Egg Tubulin Research Articles

Purification and Fluorescent Labeling of Tubulin from Xenopus laevis Egg Extracts.

For many years, microtubule research has depended on tubulin purified from cow and pig brains, which may not be ideal for experiments using proteins or extracts from non-brain tissues and cold-blooded organisms. Here, we describe a method to purify functional tubulin from the eggs of the frog, Xenopus laevis. This tubulin has many benefits for the study of microtubules and microtubule based structures assembled in vitro at room temperature. Frog tubulin lacks many of the highly stabilizing posttranslational modifications present in pig brain-derived tubulin, and polymerizes efficiently at room temperature. In addition, fluorescently labeled frog egg tubulin incorporates into meiotic spindles assembled in egg extract more efficiently than brain tubulin, and is thus superior as a probe for Xenopus egg extract experiments. Frog egg tubulin will provide excellent opportunities to identify active nucleation complexes and revisit microtubule polymerization dynamics in vitro.

Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs

Centrosomes are powerful and exclusive parthenogenetic agents in the Xenopus egg. We have previously shown that heterologous centrosomes from various vertebrate species were able to promote egg cleavage in Xenopus and that human centrosome activity was associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome. In this work, we have investigated the parthenogenetic capacity of more evolutionary distant centrosomes. We show that centrosomes devoid of centrioles, such as SPBs isolated from Saccharomyces cerevisiae, do not form asters of microtubules in cytoplasmic extracts from Xenopus eggs, and are inactive in the parthenogenetic test. We further show that Drosophila centrosomes which possess a typical centriole architecture, and are quite active to nucleate microtubules in Xenopus cytoplasmic extracts, are unable to trigger egg cleavage. This was observed both with centrosomes isolated from Drosophila syncytial embryos and nucleus-centrosome complexes from the Drosophila Kc23 cell line. We demonstrate that this inability could not be restored after pre-incubation of Drosophila centrosomes in the egg cytoplasm before injection. We conclude that the parthenogenetic activity of a centrosome is not directly linked to its capacity to nucleate microtubules from the egg tubulin, and that the evolutionary conserved nine-fold symmetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly.

Brain and egg tubulins from antarctic fishes are functionally and structurally distinct.

The multitubulin hypothesis proposes that chemically distinct tubulins may possess different polymerization properties or may form functionally different microtubules. To test this hypothesis, we have examined the functional properties and the structures of singlet-specific nonneural and neural tubulins from Antarctic fishes. Tubulins were purified from eggs of Notothenia coriiceps neglecta, and from brain tissues of N. coriiceps neglecta or N. gibberifrons, by DEAE ion-exchange chromatography and cycles of microtubule assembly/disassembly. At temperatures between 0 and 20 degrees C, each of these tubulins polymerized efficiently in vitro to yield microtubules of normal morphology. Critical concentrations for polymerization of egg tubulin ranged from 0.057 mg/ml at 3 degrees C to 0.002 mg/ml at 18 degrees C, whereas those for brain tubulin at like temperatures were 4-10-fold larger. Polymerization of both tubulins was entropically driven, but the apparent standard enthalpy and entropy changes for microtubule elongation by egg tubulin (delta Happ0 = +33.9 kcal/mol, delta Sapp0 = +151 entropy units) were significantly greater than values observed for brain tubulin (delta Happ0 = +26.5 kcal/mol, delta Sapp0 = +121 entropy units). Egg tubulin was composed of approximately six alpha and two beta chains and lacked the beta III isotype, whereas brain tubulin was more complex (greater than or equal to 10 of each chain type). Furthermore, egg alpha tubulins were more basic, and their carboxyl termini more resistant to cleavage by subtilisin, than were the alpha chains of brain. We conclude that brain and egg tubulins from the Antarctic fishes are functionally distinct in vitro, due either to qualitative or quantitative differences in isotypic composition, to differential posttranslational modification of shared isotypes, or to both.

Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.

To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.

Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin.

The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video-enhanced, differential-interference contrast (VE-DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 microM) displayed only plus-ended growth. The elongation velocity was 7.8 microns/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 microns/min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 microM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 microM) were assayed in ultrafiltrates (MW cut-off less than 30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 microns/min for 26 min before a catastrophe and rapid shortening at 11.8 microns/min. Rescue was less frequent than unfiltered extracts, minus-ended growth was observed, and self-assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non-buffer factors which stimulate elongation velocity by 6.5-fold without self-assembly, increase catastrophe frequency by 20-fold, and block minus-ended growth.

Microinjection of the monoclonal anti-tubulin antibody YL1/2 inhibits cleavage of sand dollar eggs.

Two monoclonal antibodies against alpha-tubulin (YL1/2 and D2D6) were microinjected into the egg of the sand dollar Clypeaster japonicus, and their effects on cleavage of the egg were investigated. They had already been shown by immunoblotting to react specifically with egg tubulin and by immunofluorescence to stain the mitotic apparatus [OKA et al., (1990). Cell Motil. Cytoskel. 16:239-250]. Injection of YL1/2 prevented chromosome movement and cleavage, although the cleavage furrow developed in some cases. In all eggs injected at prometaphase, metaphase, or anaphase, the birefringence of the mitotic apparatus disappeared immediately after injection. Injection of D2D6 had no significant effect on mitosis or cleavage of whole eggs injected after nuclear disappearance, although it prevented the disappearance of the nuclear envelope in 54% of the eggs injected before the disappearance. FITC-conjugated D2D6 did not accumulate in the spindle when injected into the dividing sand dollar egg. These results indicate that YL1/2 disassembled microtubules, whereas D2D6 did not bind to microtubules in the living cell.

A 70 kD microtubule‐binding protein from starfish eggs: Purification, characterization, and localization during meiosis and mitosis

AbstractA microtubule‐binding protein was purified from eggs of the starfish, Asterias amurensis, through several steps of purification including the taxol‐dependent procedure [Vallee, 1982, J. Cell Biol. 92:435–442]. This protein consists of a single polypeptide chain having an apparent molecular mass of 70 kD determined by SDS‐PAGE. The 70 kD protein was identified as a unique microtubulebinding protein, judging from electrophoretic mobility, cleavage pattern by limited proteolysis, heat stability, and immunocrossreactivity. The 70 kD protein binds to brain and egg microtubules. It does not promote assembly of brain tubulin, but promotes that of egg tubulin in vitro in a concentration‐dependent manner.Using indirect immunofluorescence and immunoelectron microscopy with the anti‐70 kD protein antibody, we analyzed the cellular localization of the 70 kD protein in starfish oocytes and eggs during both meiotic maturation (meicsis) and first cleavage (mitosis). Immunofluorescence studies showed that the 70 kD protein localized on microtubule structures spread widely throughout the cytoplasm, the sperm aster, and the microtubules making up the mitotic apparatus through both meiosis and mitosis. The antibody, however, did not recognize sperm axonemes. These results were confirmed by immunoelectron microscopy. Using a colloidal gold technique, the 70 kD protein was localized along the microtubules in vivo.This 70 kD protein is the first microtubule‐binding protein that has been shown to localize along the microtubules in oocytes and eggs throughout meiosis and mitosis and to promote microtubule assembly. The 70 kD protein may be involved in the dynamic changes of microtubule structures occurring within oocytes and eggs.

Molecular basis of physical and chemical probes for spindle assembly.

Microtubules are major cytoskeletal components of the mitotic spindles in echinoderm eggs. Well preserved isolated spindles from the first cleavage of sea urchin eggs reveal that 3, 000 to 5, 000 orderly arranged microtubules are present. Considering the sensitive nature of spindle bi-refringence, it is highly probable that the spindle assembly is regulated by the labile association of tubulin molecules and the involvement of kinetic centers such as the kinetochores and MTOCs in the centrosome region.Heavy water (D2O) increases the spindle volume and birefringence in living and dividing sea urchin eggs. Through the in vitro experiments, we confirmed that the initial rate and the final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of D2O. Yields were higher as the D2O concentration increased. D2O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concent-ration in the presence of D2O. We obtained linear van't Hoff plots and calculated thermodynamic parameters which were positive and increased with the elevation of the D2O concentration. The enhancement of the polymerization of tubulin by D2O in vitro could, therefore, be the result of the strengthening of intra-and/or inter-molecular hydrophobic interactions of tubulin molecules. We believe that the increase in length and number of microtubules of the mitotic spindles in dividing cells of eukaryotes with D2O is caused by the direct involvement of D2O in the polymerization of tubulin.When fertilized sea urchin eggs were exposed to dilute solutions of alkyl-resorcinols, i.e. T-1, an isolate from the culture medium of Pseudomonas sp., its derivatives or analogs such as Ansamycin, Curvularin or Macbecin I, spindles were more or less arrested in metaphase and became either barrel-shaped or ex-tremely enhanced. T-1 and Curvularin, which induced barrel-shaped spindles, possessed the same structural features with respect to 1, 3-benzenediol system with long lipophilic side chains. Structural modification of one of the hydroxyl groups of Curvularin provides a remarkable increase in chemical efficacy. This effect can be considered to be a freezing of the state of dynamic equilibrium or partial association/dissociation of tubulin molecules along with the microtubules by the given drugs. The barrel-shaped spindles consisted of bundles of straight microtubules of equal length, and we believe this might reflect the stabilized state of tubulin polymers caused by the local increase of molecular hydrophobicity. The anaphase sequence was usually delayed and chromosomes moved very slowly. We found that this unusual spindle shape was induced by the spread, discoidal redistribution of centrosomal MTOCs, and comparable with the irregular sea urchin spindles induced with mercaptoethanol.The finding of the TO series compounds turned our attention to 1, 4-benzo-quinones with long hydrophilic side chains, and Macbecin I was recognized to be a unique mitotic arrester. This compound could be a lead compound to search new mitotic arresters and its analogues obtained by synthesis. The unique in vivo assay system developed by us using the mitosis in dividing sea urchin eggs can be widely applied for the experimental survey of new mitotic arresters. Fur-ther massive screening effort would provide us more opportunity to find new drugs which can be used as the molecular probes to analyze the physico-chemical basis of the assembly and/or disassembly of cytoskeleton.

A microtubule-activated ATPase from sea urchin eggs, distinct from cytoplasmic dynein and kinesin.

We report an ATPase activity, present in sea urchin egg cytosol, that is activated by microtubules. The activity sediments at 10 S in sucrose gradients and is clearly distinct from activities at 12 S and 20 S due to cytoplasmic dynein. Potent activation of the ATPase is observed when endogenous egg tubulin is induced to assemble with taxol or when exogenous taxol-stabilized pure brain tubulin microtubules or flagellar outer-doublet microtubules are added. No activation by tubulin subunits or taxol alone is detectable. In contrast to flagellar or cytoplasmic dynein, the microtubule-activated enzyme is unaffected by vanadate or by nonionic detergents and hydrolyzes GTP in addition to ATP. In contrast to kinesin, it cosediments with microtubules in the presence or absence of ATP. The microtubule-activated enzyme may have a role in microtubule-based motility.

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin.

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

The effects of deuterium oxide ( 2H 2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of 2H 2O. The yields were increased in association with the elevation of the 2H 2O concentration. 2H 2O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of 2H 2O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the 2H 2O concentration. The enhancement of the polymerization of tubulin by 2H 2O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with 2H 2O may be caused by the direct involvement of 2H 2O in the polymerization of tubulin.

Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs

Sedimentation studies and [ 3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20–25 % of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles.

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus.

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.

Assembly of unfertilized sea urchin egg tubulin at physiological temperatures.

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by DEAE-column chromatography and cycles of temperature-dependent assembly and disassembly. Tubulin-containing column fractions self-assemble into intact microtubules in the absence of high molecular weight microtubule-associated proteins. Egg microtubules assembled during the third cycle of assembly following DEAE-chromatography are composed of 2 or 3 alpha tubulins and 2 beta tubulins as assayed by isoelectric focusing and two-dimensional electrophoresis. The critical protein concentrations necessary for assembly of egg tubulin at 37 and 25 degrees C are 0.15-0.24 and 0.24-0.28 mg/ml, respectively. At physiological temperatures, the critical protein concentrations are 0.81 mg/ml at 15 degrees C and 0.70-0.79 mg/ml at 18 degrees C. At 18 degrees C, bovine brain microtubule-associated proteins stoichiometrically stimulate the initial rate and final extent of egg tubulin assembly. These hybrid microtubules assemble at 18 degrees C at a critical protein concentration of 4-20 micrograms/ml.

An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis.

Tubulin can be isolated and purified from Xenopus laevis eggs through modification of Olmstedt's (1970) tubulin isolation method, viz. by repeating the vinblastin precipitation step after resuspension of the sediment in a detergent-containing stabilizing medium. By this we overcome the deleterious influence of the yolk granules in the isolation procedure. From 11 of Xenopus laevis eggs 25 mg VB-paracrystals can be obtained. The apparent molecular weight of the purified tubulin is 52,800. Antiserum against the purified Xenopus VB-paracrystals, raised in 2 Chinchilla rabbits, cross-reacts in immunodiffusion tests in agar gels with rat brain tubulin and with tubulin isolated from Xenopus laevis eggs by the described procedure. Specific indirect fluorescence staining and appropriate control reactions reveal that cilia of Tetrahymena pyriformis, cytoplasmic networks in cultured mouse Leydig cells, as well as mitotic spindles and nuclear regions in paraffin sections of Xenopus laevis blastulae, react with the antibodies against Xenopus laevis egg tubulin as well as with monoclonal antibodies against pig brain tubulin. These results provide additional evidence for the view that tubulin antibodies are neither species nor tissue specific and show that under appropriate conditions tubulin containing structures can be visualized in paraffin sections.

Microtubule proteins in axolotl eggs and developing embryos

Tubulin from eggs and embryos of the Mexican axolotl was characterized by electrophoresis and colchicine binding. In urea-polyacrylamide gel electrophoresis, soluble axolotl egg tubulin migrated as two bands, identical to tubulins from sea urchin sperm and Drosophila eggs. However, in SDS-containing gels, on which the α and β subunits of standard tubulins were well resolved, axolotl egg tubulin migrated as a single band with an apparent molecular weight of 53,500. The method of disruption of the eggs affected both yield of tubulin from vinblastine sulfate precipitates and stability of the colchicine binding activity. The colchicine binding activity of soluble tubulin from gently disrupted eggs was specific and of high affinity, with properties similar to those reported for other tubulins. The tubulin pool in unfertilized eggs was determined to be approximately 2 μg/egg; the level decreased 20% after initiation of cleavage and then remained constant through development to postneurula stages. The colchicine binding activity of soluble tubulin from embryos was much less stable than that of unfertilized eggs and decreased further during development. No differences were found in properties of tubulin from eggs of several strains of normally pigmented axolotls; however, tubulin from albino eggs showed slightly different properties in both electrophoresis and colchicine binding. The colchicine binding activity of soluble tubulin accounts for only half the total activity in axolotl eggs; they possess, in addition, a particulate nontubulin colchicine binding activity.

In Vitro Polymerization of Marine Egg Tubulin into Microtubules

In Vitro Polymerization of Marine Egg Tubulin into Microtubules

Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction.

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.