Calcium binding to the tubulin-colchicine complex in the state of GTP in a BES buffer

Magnesium and calcium binding were assayed using the tubulin-colchicine complex in a BES buffer, in which the calcium binding to tubulin had been measured by Grisham et al. (Fed. Proc. 39, 2162). In the previous paper, an imidazole buffer was used as the buffer which does not chelate to calcium and which is substituted to phosphate buffer. The result of calcium binding measurement indicated the same binding constant between at pH 7.0 and at pH 6.5 in the absence of magnesium (1.08×10^ M at pH 7.0 and 1.10×10^ M at pH 6.5). Also, the calcium binding constant of the tubulin-colchicine complex was the same as that of tubulin in a BES buffer, pH 7.0. The increase of magnesium concentration inhibited calcium binding to the tubulin-colchicine complex. The affinity of calcium at pH 7.0 was lower than that at pH 6.5 in the presence of magnesium. This leads the effect of calcium on the polymerization of the tubulin-colchicine complex is small at pH 6.5 rather than at pH 7.0. The results obtained in the polymerization experiment were in good agreement with the results of the calcium binding experiment

Studies on the Structural and Colloidal Behavior of Polyvinyl Alcohol-Acetate Copolymers and of Egg Albumin

Abstract not availabl

Inhibition of Tubulin Self-Assembly and Tubulin-Colchicine GTPase Activity by Guanosine 5’-γ-Fluorotriphosphate)

The inhibitory effects of guanosine 5'-(ᵧ-fluorotriphosphate) [GTP(ᵧF)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the ᵧ-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 ± 0.6) × 10-4 M. GTP(ᵧF) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(ᵧF) on the assembly led to a value of Kdis = 1.1 × 10-6 M for the complex GTP(ᵧF)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 °C was arrested by GTP(ᵧF), and only limited depolymerization was induced by the

The Structure of High Molecular Weight Ribonucleic Acid in Solution: A Small-Angle X-Ray Scattering Study

Small-angle x-ray scattering studies on an absolute scale have been carried out on isotropic solutions of high molecular weight RNA obtained from ascites tumor cells, E. coli, and yeast. It was found that in all three cases the RNA is composed of short rigid rods (50 to 150Å in length) joined by small flexible regions. The rods account for almost the entire structure (at least 90 per cent); their radius of gyration about the axis and their mass per unit length are similar to those of DNA, suggesting a double-stranded helical structure. The rods are joined in an array forming the compact RNA molecule. On thermal degradation, the molecular superstructure disappears while the rods persist

Ultracentrifugal Behaviors of the Tubulin-Colchicine Complex in the State of GTP in Different Buffers

The behavior of the tubulin-colchicine complex was investigated by using an analytical ultracentrifugation technique. The partial specific volumes of the tubulin-colchicine complex in the state of GTP were 0.739 and 0.744 ml/g in imidazole and BES buffers, respectively. The values S 0 20.w were 5.82 and 6.06 S in an imidazole buffer in the presence of 0 and 2 mM MgCl 2 , respectively. In the BES buffer, however, very interesting results were obtained. The sedimentation coefficient S 0 20.w increased from 5.38 S with a calcium concentration of 0 μM to 5.75, 6.08 S with calcium concentration of 100 and 500 μM, respectively, in the absence of magnesium. This means that the protein molecule has a very flexible shape in the BES buffer and that the shape shrinks by the addition of calcium. The increase of either the calcium or the magnesium ion concentration induced the increase of the sedimentation coefficient. This result was in good agreement with the inducing effect of calcium on the polymerization of the tubulin-colchicine complex in the state of GDP. The sedimentation coefficient of the GDP state was higher than that of the GTP state. Many studies on the conformation of tubulin have been done by using an analytical ultracentrifugation techniqu

Études du dichroïsme circulaire et des spectres infrarouges des polypeptides et protéines en solution aqueuse

Des polypeptides de synthèse en diverses conformations ont été étudiés par Ia spectroscopie infrarouge dans la zone spectrale de la bande amide I, aussi bien que par le dichroïsme circulaire dans la région de l’ultraviolet (entre 185 et 240 mμ), en solution aqueuse. Les résultats de ces études indiquent qu’il devrait être possible d’effectuer des études similaires sur les protéines globulaires en solution