Calorimetric studies of the in vitro polymerization of brain tubulin.

Abstract

The enthalpy change for chain propagation in the polymerization of bovine tubulin has been studied directly by stopped-flow microcalorimetry at 170 and 250, and found to be 0 + 1 kcal per mol of 6S tubulin dimer at both temperatures.Substantial heat evolution with a half-time of decay of approximately 1 hr was observed when tubulin was injected into the calorimeter. This heat was shown to result from contamination of the tubulin by small amounts of some material from the crude brain homogenate from which the tubulin was prepared, and to be totally unconnected with microtubule assembly. Model In the following discussion, it should be remembered that thermodynamics strictly applies only to reversible processes between well defined states. Microtubule assembly is only quasi-reversible (3) and may involve nucleotide hydrolysis (4). Moreover, the intermediate species and even the polymers may vary with the biological system or from one preparation to another. We therefore attempted to keep our experimental procedure as nearly constant as possible. Of course, the same restrictions apply as well to van't Hoff measurements but for these measurements the interpretation is complicated by the existence of states intermediate between the polymerizing unit and the final polymer, as will be discussed below.Although the measurement discussed herein applies primarily to the chain lengthening process and not to the nucleation process, the amount of tubulin in the form of nuclei is likely to comprise at most only a small percentage of the total mass of tubulin in any microtubule preparation. Therefore, the overall enthalpy change involved in the formation of microtubules reflects primarily this enthalpy and not the enthalpy of nucleation. MATERIALS AND METHODSTubulin, twice polymerized and depolymerized, was prepared by the method of Shelanski et al. (5) The GTP was purchased from Sigma (Type II-S) and fl,ymethyleneguanosine triphosphate (GMPPCP) was from P-L Biochemicals. Protein concentrations were determined by a modified Lowry procedure (6), using a bovine serum albumin standard.Flow Calorimetry. Our most reliable thermochemical data were obtained by means of flow calorimetry. The instrument employed is a modification of the Beckman model 190 batch calorimeter, and was previously described (7). The flow tubing is platinum of 1 mm internal diameter, and a total volume of 1 ml in thermal contact with the measuring thermopile. The volume of delivery tubing from each syringe to the mixer is 0.58 ml; part of this tubing is Teflon in good thermal contact with the constant temperature water bath surrounding the calorimeter submarine, and part of it is platinum in good thermal contact with the massive aluminum heat sink of the calorimeter.Various experimental formats were tried with this instrument, of which the only successful one may be described as a 3565 Abbreviations: DSC, differential scanning calorimetry; GMPPCP, f3, 'y-methyleneguanosine triphosphate.

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules

“…The distinct possibility of such a mechanism can be inferred from the report by Weisenberg et al (1976) that GTP hydrolysis occurs during or immediately following the tubulin addition step to a growing microtubule, as well as from those of David-Pfeuty et al (1977,1978) and of MacNeal & Purich (1978) that the hydrolysis of at least one GTP molecule per tubulin dimer incorporated into microtubules occurs during microtubule growth. While a direct comparison between our study and that of Sutherland & Sturtevant (1976) is not possible since they worked with partially purified tubulin in the presence of microtubule-associated proteins which copurify with tubulin in the Shelanski et al (1973) cycle procedure (Sloboda et al, 1976), such a situation could account for the observed heat capacity change pattern. In analogy to ATP hydrolysis, GTP hydrolysis may be assumed to be an exo- thermic reaction with a AH value in the vicinity of -5 kcal/mol (Sutherland & Sturtevant, 1976;Alberty, 1969;Gellert & Sturtevant, 1960;Gerlt et al, 1975).…”

“…While a direct comparison between our study and that of Sutherland & Sturtevant (1976) is not possible since they worked with partially purified tubulin in the presence of microtubule-associated proteins which copurify with tubulin in the Shelanski et al (1973) cycle procedure (Sloboda et al, 1976), such a situation could account for the observed heat capacity change pattern. In analogy to ATP hydrolysis, GTP hydrolysis may be assumed to be an exo- thermic reaction with a AH value in the vicinity of -5 kcal/mol (Sutherland & Sturtevant, 1976;Alberty, 1969;Gellert & Sturtevant, 1960;Gerlt et al, 1975). The polymerization of tubulin into microtubules is, however, endothermic and has a large negative change in heat capacity.…”

Biophysical studies of protein solubility and amorphous aggregation by systematic mutational analysis and a helical polymerization model

“…The HPM provides a theoretical framework for systems that undergo an abrupt transition from a monomeric state to an oligomeric or colloidal state (Oosawa and Kasai 1962). Besides actin (Arisaka et al 1975), HPM-derived models have been applied to various systems, such as tobacco mosaic virus (Klug and Durham 1972) and tubulin (Sutherland and Sturtevant 1976).…”

“…In the linear model (Fig. 5a), the equilibrium constant (Sutherland and Sturtevant 1976) for adding a single monomer to the aggregate is k a . According to the linear model, the concentration (M i ) of aggregates containing i molecules is written in terms of the free monomer concentration in the supernatant m as:…”

Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis

“…The most fundamental way of investigating the nature of the interactions responsible for macromolecular polymer formation is by thermodynamic analysis (Wyman and Gill, 1990). There have been extensive van't Hoff and calorimetric studies on the thermodynamics of tubulin assembly under various ligand and buffer conditions (Frigon and Timasheff, 1975a,b;Sutherland and Sturtevant, 1976;Lee and Timasheff, 1977;Hinz et al, 1979;Johnson and Borisy, 1979;Johnson, 1980;Berkowitz et al, 1980;Andreu et al, 1983;Melki and Carlier, 1993;Diaz et al, 1993). However, a quantitative thermodynamic study of the contribution of GTP hydrolysis to the assembly reaction has not been conducted.…”