Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin

In mammalian brain, beta-tubulin occurs as a mixture of four isotypes designated as types I, II, III, and IV. It has been speculated in recent years that the different tubulin isotypes may confer functional diversity to microtubules. In an effort to investigate whether different tubulin isotypes differ in their functional properties we have studied the colchicine binding kinetics of bovine brain tubulin upon removal of the beta III isotype. We found that the removal of the beta III isotype alters the binding kinetics from biphasic to monophasic with the disappearance of the slow phase. The kinetics become biphasic with the reappearance of the slow phase when the beta III-depleted tubulin was mixed with the beta III fraction eluted from the affinity column with 0.5 M NaCl. The analysis of the kinetic data reveals that the tubulin dimers containing beta III bind colchicine at an on-rate constant of 35 M-1 s-1 while those lacking beta III bind at 182 M-1 s-1. Our results strongly suggest that the beta-subunit plays a very important role in the interaction of tubulin with colchicine.     

Comparative molecular field analysis of colchicine inhibition and tubulin polymerization for combretastatins binding to the colchicine binding site on beta-tubulin

A molecular modeling study using Comparative Molecular Field Analysis (CoMFA) was undertaken to develop a predictive model for combretastatin binding to the colchicine binding site of tubulin. Furthermore, we examined the potential contribution of lipophilicity (log P) and molecular dipole moment and were unable to correlate these properties to the observed biological data. In this study we first confirmed that tubulin polymerization inhibition (IC50) correlated (R2 = 0.92) with [3H]colchicine displacement. Although these data correlated quite well, we developed two independent models for each set of data to quantify structural features that may contribute to each biological property independently. To develop our predictive model we first examined a series of molecular alignments for the training set and ultimately found that overlaying the respective trimethoxyphenyl rings (A ring) of the analogues generated the best correlated model. The CoMFA yielded a cross-validated R2 = 0.41 (optimum number of components equal to 5) for the tubulin polymerization model and an R2 = 0.38 (optimum number of components equal to 5) for [3H]colchicine inhibition. Final non-cross-validation generated models for tubulin polymerization (R2 of 0.93) and colchicine inhibition (R2 of 0.91). These models were validated by predicting both biological properties for compounds not used in the training set. These models accurately predicted the IC50 for tubulin polymerization with an R2 of 0.88 (n = 6) and those of [3H]colchicine displacement with an R2 of 0.80 (n = 7). This study represents the first predictive model for the colchicine binding site over a wide range of combretastatin analogues.     

Functional reconstitution of a GABAA receptor purified from bovine brain.

The GABAA/benzodiazepine receptor has been solubilized from bovine brain membranes and purified by benzodiazepine affinity chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two major protein species of 53 and 56 kDa. The purified protein has been reconstituted, in a functionally active form, into phospholipid vesicles. Chloride flux responses of the reconstituted preparations were investigated in stopped-flow experiments by monitoring fluorescence changes of a chloride-sensitive dye trapped within the vesicles. Flux was rapidly stimulated by muscimol and this response was potentiated by diazepam and blocked by desensitization of the receptor and by preincubation with the channel blocker, picrotoxin.     

Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.     

Properties of heparin binding to purified plasma membranes from bovine granulosa cells

Bovine granulosa cells were disrupted by nitrogen cavitation and the resulting membrane vesicles were isolated by centrifugation using a self-generating Percoll gradient. Transmission electron microscopy and marker enzyme assays revealed a highly enriched preparation of plasma membrane vesicles with little contamination from intracellular organelles. The membranes were examined for their ability to bind [3H]heparin under a variety of physical conditions. Binding was dependent largely on electrostatic interactions which were sensitive to alterations in the ionic strength and pH of the medium. Optimal binding was obtained in the absence of added salt and at pH 6.5 but reduced by 50% at 150 mM-NaCl or at pH values above 7.5. Heparin binding to the membranes was abolished by a 1-h pretreatment with chymotrypsin, plasmin, pronase or trypsin. Detergent treatment of the membranes had various effects, depending on the ionic characteristics of the detergents used. Sodium dodecyl sulphate-polyacrylamide gels of plasma membrane proteins revealed a complex pattern of polypeptides with Mr of 10,000-120,000. Autoradiographic analysis of plasma membrane proteins on Western blots labelled with 125I-labelled heparin revealed 3 major heparin-binding proteins with molecular weights of 14,000-16,000. These studies report a new method of rapidly obtaining purified membranes from a limited population of granulosa cells. The characterization of the binding domains as membrane-associated proteins provides opportunities for numerous additional studies. Detergent solubilization of the membranes without appreciable loss in binding activity should simplify attempts to purify the binding proteins. Further analysis of the interactions of these molecules with native follicular fluid GAGs at various stages of granulosa cell development should provide useful insights into the role of complex carbohydrates in follicular maturation.     

Oligomeric properties of alpha-dendrotoxin-sensitive potassium ion channels purified from bovine brain.

Neuronal acceptors for alpha-dendrotoxin (alpha-DTX) have recently been purified from mammalian brain and shown to consist of two classes of subunit, a larger (approximately 78,000 M(r)) protein (alpha) whose N-terminal sequence is identical to that of a cloned, alpha-DTX-sensitive K+ channel, and a novel M(r) 39,000 (beta) polypeptide of unknown function. However, little information is available regarding the oligomeric composition of these native molecules. By sedimentation analysis of alpha-DTX acceptors isolated from bovine cortex, two species have been identified. A minority of these oligomers contain only the larger protein, while the vast majority possess both subunits. Based on accurate determination of the molecular weights of these two forms it is proposed that alpha-DTX-sensitive K+ channels exist as alpha 4 beta 4 complexes because this combination gives the best fit to the experimental data.     

Differential utilization of beta-tubulin isotypes in differentiating neurites

beta-Tubulin is encoded in vertebrate genomes by a family of six to seven functional genes that produce six different polypeptide isotypes. We now document that although rat PC-12 cells express five of these isotypes, only two (classes II and III) accumulate significantly as a consequence of nerve growth factor-stimulated neurite outgrowth. In contrast to previous efforts that have failed to detect in vivo distinctions among different beta-tubulin isotypes, we demonstrate using immunoblotting with isotype-specific antibodies that three beta-tubulin polypeptides (classes I, II, and IV) are used preferentially for assembly of neurite microtubules (with approximately 70% of types I and II assembled but only approximately 50% of type III in polymer). Immunofluorescence localization shows that an additional isotype (V) is partially excluded from neurites. Distinctions in in vivo localization of the neuron-specific, class III isotype have also been directly observed using immunofluorescence and immunogold electron microscopy. The sum of these efforts documents that some in vivo functional differences between tubulin isotypes do exist.     

Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.     

Properties of partially purified bovine brain dopamine beta-hydroxylase.

Dopamine beta-hydroxylase has been partially purified from bovine brain. A 140-fold purification factor was achieved using solubilization with Triton X-100, ammonium sulphate fractionation between 20-50 per cent saturation, affinity chromatography on concanavalin A-Sepharose 4 B and then filtration through Sephadex G200. The specific activity at the end was 51 nmoles/h/mg protein. The majority of endogenous inhibitors were lost. Immunological studies, kinetic studies, studies on the interaction with lectins and the effect of carboxylic acids on enzyme activity were carried out. Our data are in favour of the close similarity between the bovine brain and adrenal enzymes. No major differences could be found, at least with the characterization experiments using in the present study.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

Production and properties of antibody to soluble guanylate cyclase purified from bovine brain

Guanylate cyclase was purified 12,700-fold from bovine brain supernatant, and the purified enzyme exhibited essentially a single protein band on polyacrylamide gel electrophoresis. Repeated injection of the purified enzyme into rabbits produced an antibody to guanylate cyclase. The immunoglobulin G fraction from the immunized rabbit gave only one precipitin line against the purified guanylate cyclase and the crude supernatant of bovine brain on double immunodiffusion and immunoelectrophoreis. The antibody completely inhibited the soluble guanylate cyclase activity from bovine brain, various tissues of rat and mouse and neuroblastoma N1E 115 cells, whereas the Triton-dispersed particulate guanylate cyclase from these tissues was not inhibited by the antibody.     

Nucleotide binding and cofactor activities of purified bovine brain and bacterially expressed ADP-ribosylation factor

The ADP-ribosylation factor (ARF) is a member of the small molecular weight GTP-binding protein family and serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory regulatory subunit (Gs) of adenylate cyclase. Bovine Arf1 has been expressed at high levels and purified from bacteria. The recombinant Arf1 was compared with purified bovine brain Arf and shown to be nearly identical with respect to immunoblotting, guanine nucleotide binding, GTP hydrolysis, and cholera toxin cofactor activities. The only known chemical difference between the recombinant and brain proteins is the lack of myristic acid at the amino terminus of the expressed protein. The preparation of nucleotide-free Arf1 has allowed a more accurate determination of the binding constants for guanine nucleotides and revealed a significantly higher affinity for GDP than was previously determined. The effect of magnesium ions on nucleotide affinities was also determined and found to be quite different for the different guanine nucleotides. We have shown that GDP binds to the protein in the absence of magnesium, while GTP or guanosine 5'-O-(thiotriphosphate) can only bind to Arf1 in the presence of nanomolar (or higher) levels of the free metal. This characterization of the nucleotide binding and the ability to produce large amounts of a single species of ARF with full retention of a range of activities should greatly facilitate subsequent studies on the structure and function of ARF.     

Kinetics of [3H]muscimol binding to the GABAA receptor in bovine brain membranes

The binding of the GABA receptor agonist [3H]muscimol to membrane preparations from bovine cerebral cortex has been investigated in equilibrium and kinetic experiments. Equilibrium binding curves are biphasic and suggest that [3H]muscimol binds to both high-affinity (Kd approximately 10 nM) and low-affinity (Kd approximately 0.5 microM) sites. Binding to each class of sites is inhibited by GABA and by the specific GABAA receptor antagonist bicuculline. The kinetics of [3H]muscimol binding have been measured by using both manual filtration assays and an automated rapid filtration technique which permits the measurement of ligand dissociation on subsecond time scales. Association and dissociation curves are biphasic at all concentrations of [3H]muscimol studied, even under conditions of low receptor saturation when no significant occupancy of the low-affinity sites would be expected. These results cannot be simply explained by the presence of two populations of binding sites in the membrane preparations but suggest the existence of two forms of the monoliganded receptor. Dissociation constants for these two forms have been estimated to be 16 and 82 nM at 23 degrees C. At higher ligand concentrations, kinetic measurements have suggested that the binding of [3H]muscimol to low-affinity sites is accompanied by a slow conformational change of the receptor-ligand complex.     

Conformational analysis of the carboxy-terminal tails of human beta-tubulin isotypes

Several isotypes of the structural protein tubulin have been characterized. Their expression offers a plausible explanation for differences regarding microtubule function. Although sequence variation between tubulin isotypes occurs throughout the entire protein, it is the extreme carboxy-terminal tails (CTTs) that exhibit the greatest concentration of differences. In humans, the CTTs range in length from 9 to 25 residues and because of a considerable number of glutamic acid residues, contain over 1/3 of tubulin's total electrostatic charge. The CTTs are believed to be highly disordered and their precise function has yet to be determined. However, their absence has been shown to result in altered microtubule stability and a reduction in the interaction with several microtubule-associated proteins (MAPs). To characterize the role that CTTs play in microtubule function, we examined the global conformational differences within a set of nine human β-tubulin isotypes using replica exchange molecular dynamics simulations. Through the analysis of the resulting configuration ensembles, we quantified differences such as the CTTs sequence influence on overall flexibility and average secondary structure. Although only minor variations between each CTT were observed, we suggest that these differences may be significant enough to affect interactions with MAPs, thereby influencing important properties such as microtubule assembly and stability.