Tubulin Complex Research Articles

Mutations of oncoprotein 18/stathmin identify tubulin-directed regulatory activities distinct from tubulin association.

Oncoprotein 18/stathmin (Op18) is a recently identified phosphorylation-responsive regulator of the microtubule (MT) system. It was originally proposed that Op18 specifically regulates dynamic properties of MTs by associating with tubulin, but it has subsequently been proposed that Op18 acts simply by sequestering of tubulin heterodimers. We have dissected the mechanistic action of Op18 by generation of two distinct classes of mutants. One class has interruptions of the heptad repeats of a potential coiled-coil region of Op18, and the other involves substitution at all four phosphorylation sites with negatively charged Glu residues. Both types of mutation result in Op18 proteins with a limited decrease in tubulin complex formation. However, the MT-destabilizing activities of the coiled-coil mutants are more severely reduced in transfected leukemia cells than those of the Glu-substituted Op18 derivative, providing evidence for tubulin-directed regulatory activities distinct from tubulin complex formation. Analysis of Op18-mediated regulation of tubulin GTPase activity and taxol-promoted tubulin polymerization showed that while wild-type and Glu-substituted Op18 derivatives are active, the coiled-coil mutants are essentially inactive. This suggests that Op18-tubulin contact involves structural motifs that deliver a signal of regulatory importance to the MT system.

Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p.

Previously, we have shown that the yeast gamma-tubulin, Tub4p, forms a 6S complex with the spindle pole body components Spc98p and Spc97p. In this paper we report the purification of the Tub4p complex. It contained one molecule of Spc98p and Spc97p, and two or more molecules of Tub4p, but no other protein. We addressed how the Tub4p complex binds to the yeast microtubule organizing center, the spindle pole body (SPB). Genetic and biochemical data indicate that Spc98p and Spc97p of the Tub4p complex bind to the N-terminal domain of the SPB component Spc110p. Finally, we isolated a complex containing Spc110p, Spc42p, calmodulin and a 35 kDa protein, suggesting that these four proteins interact in the SPB. We discuss in a model, how the N-terminus of Spc110p anchors the Tub4p complex to the SPB and how Spc110p itself is embedded in the SPB.

Interaction of chicken gizzard smooth muscle calponin with brain microtubules.

Calponin, a major actin-, tropomyosin-, and calmodulin-binding protein in smooth muscle, interacted with tubulin, a main constituent of microtubules, in a concentration-dependent fashion in vitro. The apparent K(d) value of calponin to tubulin was calculated to be 5.2 microM with 2 mol of calponin maximally bound per 1 mol of tubulin. At low ionic strength, tubulin bound to calponin immobilized on Sepharose 4B, and the bound protein was released at about 270 mM NaCl. Chemical cross-linking experiments showed that a 1:1 molar covalent complex of calponin and tubulin was produced. The amount of calponin bound to microtubules decreased with increasing ionic strength or Ca2+ concentration. The addition of calmodulin or S100 to the mixture of calponin and microtubule proteins caused the removal of calponin from microtubules in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including tropomyosin, SM22, and caldesmon had little effect on the calponin binding to microtubules, whereas MAP2 inhibited the binding. Interestingly, there was little, if any, effect of mycalolide B-treated actin on the binding of calponin to microtubules. Furthermore, only about 20% of calponin-F-actin interaction was inhibited in the presence of an excess amount of tubulin (4 mol per mol of calponin), indicating that tubulin binds to calponin at a different site from that of actin. Compared with MAP2, calponin had little effect on microtubule polymerization.

Synaptic membrane G proteins are complexed with tubulin in situ.

The G proteins G S and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express G s alpha but not G i alpha 1, responded to the addition of tubulin with a stable activation of adenyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both G s and G i 1, responded to exogenous tubulin with a stable inhibition of adenyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and G i 1 and G s from detergent extracts of these membranes. Nearly 90% of the G s alpha from Triton X-114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein-cytoskeleton link that participates in the modulation of cellular signal transduction.

A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer.

The utility of collisional quenching of energy donors in fluorescence energy transfer is described. In multi-donor single acceptor systems, which contain different classes of donors (as distinguished by their accessibility towards a collisional quencher), donor quenching may be used to assess the fraction of energy transfer from each class of donor. The tubulin-colchicine complex was used as a donor-acceptor system to show that two inaccessible tryptophans are at or near the colchicine binding site.

Differential stability of the benzimidazole (BZ)-tubulin complex in BZ-resistant and BZ-susceptible isolates of Haemonchus contortus and Trichostrongylus colubriformis

The binding of [ 3H]mebendazole ([ 3H]MBZ) to tubulin from BZ-susceptible (BZ-S) and BZ-resistant (BZ-R) isolates of Haemonchus contortus and Trichostrongylus colubriformis was investigated using charcoal extraction and gel filtration techniques. The amount of [ 3H]MBZ bound at infinite free ligand concentration ( B max) was significantly reduced for the BZ-R isolate compared with the BZ-S isolate in both species when assayed by charcoal extraction. However, B max was increased to comparable levels for both BZ-S and BZ-R isolates of each species when assayed by the less stringent gel filtration technique. These results indicate that the BZ-tubulin interaction in trichostrongylid nematodes is comprised of a minimum of two components. As similar levels of total [ 3H]MBZ binding were observed for both BZ-S and BZ-R isolates of each species when assayed by gel filtration, it is suggested that the reduction in the pseudo-irreversible BZ binding component in BZ-R isolates results in an increase in the level of reversible BZ binding and therefore provides a survival advantage to BZ-R nematodes.

Aluminum Inhibits Calpain‐Mediated Proteolysis and Induces Human Neurofilament Proteins to Form ProteaseResistant High Molecular Weight Complexes

We studied the effects of aluminum salts on the degradation of human neurofilament subunits (NF-H, NF-M, and NF-L, the high, middle, and low molecular weight subunits, respectively) and other cytoskeletal proteins using calcium-activated neutral proteinase (calpain) purified from human brain. Calpain-mediated proteolysis of NF-L, tubulin, and glial fibrillary acidic protein (GFAP), three substrates that displayed constant digestion rates in vitro, was inhibited by AlCl3 (IC50 = 200 microM) and by aluminum lactate (IC50 = 400 microM). Aluminum salts inhibited proteolysis principally by affecting the substrates directly. After exposure to 400 microM aluminum lactate and removal of unbound aluminum, human cytoskeletal proteins were degraded two- to threefold more slowly by calpain. When cytoskeleton preparations were exposed to aluminum salt concentrations of 100 microM or higher, proportions of NF-M and NF-H formed urea-insoluble complexes of high apparent molecular mass, which were also resistant to proteolysis by calpain. Complexes of tubulin and of GFAP were not observed under the same conditions. Aluminum salts irreversibly inactivated calpain but only at high aluminum concentrations (IC50 = 1.2 and 2.1 mM for aluminum lactate and AlCl3, respectively), although longer exposure to the ion reduced by twofold the levels required for protease inhibition. These interactions of aluminum with neurofilament proteins and the effects on proteolysis suggest possible mechanisms for the impaired axoplasmic transport of neurofilaments and their accumulation in neuronal perikarya after aluminum administration in vivo.

Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: Evidence for an inhibitor of vincristine binding

In cytosols from human rhabdomyosarcoma xenografts, the formation of vincristine (VCR)-tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Biophys. Res. Commun., 135:695-700, 1986). We have further examined this modulation to determine whether a) GTP was protecting the VCR binding site from denaturation, b) the enhancement of complex formation was guanosine specific, and c) whether this influence was a direct interaction between GTP, VCR, and tubulin, or was mediated through another factor. In GTP-depleted cytosols from tumor xenografts HxRH18 and HxRh12, VCR binding activity was stable for at least 2 hours at 37 degrees C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 nM GTP increased complex formation slightly, with complex formation increasing as GTP concentrations were increased to 5 microM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation three-fold, while GMP, GMP-PNP, and ITP increased formation 1.5-fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific for the guanine nucleotides GDP and GTP. Microtubule protein, purified from Rh18 and Rh12 tumors by cycles of polymerization-depolymerization, bound VCR rapidly and binding was not influenced by GTP. This suggested that GTP modulation of VCR binding in cytosols was through a soluble factor lost in tubulin purification. In experiments with cytosol fractionated by molecular weight, there was inhibition of VCR binding activity by fractions with an mw range 20-50 kD. This inhibition was decreased by 25% by the addition of GTP. These data suggest that in tumor cytosols there may be competition between VCR and a natural ligand that is modulated by GTP. Two potential models for VCR binding are proposed.

Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex.

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.

The plant cytoskeleton

The plant cytoskeleton is a dynamic, three dimensional array of filamentous protein polymers, consisting of microtubules (MTs) and microfilaments (MFs). These cytoskeletal components play active roles in most, if not all aspects of plant cell growth, development and interaction with the environment. Recent advances in molecular genetics, cell imaging and the exploitation of plant systems, such as Arabidopsis thaliana have resulted in substantial advances in our understanding of the protein composition, regulation, dynamics of the cytoskeleton and interactions between MTs and MFs. MTs consist of a variety of tubulin isotypes (coded for by an array of tubulin genes and subsequently altered through post-translational modifications) and a large number of MT-associated proteins (MAPs). The complexity of MT chemistry (tubulins and MAPs) has helped explain how very similarly looking MTs can accomplish the wide array of observed stabilities, dynamics and functions. Although plants lack the recognizable MT organizing centers (MTOCs) found in other eukaryotes, MT assembly and reorganization is accomplished through complexes of gamma tubulin (γ-tubulin) and gamma tubulin complex proteins (GCPs) which regulate when, where, and in which pattern MTs are formed. Once formed, MT dynamics and function is controlled by a wide-array of MAPs, many of which have comparable homologs in other eukaryotic systems while other MAPs are plant-specific. Similar to MTs, MFs in plants exhibit a variety of actin isotypes (coded by an array of actin genes) and a large number of actin-binding proteins (ABPs). Actin proteins are divided into those involved in vegetative processes and those involved in reproductive processes. The specific functions of MFs are controlled by the activities of various ABPs. Many of the process originally thought to involve only MTs, have now been shown to require the proper functioning of both MTs and MFs. All aspects of cell reproduction (establishment of division plane, mitosis and cytokinesis) involve both MTs and MFs. Cell growth and shape is also controlled by the cytoskeleton. While MTs play active roles in regulating wall patterning and subsequent shape of cells growing via a diffuse growth mechanism, MFs are the key cytoskeletal elements important in defining and controlling cell expansion and shape in tip-growing systems. While great strides have been made in unraveling the complexities of cytoskeletal function in plants, much more needs doing. MTs and MFs appear to play a much more integrative role in plant development than previously believed. It is predicted that many more MAPs and ABPs await discovery, resulting more clarity in our understanding of the role of MTs and MFs in plant growth, development and interaction with the environment.

Mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate: alkylation of sulfhydryl group(s) of beta-tubulin.

The compound, 2,4-dichlorobenzyl thiocyanate (DCBT) was previously shown to cause mitotic arrest, disruption of intracellular microtubules, and inhibition of tubulin polymerization, with resistance to the drug conferred by a mutation in a beta-tubulin gene (Abraham, I., Dion, R.L., Duanmu, C., Gottesman, M.M. and Hamel, E. (1986) Proc. Natl. Acad. Sci. USA 83, 6839-6843). We have now examined its mechanism of action in further detail and conclude that DCBT acts as a sulfhydryl alkylating reagent. A mixed disulfide forms between the 2,4-dichlorobenzyl mercaptan moiety of DCBT and protein sulfhydryl groups with release of cyanate anion to the medium. Gel filtration and dialysis of complexes of tubulin formed with either [nitrile-14C]DCBT, [35S]DCBT or [benzyl-3H]DCBT demonstrated persistent association of 35S and 3H with denatured tubulin, but no binding of 14C to the protein even under native conditions. With equimolar tubulin and DCBT, beta-tubulin is the predominant alkylated species. At high drug concentrations, superstoichiometric amounts of DCBT react with tubulin, and both subunits are alkylated almost equally. When extracts of drug-treated L1210 murine leukemia cells were examined by polyacrylamide gel electroporesis, we found that multiple proteins were alkylated by DCBT, but the most prominent radiolabeled band was that corresponding to beta-tubulin. Dithiothreitol partially reverses inhibition of tubulin polymerization by DCBT and removes almost all the 2,4-dichlorobenzyl mercaptan moiety covalently bound to tubulin. Mitotic arrest occurs with DCBT because tubulin is the cellular protein most sensitive to the agent, probably because of its high cysteine content (20/mol).

Interzone microtubule behavior in late anaphase and telophase spindles.

We have studied microtubule behavior in late anaphase and telophase spindles of PtK1 cells, using fluoresceinated tubulin (DTAF-tubulin), microinjection, and laser microbeam photobleaching. We present the results of two novel tests which add to the evidence that DTAF-tubulin closely mimics the behavior of native tubulin in vivo. (a) Microinjected DTAF-tubulin was as effective as injected native tubulin in promoting division of taxol-dependent mitotic mutant cells that had been deprived of taxol. (b) Microinjected colchicine-DTAF-tubulin complex was similar to injected colchicine-native tubulin complex in causing depolymerization of spindles. Immediately after microinjection of DTAF-tubulin into wild-type cells during late anaphase or telophase, fluorescence incorporation by microtubules was seen in chromosomal half-spindles and just behind the chromosomes, but there was no fluorescence incorporation near the middle of the interzone. Over the next few minutes, tubulin fluorescence accumulated at the center of the interzone (the equator), becoming progressively more intense. In other experiments, cells were microinjected with DTAF-tubulin at prophase and allowed to equilibrate for 30 min. Cells that had progressed to late anaphase were then photobleached to reduce the fluorescence in the central portion of the interzone. Over a period of several minutes, the only substantial redistribution of fluorescence was the appearance of a bright area at the equator of the interzone. Both the site of fluorescence incorporation and the photobleaching data suggest that tubulin adds to the elongating spindle interzone near the equator where the plus ends of the interdigitated microtubules are located. In further experiments, several dark lines were photobleached perpendicular to the pole-to-pole axis of fluorescent anaphase-telophase spindles. Time-dependent changes in the spacings between the lines indicated that the two halves of the interzone lying on opposite sides of the spindle equator moved away from one another. This shows that the interdigitated microtubules, which make up most of the interzone, can undergo antiparallel sliding. Our data support a model for anaphase B in which plus end elongation of interdigitated microtubules and antiparallel sliding contribute to chromosome separation.

GTP influences the binding of vincristine in human tumor cytosols

The influence of GTP on the formation and stability of [ 3H]vincristine (VCR)-tubulin complexes in cytosols from two human rhabdomyosarcoma xenografts which have different sensitivities to VCR has been evaluated. After removal of endogenous GTP the initial rate of [ 3H]VCR binding and the maximal level of bound drug were 2- to 3-fold higher in the presence of 0.1 mM GTP than in its absence. Similarly, the stability of complexes was GTP-dependent. Complex formed from Rh18 tumors, only moderately sensitive to VCR, dissociated at 37° in the absence of GTP with a half-time of 67 min; complex from Rh12 tumors (exquisitely sensitive to VCR) was more stable. Neither complex dissociated in the presence of 0.1 mM GTP over 2 hr examined.

Interactions of combretastatin, a new plant-derived antimitotic agent, with tubulin

Tubulin is an important cancer drug target. Compounds that bind at the colchicine site in tubulin have attracted significant interest as they are generally less affected by multidrug resistance than other potential drugs. Modeling is useful in understanding the interactions between tubulin and colchicine binding site inhibitors (CBSIs), but because the colchicine binding site contains two flexible loops whose conformations are highly ligand-dependent, modeling has its limitations. X-ray crystallography provides experimental pictures of tubulin–ligand interactions at this challenging colchicine site. Since 2004, when the first X-ray structure of tubulin in complex with N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) was published, many X-ray crystal structures have been reported for tubulin complexes involving the colchicine binding site. In this review, we summarize the crystal structures of tubulin in complexes with various CBSIs, aiming to facilitate the discovery of new generations of tubulin inhibitors.

Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging.

Mammalian neurofilaments prepared from brain and spinal cord by either of two methods partially inhibit the in vitro assembly of microtubules. This inhibition is shown to be due to the association of a complex of high molecular weight microtubule-associated proteins (MAP1 and MAP2) and tubulin with the neurofilament. Further analysis of the association reveals a saturable binding of purified brain MAPs to purified neurofilaments with a Kd of 10(-7) M. Purified astroglial filaments neither inhibit microtubule assembly nor show significant binding of MAPs. It is proposed that the MAPs might function as one element in a network of intraorganellar links in the cytoplasm.

Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain.

1. The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol has been studied by near-u.v. circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 have been correlated with the known oligomeric composition of microtubule protein preparations, as determined by the sedimentation behaviour of this preparation [Bayley, Charlwood, Clark & Martin (1982) Eur. J. Biochem.121, 579-585]. 2. The formation of 30S oligomeric ring species, either by decreasing ionic strength at pH6.5 or by changing pH in the presence of 0.1m-NaCl, correlates with a significant change in tubulin c.d. Formation of 18S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. 3. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. 4. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH6.4 than at pH6.9. 5. Oligomeric complexes of tubulin with microtubule-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein.

Interactions of the tau-tubulin-vinblastine complex with colchicine, podophyllotoxin, and N,N'-ethylenebis(iodoacetamide).

Microtubule assembly is inhibited by anti-mitotic drugs such as colchicine or podophyllotoxin and also by sulfhydryl-oxidizing reagents, but it is not known which tubulin-tubulin interactions are disrupted by these agents. We have studied the interactions of a complex of tubulin, vinblastine, and tau protein with these agents. This complex has the form of a spiral filament and may consist of tubulin dimers joined end to end as in a protofilament; presumably, therefore, the lateral interaction sites should be accessible in this structure but not in the intact microtubule. Unlike the microtubule, the complex binds to colchicine and podophyllotoxin with high affinity. Again, unlike intact microtubules, the complex reacts with N,N'-ethylene-bis(iodoacetamide) to generate an intra-chain cross-link in beta-tubulin. Tubulin molecules containing this cross-link are unable to polymerize, suggesting that formation of this cross-link involves sulfhydryl groups that are critical for assembly. These results are consistent with a model whereby colchicine-, podophyllotoxin-, and sulfhydryl-oxidizing agents inhibit microtubule assembly by preventing lateral interactions between tubulin molecules in adjacent protofilaments.

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.

Tubulin‐Associated Nucleoside Diphosphokinase

Microtubule protein, prepared by cycles of polymerisation and dissociation, contained a nucleoside diphosphokinase (NDP kinase) activity (EC 2.7.4.6). This activity was not intrinsic to the tubulin dimer or the so-called microtubule-associated proteins. The NDP kinase had the following properties. (1) The enzyme existed in a low-molecular-weight form and in association with the complex of microtubule-associated proteins and tubulin (i.e. multimeric tubulin). (2) The low-molecular-weight species was also formed by dissociation of multimeric tubulin by salt or by removal of microtubule-associated proteins on phosphocellulose. (3) GDP bound to the exchangeable site of multimeric tubulin and also GDP derived from the E site of the tubulin dimer was a substrate for the NDP kinase. (4) The NDP kinase showed a 7-fold increase in activity during ATP-dependent microtubule assembly. On the basis of these properties, it is proposed that microtubule protein contains an NDP kinase specifically associated with tubulin and its functions.

Enzyme which specifically adds tyrosine to the alpha chain of tubulin.

A posttranslational modification of tubulin with potential significance for the regulation of microtubule assembly or function has been revealed by the discovery of an enzyme (tubulin--tyrosine ligase) which can add a tyrosine residue to the alphachain, apparently through peptide bond linkage to a C-terminal glutamate or glutamine. The ability to add tyrosine in the presence of ATP and to release it agiain in the presence of ADP and inorganic phosphate (or arsenate) appear to be functions of the same enzyme, as judged by the relative rates at which these reactions are catalyzed over a 20-fold enzyme purification. The apparent size of the enzyme from bovine brain is 150 000 daltons in extracts and after ammonium sulfate fractionation, but 35 000 after elution from anion-exchange columns. Addition of pure dimeric tubulin to the latter species converts it back to the larger form, which is apparently a stoichiometric 1:1 complex of tubulin and the 35 000-dalton enzyme. Tubulin--tyrosine ligase is specific for tubulin; other proteins with C-terminal glutamate or glutamine do not substratesmate or glutamine do not act as substrates or inhibitors. It is less specific for tyrosine; five out of six tyrosine dipeptides were inhibitors and competed with tyrosine.