FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1997219785> ?p ?o ?g. }

• W1997219785 endingPage "29027" @default.
• W1997219785 startingPage "29018" @default.
• W1997219785 abstract "Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide,N-[14C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios ≪1 cysteine/dimer always leads to labeling of 6–7 cysteine residues. These have been identified as Cys-305α, Cys-315α, Cys-316α, Cys-347α, Cys-376α, Cys-241β, and Cys-356β by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347α as the most reactive cysteine of tubulin. Using the 3.5-Å electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 Å of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity. Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide,N-[14C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios ≪1 cysteine/dimer always leads to labeling of 6–7 cysteine residues. These have been identified as Cys-305α, Cys-315α, Cys-316α, Cys-347α, Cys-376α, Cys-241β, and Cys-356β by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347α as the most reactive cysteine of tubulin. Using the 3.5-Å electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 Å of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity. 5,5′-dithiobis(2-nitrobenzoic acid) or Ellman's reagent 5-((((2-iodoacetyl)amino)ethyl)-amino) naphthalene-1-sulfonic acid (AEDANS is the fluorescent moiety that is bound to protein and lacks the iodine) N-ethylmaleimide high performance liquid chromatography l-1-tosylamide-2-phenethyl chloromethyl ketone TPCK-treated trypsin 3-[cyclohexylamino]-1-propanesulfonic acid 4- morpholineethanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine matrix-assisted laser desorption/ionization-time of flight The 20 SH groups of the tubulin dimer have long led to speculation as to their function. Requirements for a few of the SH groups have been identified. Thus, Cys-12β is near the binding site of the exchangeable GTP of β-tubulin (1Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar), and a C12Sβ mutation is lethal in haploid yeast, although a C12Aβ mutation is survivable (2Gupta M.L. Bode C.J. Dougherty C.A. Marquez R.T. Himes R.H. Cell Motil. Cytoskeleton. 2001; 49: 67-77Crossref PubMed Scopus (41) Google Scholar). Cys-241β 1Because of a difference in alignment in the electron diffraction structure of β-tubulin, Cys-241 corresponds to Cys-239, and Cys-356 corresponds to Cys-354 in the linear sequence. and Cys-356β are near or are part of the binding site for colchicine and other agents (3Uppuluri S. Knipling L. Sackett D.L. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11598-11602Crossref PubMed Scopus (162) Google Scholar, 4Bai R. Lin C.M. Nguyen N.Y. Liu T.-Y. Hamel E. Biochemistry. 1996; 28: 5606-5612Crossref Scopus (53) Google Scholar, 5Shan B. Medina J.C. Santha E. Frankmoelle W.P. Chou T.-C. Learned R.M. Narbut M.R. Stott D. Wu P. Jaen J.C. Rosen T. Timmermans P.B.M.W.M. Beckmann H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5686-5691Crossref PubMed Scopus (166) Google Scholar). What the precise role of the involvement of these cysteines may be is for the most part not clear. Some of the SH groups of tubulin form thioesters with palmitic acid both in vivo and in vitro; these may be responsible for membrane localization of tubulin (6Zambito A.M. Wolff J. Biochem. Biophys. Res. Commun. 1997; 239: 650-654Crossref PubMed Scopus (25) Google Scholar, 7Caron J.M. Mol. Biol. Cell. 1992; 8: 621-636Crossref Scopus (61) Google Scholar, 8Wolff J. Zambito A.M. Britto P.J. Knipling L. Protein Sci. 2000; 9: 1357-1364Crossref PubMed Scopus (19) Google Scholar, 9Zambito A.M. Wolff J. Biochem. Biophys. Res. Commun. 2001; 283: 42-47Crossref PubMed Scopus (16) Google Scholar). One of these has been located as Cys-376α (10Ozols J. Caron J.M. Mol. Biol. Cell. 1997; 8: 637-645Crossref PubMed Scopus (43) Google Scholar). Except for this palmitoylation site, no specific functions have been identified for the 12 SH groups of α-tubulin, and the order of reactivities of the SH groups has not been definitively established. It has been repeatedly demonstrated (11Luduena R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (194) Google Scholar) that reaction of an equivalent of 1 or 2 SH groups with the usual alkylating agents abolished polymerization competence, but their location in the sequence has not been unambiguously determined. Loss of colchicine binding requires modification of additional SH groups by these nonspecific SH reagents. For this reason we have approached the reactivity of tubulin SH groups in a more general sense, comparing the effects of thioether, disulfide, and thioester formation as well as their location in α-tubulin and β-tubulin. Protein sulfhydryl groups can be involved in numerous reactions such as oxidation, disulfide interchange, thioether, and thioester formation. For the purpose of this discussion, we shall exclude oxidation. Although free radical reactions of SH groups are known, the remaining reactions all involve the thiolate anion as the reactive species, whereas the thiol group has very much lower reactivity (12Friedman M. The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides, and Proteins. Pergamon Press Ltd., Oxford1973Google Scholar). Cysteine reactivity toward various sulfhydryl reagents is regulated by a number of factors including first, exposure to the solvent, and second, dissociation of the thiol to the thiolate anion. RS− is a strong nucleophile (stronger than RO−) normally leading to SN2 reactions. Ionization is suppressed by neighboring acidic groups and enhanced by basic amino acids (12Friedman M. The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides, and Proteins. Pergamon Press Ltd., Oxford1973Google Scholar). Although the great preponderance of SH groups involved catalytically in enzyme reactions have low pK a values for dissociation to the thiolate anion, less is known about pK a values of SH groups of cysteines not directly involved in catalysis. The tubulin cysteine pK a values are not known. In general it is assumed and has been shown in certain cases that these approach the “normal” SH pK a values near pH 8.5–9.0. Third, cysteine reactivity is regulated by the reactivity of the SH reagent. For disulfide interchange, the pK a is aryl-SH ≪ alkyl-SH, making the former more reactive. Thus, DTNB2 is highly reactive with respect to rate and extent of reaction with native tubulin. Factors outlined fourth and sixth (in the next paragraphs) also contribute to this high reactivity of DTNB. It must be remembered that many of the SH reagents can also react with undissociated thiols albeit at a much lower rate. This must be kept in mind when ascribing low pK a values for SH groups from reactivity with a thiol reagent. Fourth, cysteine reactivity is regulated by charge compatibility between the reagent and the cysteine environment, e.g.iodoacetate versus iodoacetamide or DTNB versus2,2′-dipyridyl disulfide (13Brocklehurst K. Little G. Biochem. J. 1973; 133: 67-80Crossref PubMed Scopus (124) Google Scholar). 2,2′-Dipyridyl disulfide yields significant reaction with cysteine at pH 2. Because the tubulin pK a values are not known, we tested cysteine (assuming a pK a ∼ 8.5) reactivity at pH 2.0 and found a brisk reaction with this reagent. Presumably, this indicates that the weakly nucleophilic, undissociated thiol was one of the reactants. Fifth, cysteine reactivity is regulated by the stability of the bonds formed (in decreasing order), thioether > disulfide > thioester. Most studies on tubulin SH groups have used thioether formation with iodoacetate or iodoacetamide or their derivatives or maleimides. The former react relatively slowly with native protein and with a rather limited number of cysteines (11Luduena R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (194) Google Scholar). Sixth, cysteine reactivity is regulated by the nature of the leaving group of the sulfhydryl reagent, e.g. a thiolate, as found in DTNB, is a good leaving group as are other negatively charged species. The great difficulty in analyzing very hydrophobic peptides produced by palmitoylation led us to take advantage of the greater stability and hydrophilicity of the thioether bond for subsequent manipulations such as the analysis of tryptic peptides. In the present study we have focused on the comparative reactivities for thioether formation of the SH groups of tubulin. In a subsequent study we shall compare this with disulfide and thioester bond formation, the effect of the loss of the fast or slow reacting cysteines, and the effect of the size of the substituents on the ability of tubulin to polymerize and to react with ligands. N-[ethyl-1-14C]Ethylmaleimide (50 mCi/mmol) in n-pentane, [1-14C]iodoacetamide (50 mCi/mmol) in ethanol, and [carbonyl-14C]chloroacetamide (55 mCi/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). N-Ethylmaleimide and iodoacetamide were from Sigma, and 1,5-IAEDANS [5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid] was from Molecular Probes.syn-Monobromobimane (Molecular Probes) and Thioglo 1 (Calbiochem) were used from their acetonitrile stock solutions. Trypsin-TPCK was obtained from Worthington. All other reagents were the highest grade available from Sigma unless otherwise noted. Pure (>99%) rat brain tubulin was prepared as described (14Wolff J. Knipling L. Sackett D.L. Biochemistry. 1996; 35: 5910-5920Crossref PubMed Scopus (24) Google Scholar). All the experiments were performed with buffer (0.3 m Mes, pH 6.9, 1.0 mm EGTA, and 1.0 mm MgCl2) in the dark, and tubulin concentration was 30 μm in all experiments. Stock solutions of the sulfhydryl reagents were prepared fresh in Mes assembly buffer (0.1 m Mes, 1.0 mmEGTA, 1.0 mm MgCl2, pH 6.9). The specific activities of 14C-labeled reagents were adjusted with unlabeled compounds whenever necessary. The reverse phase HPLC columns were obtained from Phenomenex. Two types of experiments were performed, 1) a time course of tubulin sulfhydryl modification at 37 °C at low (1:2) and high (50:1) molar ratios of reagent to tubulin and 2) an 8-h incubation at 4 °C with varying molar ratios (1:5, 1:2, 1:1, 3:1, 5:1, and 10:1) of reagent to tubulin. Reactions were stopped by adding β-mercaptoethanol to a final concentration of 5 mm, and samples were sonicated and placed on ice. The modified tubulin was separated immediately from the unreacted reagent and β-mercaptoethanol by passing through a Sephadex G25 medium column (25 × 0.7 cm) equilibrated with 10 mm Tris-HCl buffer, pH 8.5. The protein fractions were pooled, and protein was estimated using the bicinchoninic acid assay (15Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18709) Google Scholar). The radioactivity was measured using a TRI-CARB liquid scintillation analyzer (model 1900CA) with 5 ml of scintillation liquid (Ultima GOLD, Packard) plus 5–20 μl of sample. The mole ratio of14C bound per tubulin dimer was calculated for each sample. For 1,5-IAEDANS-modified tubulin samples, 8 or 10 μmmodified tubulin solutions were made (from the pooled fraction) in 0.1m Tris-HCl, pH 8.0 buffer, and the absorbance spectra (Cary 300 spectrophotometer) were recorded for each sample. AEDANS has an absorbance maximum at 330 nm at pH 8.0 with an ε = 5.7 × 103m−1 cm−1. From the absorbance at 330 nm and the extinction coefficient, the concentration of bound AEDANS to tubulin dimer was estimated. In addition, time courses of syn-monobromobimane (λex = 392 nm, λem = 480 nm, at a mole ratio of 67:1) (16Radkowski A.E. Kosower E.M. J. Am. Chem. Soc. 1986; 108: 4527-4531Crossref Scopus (38) Google Scholar) and Thioglo 1 (λex = 379 nm, λem = 510 nm, at a mole ratio of 40:1) (17Wright S.K. Viola R.E. Anal. Biochem. 1998; 265: 8-14Crossref PubMed Scopus (102) Google Scholar) modifications of tubulin were done at room temperature by following the fluorescence of the product in a PerkinElmer LS-50B fluorimeter using 3-mm masked cells. The14C-modified tubulin samples were subjected to electrophoresis in 10% polyacrylamide gels (1.0-, 2.0-, and 3.0-mm thick gels were used) to separate α- and β-subunits as described by Knipling et al. (18Knipling L. Hwang J. Wolff J. Cell Motil. Cytoskeleton. 1999; 43: 63-71Crossref PubMed Scopus (35) Google Scholar). After the run, the gels were equilibrated in the transfer buffer (10 mm CAPS in 20% methanol, pH 11) for 1–2 h and transferred to polyvinylidene difluoride membranes (Immobilon PVDF from Millipore) by applying 1–1.5 mA/cm2 over 5–8 h using a Amersham Biosciences Multiphor II unit. The membranes were air dried overnight and exposed to phosphorimaging plates (BAS-IP MS 2340) for 10–20 days at room temperature. Then the imaging plates were scanned in an FLA3000G image analyzer (FujifilmTM I&I Imaging, Fuji Medical Systems). The IAEDANS-modified tubulin samples were subjected to electrophoresis in 10% polyacrylamide gels (3.0 mm thick). The fluorescent gel bands were imaged using FluorChemTM8000 Advanced Fluorescence, Chemiluminescence, and Visible Imaging Software. The gel was excited at 302 nm to see the emission at 490 nm. The modified tubulin samples (0.5–1.0 mg) were digested with trypsin-TPCK (1:20 weight ratio) at 37 °C for 15–24 h in 0.1 m Tris buffer, pH 8.5, with 5 mm CaCl2. The trypsin digests of modified tubulin were subjected to electrophoresis in 16% or 10–20% Tris-Tricine Novex precast gels. To obtain a higher yield of labeled peptides, we used a preparative (250 × 10 mm, pore size 300 Å, particle size 10 μm) C18 reverse phase column. 500–700 μg of the protein digest (in 150–200 μl) containing 4–6m guanidine-HCl was sonicated and centrifuged before injection into the column. A PerkinElmer series 410 LC pump with a LC-95 UV-visible spectrophotometer was used to apply solvent gradients. We used methanol instead of acetonitrile, and the fractionation of labeled tubulin digest was achieved by applying the following gradient (flow rate, 1.0 ml/min): 5% methanol plus 95% water, 5 mmammonium acetate for 20 min; 5% methanol plus 95% water, 5 mm ammonium acetate to 50% methanol plus 50% water, 5 mm ammonium acetate over a period of 150 min; 50% methanol plus 50% water, 5 mm ammonium acetate to 95% methanol plus 5% water, 5 mm ammonium acetate over a period of 45 min; 95% methanol plus 5% water, 5 mm ammonium acetate for 40 min. This gradient gave us good reproducibility and recovery of radioactivity (greater than 80%). A C12 reverse phase column (50 × 4.6 mm, pore size 90 Å, particle size 4 μm) was used for further purification of peptides. The absorbance at 214 nm was monitored; the peaks were collected manually and counted. The radioactive and the fluorescent peaks were concentrated on a Speedvac instrument and sent for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and N-terminal sequence analysis to the Macromolecular Structure Facility, Michigan State University. The masses of the peptides were calculated from the Protein and Peptide Software developed by Dr. Lewis Pannell, NIH (sx102a.niddk.nih.gov/peptide). RASMOL (19Sayle R. Milner-White E.J. Trends Biochem. Sci. 1995; 20: 374-376Abstract Full Text PDF PubMed Scopus (2323) Google Scholar) was used to visualize and generate tubulin structure (Protein Data Bank code1JFF). It has been proposed that several SH groups of tubulin may form disulfide bonds (20Chaudhuri A.R. Khan I.A. Luduena R.F. Biochemistry. 2001; 40: 8834-8845Crossref PubMed Scopus (45) Google Scholar), but in the hands of most investigators the 20 SH groups (12 in α- and 8 in β-tubulin) are readily shown to be reduced, and the rat brain tubulin used here consistently yields >19 SH groups by titration with excess DTNB in the absence of any denaturing agent. Moreover, the electron diffraction structure shows no disulfide bonds (21Löwe J. Li H. Downing K.H. Nogales E. J. Mol. Biol. 2001; 313: 1045-1057Crossref PubMed Scopus (998) Google Scholar). The time courses of thioether formation with iodoacetamide, IAEDANS, or the maleimides were compared in an excess of reagent (reagent to tubulin = 50:1 or 2.5:1 per SH group at pH 6.9 and 37 °C (Fig.1 A). Both SN2 displacing reagents, iodoacetamide and IAEDANS, showed a slow progressive increase in the number of cysteines reacting over a period of 3 h. Of considerable interest is the finding that the bulky IAEDANS reacted at the same rate as iodoacetamide. Another reagent in this class is syn-monobromobimane (16Radkowski A.E. Kosower E.M. J. Am. Chem. Soc. 1986; 108: 4527-4531Crossref Scopus (38) Google Scholar), whose progress curve can by followed by fluorescence (excitation = 392 nm, emission = 480 nm, quantum yield 0.2–0.3), as the reagent itself is negligibly fluorescent. The initial rate of reaction is not much faster than iodoacetamide, and the extent of reaction is comparable. As has been reported for small thiols (16Radkowski A.E. Kosower E.M. J. Am. Chem. Soc. 1986; 108: 4527-4531Crossref Scopus (38) Google Scholar), the reaction is pH-sensitive and is a linear function of the OH− concentration (over the pH range available due to the pI of the protein) (data not shown). As might be expected, monochlorobimane reacts much more slowly than its bromo congener (data not shown). Unfortunately, monobromobimane is subject to photolysis leading to fluorescent products; hence, minimal light exposure and careful blank corrections are critical at all time points. Because separation of excess reagent is required, we have not further pursued this labeling procedure. Nevertheless, all three reagents react with ∼9 of the 20 SH residues in 3 h (Fig.1 A). Thioether formation with maleimides occurs by nucleophilic addition to a double bond rather than by nucleophilic displacement. As shown in Fig. 1 A (top curves), this is a much faster and more extensive reaction. Thus, at 15 min and 37 °C, virtually all of the cysteines have reached the plateau value seen at 2 h. 3–4 SH groups did not react over the 2-h time period. Interaction with a fluorescent maleimide analogue, Thioglo 1 (17Wright S.K. Viola R.E. Anal. Biochem. 1998; 265: 8-14Crossref PubMed Scopus (102) Google Scholar), occurs at an equally rapid rate under these conditions, and again, ∼3–4 cysteines failed to react in the time allowed. It is of interest that here, too, a bulky fluorophore in no way impeded access to 16–17 cysteines of tubulin nor does the fluorophore significantly accelerate interaction, as has been noted for long chain alkylmaleimides on proteins but not small thiols (22Denicola-Seoane A. Anderson B.M. Biochim. Biophys. Acta. 1990; 1040: 84-88Crossref PubMed Scopus (2) Google Scholar). Thus, although reaction conditions are not identical, it is apparent that thioether formation by nucleophilic displacement is substantially slower than by the addition to double bonds. This difference in rates has been observed previously for small thiols; second order rate constants differ by between 1 and 2 orders of magnitude (23Gorin G. Martic P.A. Doughty G. Arch. Biochem. Biophys. 1966; 115: 593-597Crossref PubMed Scopus (110) Google Scholar, 24Hanson H. Hermann P. Bull. Soc. Chim. Biol. 1958; 40: 1835-1847PubMed Google Scholar, 25Steenkamp D.J. Biochem. J. 1993; 292: 295-301Crossref PubMed Scopus (20) Google Scholar). There are clearly fast and slow reacting SH groups in tubulin; the latter become fast upon denaturation with urea such that the full increase in fluorescence occurs virtually instantaneously (data not shown). With progressively increasing urea concentrations, two reaction classes (initial slopes) are observed, a relatively slow rate up to ∼1.5 m urea and a faster rate with urea >1.5m. Because the main objective of the present study is to discover the location of the most reactive cysteines of tubulin, it is necessary to devise conditions for attaining limited stoichiometries by using low mole ratios of reagent to tubulin, low temperatures, or low pH. To minimize reactions with less reactive cysteines, we used mole ratios of 1:2 at 37 °C at pH 6.9 for varying times. Under these conditions the available iodoacetamide was not used up at 3 h (Fig.1 B), and, although there was very low incorporation at early time intervals (e.g. 0.09 SH/dimer), these amounts proved to be useful for further analysis as discussed below. It is well known that tubulin decays at 37 °C (26Wilson L. Biochemistry. 1970; 9: 4999-5007Crossref PubMed Scopus (336) Google Scholar, 27Prakash V. Timasheff S.N. Arch. Biochem. Biophys. 1992; 295: 137-154Crossref PubMed Scopus (12) Google Scholar). Therefore, to minimize any contribution from denaturation to the accessibility or reactivity of tubulin cysteines, experiments were carried out at 4 °C with 30 μm tubulin at mole ratios of reagent/tubulin of 1:5 to 10:1 at pH 6.9 for prolonged periods. As shown in Fig. 1 C, the 8-h incorporation rose gradually as a function of the mole ratio for both iodoacetamide and IAEDANS but never exceeded 4 SH/dimer. These samples also served as samples for tryptic peptide analysis. About twice as much substitution occurred with NEM. In preliminary experiments to compare the distribution of label between the two tubulin monomers, we used 0.06–0.6 mol of [14C]NEM/dimer, 0.05–0.3 mol of [14C]iodoacetamide/dimer, or 0.5–4.0 mol of IAEDANS/dimer. 10% sodium dodecyl sulfate polyacrylamide gels were used to separate α- and β-tubulin followed by transfer to polyvinylidene difluoride membranes, phosphorimaging (Figs.2, A and B), or fluorescence analysis (Fig. 2 C). Under various incubation conditions using different temperatures, times, or mole ratios, no conditions could be found that led to unique labeling of only one monomer at the expense of the other. Even with mole ratios as low as 0.06 (Fig. 2 A) of label/dimer both monomers were labeled. Identical results were obtained with [14C]NEM (Fig.2 B) or with IAEDANS (Fig. 2 C). Although the literature deals almost exclusively with modification of SH groups on β-tubulin, the α-tubulin SH groups appear to react at least as vigorously, and in the case of IAEDANS, α-tubulin labeling exceeds that of β-tubulin. This suggests possibly fast, but partial, reactions on several cysteine residues whose reactivity cannot be distinguished by these reagents; moreover, it is clear that incorporation of 1 eq of any of these three sulfhydryl reagents cannot be unambiguously interpreted in terms of a single cysteine residue. The modified tubulin, digested with trypsin-TPCK for 24 h at 37 °C, was separated on a 16% Tris-Tricine gel. 12 Cysteines of 20 are present in larger (≥2.4 kDa) tryptic peptides that could be identified in 16% Tris-Tricine gels. The following 4 α-tubulin tryptic peptides contain 7 cysteines: 1) residues 3–40 (3.8 kDa), including Cys-4, Cys-20, and Cys-25; 2) residues 125–156 (3.2 kDa), including Cys-129; 3) residues 167–214 (4.8 kDa), including Cys-200 and Cys-213; 4) residues 281–304 (2.4 kDa), including Cys-295. The following three β-tubulin tryptic peptides contain 5 cysteines: 1) residues 123–154 (3.2 kDa), including Cys-129 and Cys-131; 2) residues 175–213 (3.9 kDa), including Cys-201 and Cys-211; 3) residues 217–241 (2.5 kDa), including Cys-241. Most of the bound radioactivity (75 to 80%) was lost during the electrophoresis. This clearly indicates that the smaller tryptic peptides contained the most reactive cysteines. Therefore, the following 11 tubulin cysteines could be eliminated safely as the reactive cysteines: Cys-4α, Cys-20α, Cys-25α, Cys-129α, Cys-200α, Cys-213α, Cys-295α, Cys-129β, Cys-131β, Cys-203β, and Cys-213β. Nine cysteines, Cys-305α, Cys-315α, Cys-316α, Cys-347α, Cys-376α, Cys-12β, Cys-305β, and Cys-356β plus Cys-241β, should contain the fast reacting cysteines of tubulin. Tryptic peptides from tubulin, labeled at low mole ratios, were analyzed by HPLC, mass spectroscopy, and N-terminal sequencing. To identify the most reactive tubulin cysteines with iodoacetamide, the following two samples were used. Tubulin (30 μm) was incubated 1) with [14C]iodoacetamide (15 μm, 56 dpm/pmol) and 2) with [14C]iodoacetamide (1.5 mm, 2.44 dpm/pmol) for 60 min at 37 °C. The samples were processed according to the procedure described under “Experimental Procedures.” The iodoacetamide to tubulin ratio was 1:2 in the former case and 50:1 in the latter, and the moles of 14C incorporated were 0.16 and 5.6, respectively. To obtain a higher yield of labeled peptides, 1) we digested the whole tubulin rather than separating and purifying α- and β-tubulins and 2) used a preparative (250 mm × 10 mm) C18 column. The formation of soluble peptide aggregates impeded the progress of the experiment, so a high concentration of about 4–6 m guanidine-HCl was used for sample preparations. The common acetonitrile gradient did not give reproducible results with good recovery of radioactivity. We used methanol instead of acetonitrile for the fractionation of labeled tubulin digest. This gradient yielded good reproducibility and recovery of radioactivity (>80%). Fig. 3 A shows the HPLC chromatogram of a digest of tubulin modified with [14C]iodoacetamide to a mole ratio of 0.16/dimer. We identified five peaks, labeled as 1*, 2*, 3*, 4*, and 5*, with significant radioactivity (Fig. 3 A). The inset in Fig. 3 A shows the radioactivity of the peaks. Even though the mole ratio of 14C bound per tubulin dimer was 0.16, we observed 5 radioactive peaks, later identified as 3 coming from α-tubulin and 2 from β-tubulin (see below). Fig.3 B shows the HPLC chromatogram of [14C]iodoacetamide-reacted tubulin with a mole ratio of 5.6 per dimer. Again the radioactivity was localized on the same five peaks as at low mole ratios. Thus, there are at least five fast-reacting cysteines present in tubulin, and all of them react with iodoacetamide even at substoichiometric labeling. Similar results were obtained when tubulin was labeled with [14C]NEM. Fig.4 A shows chromatograms of tubulin modified at a mole ratio of 14C to dimer of 0.45. Under these conditions seven labeled peaks could be identified (*) despite a total labeling stoichiometry of ≪1 NEM/dimer. This greater number of modified cysteines is expected because of the greater reactivity of NEM. When a much higher mole ratio of 5.1 NEM/dimer was used, only 1 additional labeled peptide was obtained as shown in Fig.4 B. In addition to the peaks labeled with iodoacetamide above, several of these peaks could not be identified on the basis of their masses, and these were not pursued further. Our failure to identify a single most reactive cysteine using either iodoacetamide or N-ethylmaleimide suggested the use of a less potent reagent such as chloroacetamide. When [14C]chloroacetamide (1.5 mm with 30 μm tubulin for 3 h at 37 °C) was used for kinetic analysis, the moles of 14C incorporated per dimer were only 0.2 as compared with 8 when iodoacetamide was used under the same conditions. When 1/10 as much chloroacetamide was used (Fig. 5), 0.02 mol of 14C were incorporated per dimer. Analysis of the tryptic peptides revealed a single radioactive peak corresponding to Cys-347α (that was also labeled by the other reagents as shown above) (Fig. 5, inset). It is clear that by this approach the one most reactive cysteine could be selected from the other fast-reacting cysteine residues. The tryptic peptides obtained from IAEDANS-, iodoacetamide-, and NEM-modified tubulin after HPLC separation (TableI, actual mass) are compared with their calculated masses (Table I, calculated mass). These values are in good agreement. Subsequent N-terminal sequencing revealed that each peptide had one or two unidentified residues in the cysteine position of the primary sequence. This accounted for the expected mass of the particular modification of the peptide. Table IA lists the four IAEDANS-modified peptides containing five cysteines, Cys-305α, Cys-315α-Cys-316α, Cys-347α and Cys-356β. For reasons we don't understand at present, iodoacetamide modified five peptides (see also Fig. 3, A and B), but one (peak 1) could not be identified. The modified cysteines are Cys-305α (peak 2), Cys-347α (peak 4), Cys-241β (peak 5), and Cys-356β (peak 3)." @default.
• W1997219785 created "2016-06-24" @default.
• W1997219785 creator A5018229511 @default.
• W1997219785 creator A5031362875 @default.
• W1997219785 creator A5061355891 @default.
• W1997219785 date "2002-08-01" @default.
• W1997219785 modified "2023-09-26" @default.
• W1997219785 title "The Local Electrostatic Environment Determines Cysteine Reactivity of Tubulin" @default.
• W1997219785 cites W105839754 @default.
• W1997219785 cites W1514869822 @default.
• W1997219785 cites W1570892680 @default.
• W1997219785 cites W1887614213 @default.
• W1997219785 cites W1971913380 @default.
• W1997219785 cites W1986318090 @default.
• W1997219785 cites W1987137042 @default.
• W1997219785 cites W2005038450 @default.
• W1997219785 cites W2005089124 @default.
• W1997219785 cites W2010870281 @default.
• W1997219785 cites W2021057279 @default.
• W1997219785 cites W2026258231 @default.
• W1997219785 cites W2026987596 @default.
• W1997219785 cites W2042806231 @default.
• W1997219785 cites W2043885312 @default.
• W1997219785 cites W2046096630 @default.
• W1997219785 cites W2047068038 @default.
• W1997219785 cites W2051210276 @default.
• W1997219785 cites W2058997498 @default.
• W1997219785 cites W2059001690 @default.
• W1997219785 cites W2064096847 @default.
• W1997219785 cites W2064424443 @default.
• W1997219785 cites W2066913136 @default.
• W1997219785 cites W2068702966 @default.
• W1997219785 cites W2071147906 @default.
• W1997219785 cites W2072034233 @default.
• W1997219785 cites W2073285365 @default.
• W1997219785 cites W2080873944 @default.
• W1997219785 cites W2083730820 @default.
• W1997219785 cites W2084546943 @default.
• W1997219785 cites W2086016933 @default.
• W1997219785 cites W2086526874 @default.
• W1997219785 cites W2090781231 @default.
• W1997219785 cites W2091097441 @default.
• W1997219785 cites W2099057076 @default.
• W1997219785 cites W2101600612 @default.
• W1997219785 cites W2120293965 @default.
• W1997219785 cites W2136429273 @default.
• W1997219785 cites W2172052207 @default.
• W1997219785 cites W2314179356 @default.
• W1997219785 cites W3004743877 @default.
• W1997219785 cites W4232534952 @default.
• W1997219785 doi "https://doi.org/10.1074/jbc.m204263200" @default.
• W1997219785 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12023292" @default.
• W1997219785 hasPublicationYear "2002" @default.
• W1997219785 type Work @default.
• W1997219785 sameAs 1997219785 @default.
• W1997219785 citedByCount "99" @default.
• W1997219785 countsByYear W19972197852012 @default.
• W1997219785 countsByYear W19972197852013 @default.
• W1997219785 countsByYear W19972197852014 @default.
• W1997219785 countsByYear W19972197852015 @default.
• W1997219785 countsByYear W19972197852016 @default.
• W1997219785 countsByYear W19972197852017 @default.
• W1997219785 countsByYear W19972197852018 @default.
• W1997219785 countsByYear W19972197852019 @default.
• W1997219785 countsByYear W19972197852020 @default.
• W1997219785 countsByYear W19972197852021 @default.
• W1997219785 countsByYear W19972197852023 @default.
• W1997219785 crossrefType "journal-article" @default.
• W1997219785 hasAuthorship W1997219785A5018229511 @default.
• W1997219785 hasAuthorship W1997219785A5031362875 @default.
• W1997219785 hasAuthorship W1997219785A5061355891 @default.
• W1997219785 hasBestOaLocation W19972197851 @default.
• W1997219785 hasConcept C121332964 @default.
• W1997219785 hasConcept C12554922 @default.
• W1997219785 hasConcept C142724271 @default.
• W1997219785 hasConcept C181199279 @default.
• W1997219785 hasConcept C185592680 @default.
• W1997219785 hasConcept C20418707 @default.
• W1997219785 hasConcept C204787440 @default.
• W1997219785 hasConcept C2776910235 @default.
• W1997219785 hasConcept C2779201268 @default.
• W1997219785 hasConcept C55493867 @default.
• W1997219785 hasConcept C62520636 @default.
• W1997219785 hasConcept C71924100 @default.
• W1997219785 hasConcept C84425145 @default.
• W1997219785 hasConcept C86803240 @default.
• W1997219785 hasConcept C87716614 @default.
• W1997219785 hasConcept C95444343 @default.
• W1997219785 hasConceptScore W1997219785C121332964 @default.
• W1997219785 hasConceptScore W1997219785C12554922 @default.
• W1997219785 hasConceptScore W1997219785C142724271 @default.
• W1997219785 hasConceptScore W1997219785C181199279 @default.
• W1997219785 hasConceptScore W1997219785C185592680 @default.
• W1997219785 hasConceptScore W1997219785C20418707 @default.
• W1997219785 hasConceptScore W1997219785C204787440 @default.
• W1997219785 hasConceptScore W1997219785C2776910235 @default.
• W1997219785 hasConceptScore W1997219785C2779201268 @default.
• W1997219785 hasConceptScore W1997219785C55493867 @default.

• next