This proposal is based on two novel findings in microtubule biochemistry: 1) in the presence of the physiological buffer, HCO3-/CO2, carbamino groups are formed on tubulin, and this is associated with an altered pH-dependency of microtubule assembly; and 2) a hydrophobic (presumably membrane) tubulin readily separable from microtubule protein can be obtained from mammalian brain and cultured cells. The properties of microtubule protein in HCO3-/CO2 suggest important mechanisms for the regulation of microtubule assembly we propose to explore in detail; the existence of a bona fide hydrophobic tubulin suggests new ways in which 'microtubules' can affect cell function. Our overall objectives are to determine the mechanism of the CO2/HCO3- effects, to define the consequences of carbamate formation for microtubule function, and to determine the molecular differences between the hydrophobic and microtubule tubulins and their expression in cultured cells. Tubulin carbamate formation will be measured over the physiological pH and CO2. Carbamate is measured by proton release following the rapid mixing of CO2 and protein solutions: carbamino groups formed and the dissociation constants of the reactive amines can be calculated. CO2/HCO3- buffers appear to promote microtubule assembly at alkaline pH and we will measure carbamate formation as a function of assembly and MAP (microtubule associated protein) binding. Sites of carbamate formation are probably localized to the carboxy-terminal region and functions at this region such as tyrosination and detyrosination will be examined. Divalent cation binding in the presence of CO2 will be measured by EPR (electron paramagnetic resonance). We will attempt to analyze the structural basis of the hydrophobic properties of the membrane tubulin and begin to study its function. Differences from soluble tubulin will be sought utilizing 2-dimensional electrophoresis, and peptide mapping. The presence of covalent incorporation of fatty acid and myoinositol will be examined. Copolymerization with microtubule protein will be tested. The quantity of the hydrophobic tubulin will be measured as a function of cell-cell and cell-substrate contact.
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| Preston, S F; Volpi, M; Pearson, C M et al. (1987) Regulation of cell shape in the Cloudman melanoma cell line. Proc Natl Acad Sci U S A 84:5247-51 |
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