no information on the functional significance of the heterogeneity and the physical properties of these subspecies, e.g., affinity for ligands such as GTP and antimitotic agents. The basic approach of this study is to employ drugs to perturb the tubulin system in order to achieve the goals of this investigation and they are: (a) to develop approaches that can resolve native tubulin subspecies and quantitate the differences in ligand affinity among these subspecies; (b) to determine the thermodynamic linkages in the interactions among GTP, antimitotic agents and tubulin; and (c) to determine the chemical nature of heterogeneity in these subspecies. To accomplish Goal (a), electrophoresis and isoelectric focusing will be developed to quantitatively resolve native tubulin-ligand complexes, to identify the specific subspecies and to determine the amounts of protein and ligand present in these complexes so that a tentative estimate of ligand affinity for these specific tubulin subspecies can be conducted. The ligands of choice will be 3H-colchicine and its derivatives. To accomplish Goal (b), multiple physical approaches will be employed. Equilibrium ligand binding studies will be conducted to provide the overall thermodynamic parameters governing the interactions between tubulin and ligands. Individual steps involved in these reaction schemes, e.g., rapid ligand binding and induced structural changes, will be resolved and quantitatively defined by rapid kinetics (stopped-flow) and steady state kinetics (ligand induced GTPase activity). Structural changes in tubulin will be monitored by various types of sedimentation experiments to determine the effects of ligands on the association-dissociation of tubulin and quaternary structure of the protein. To accomplish Goal (c), the subspecies of tubulin will be resolved and isolated in denatured state employing the techniques developed in this laboratory. These isolated subspecies will be subjected to protolysis, isolation of peptides by HPLC and determination of chemical composition with an ultimate goal of obtaining the amino acid sequences of unique peptides. This investigative plan represents a concerted effort to provide insights into the physico-chemical properties of tubulin subspecies.
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