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1.
The effects of colchicine analogues on the reaction of tubulin with iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide) 总被引:1,自引:0,他引:1
We have previously found (Ludue?a, R. F., and Roach, M. C. (1981b) Biochemistry 20, 4444-4450) that colchicine and podophyllotoxin inhibit the alkylation of tubulin by iodo[14C]acetamide and the formation of an intrachain cross-link in the beta-tubulin subunit by N,N'-ethylenebis(iodoacetamide) (EBI). It was not clear whether these effects were due to conformational changes in tubulin induced by drugs or to direct steric blockage of the sulfhydryl groups involved. In an effort to characterize further these phenomena, we have examined the effects of single-ring and bicyclic analogues of colchicine on the reaction of tubulin with iodo[14C]acetamide and EBI. We have found that neither the A-ring analogues, 3,4,5-trimethoxybenzyl alcohol, 3,4,5-trimethoxybenzaldehyde, 2,3,4-trimethoxybenzaldehyde, and benzaldehyde, nor the C-ring analogues, tropolone and tropolone methyl ether, inhibited alkylation. In contrast, colchicine, podophyllotoxin, and nocodazole and the bicyclic analogues, 5-(2',3',4'-trimethoxyphenyl)-2-methoxytropone and combretastatin, inhibited tubulin alkylation. Since the presence of a bond joining the A and C rings seems to be the determining factor in the suppression of alkylation, it is likely that inhibition by colchicine of the reaction with iodo[14C] acetamide is due largely to a conformational change induced by colchicine. A different pattern was obtained when the effects on cross-link formation by EBI were examined. Here, all the A-ring analogues, the bicyclic analogues, and colchicine, podophyllotoxin, and nocodazole all inhibited formation of the cross-link, whereas the C-ring analogue tropolone methyl ether did not inhibit cross-link formation. Since compounds whose effect on alkylation is markedly different have the same effect on cross-link formation, it is possible that this effect is a steric one and that perhaps the A-ring of colchicine binds to tubulin very close to one of the sulfhydryls involved in the intrachain cross-link formed by EBI in beta-tubulin. 相似文献
2.
Different reactivities of brain and erythrocyte tubulins toward a sulfhydryl group-directed reagent that inhibits microtubule assembly 总被引:2,自引:0,他引:2
R F Ludue?a M C Roach M A Jordan D B Murphy 《The Journal of biological chemistry》1985,260(2):1257-1264
There is considerable evidence that tubulin exists in multiple isotypes, differing in amino acid sequence and tissue distribution. Little is known, however, about the functional significance of these isotypes. Chicken erythrocyte beta-tubulin has been shown by peptide mapping to differ significantly from chicken brain beta-tubulin (Murphy, D. B., and Wallis, K. T. (1983) J. Biol. Chem. 258, 7870-7875). We now find that when the two tubulins, in their native states, are incubated with N,N'-ethylenebis(iodoacetamide) (EBI), a bifunctional sulfhydryl-directed reagent, microtubule assembly by brain tubulin is much more sensitive to inhibition by EBI than is erythrocyte tubulin assembly. The resistance of erythrocyte microtubule assembly to inhibition by EBI is correlated with a low reactivity of erythrocyte tubulin with [14C]EBI. This difference is most marked in the beta subunit which reacts 15 and 17% as well, respectively, with [14C]EBI as do the beta 1 and beta 2 subunits of brain tubulin. Also, erythrocyte beta reacts about 33% as well as does brain beta with iodo[14C]acetamide. These results suggest that a reactive sulfhydryl group, whose oxidation prevents microtubule assembly, is present in brain tubulin but absent or inaccessible in erythrocyte tubulin. Since purified erythrocyte tubulin self-aggregates much more readily than does brain tubulin, it is conceivable that erythrocyte and brain tubulin may differ in that the latter may have its assembly subject to a complex regulation, while erythrocyte tubulin assembly may be regulated by a simpler mechanism. 相似文献
3.
The synthetic anti-tumor drug 3-(1-anilinoethylidene)-5-benzylpyrrolidine-2,4-dione (TN-16) is known to block microtubule assembly and colchicine binding to tubulin, although its structure does not resemble those of either colchicine, podophyllotoxin, or nocodazole (Arai, FEBS Lett. 155:273-276 (1983]. We have found that TN-16 affects the intra-chain cross-linking of beta-tubulin by N,N'-ethylene-bis(iodoacetamide) in a manner identical to that of colchicine, podophyllotoxin, and nocodazole, but different from that of vinblastine or maytansine. TN-16 also inhibits alkylation of tubulin by iodo[14C]acetamide, as do colchicine and its congeners. TN-16 appears to bind to tubulin at the colchicine binding site and one of its phenyl groups is likely to bind at the site on tubulin where colchicine's A ring binds. 相似文献
4.
P Verdier-Pinard N Sitachitta J V Rossi D L Sackett W H Gerwick E Hamel 《Archives of biochemistry and biophysics》1999,370(1):51-58
Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [(14)C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0 degrees C, with a minimum k(f) of 4.4 x 10(3) M(-1) s(-1). We were unable to demonstrate any dissociation of the [(14)C]curacin A from tubulin. Consistent with these observations, the K(a) value was so high that an accurate determination by Scatchard analysis was not possible. The [(14)C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [(14)C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a K(a) of 3.2 x 10(-5) M(-1). 相似文献
5.
Puried complex III ) ubiquinol-cytochrome c reductase) from beef heart mitochondria was alkylated with iodol [1-14C]acetamide. After 6-8 h of incubation with iodo[1-14C]acetamide, duroquinol and ubiquinol-2-cytochrome c reductase activites were inhibited approximately 50%. During this time 4.5 +/- 1.6 nmol of iodo[1-14C]acetamide reacted per mg of complex III protein. Experiments carried out over 24 h indicated that enzyme activity could be inhibited to 70% and the alkylation of complex III was proportional to inhibition. The rates of cytochrome b and c1 reduction by duroquinol are also decreased upon treatment of complex III with iodoacetamide. Separation of the peptides of complex III by electrophoresis in sodium dodecylsulfate shows that all of the radioactivity is located in a single peptide of 50 000 molecular weight, which has been identified as one of the two core proteins. The possible functions of core protein are discussed. 相似文献
6.
Cysteine residues in tubulin are actively involved in regulating ligand interactions and microtubule formation both in vivo and in vitro. These cysteine residues are sensitive reporters in determining the conformation of tubulin. Although some of the cysteines are critical in modulating drug binding and microtubule assembly, it is not clear how many of these normally exist as disulfides. The controversy regarding the disulfide bonds led us to develop a disulfide detection assay to reexamine the presence of the disulfide linkages in purified alphabeta tubulin and explore their possible biological functions in vitro. The accessible cysteine residues in alphabeta tubulin were alkylated with an excess of iodoacetamide to prevent artifactual generation of disulfide linkages in tubulin. After removal of excess iodoacetamide, tubulin was unfolded in 8 M urea. Half of the unfolded tubulin was treated with dithiothreitol to reduce any disulfide bonds present. The aliquots were then treated with iodo[(14)C]acetamide and the incorporation of radioactivity was measured. We also used the same approach to detect the disulfide linkages in the tubulin in a whole-cell extract. We found in both cases that the samples which were not treated with dithiothreitol had little or no incorporation of iodo[(14)C]acetamide, while the others that were treated with dithiothreitol had significant amounts of (14)C incorporation into tubulin. Moreover, the reduction of the disulfide linkages in tubulin resulted in inhibition of microtubule assembly (29-54%) and markedly affected refolding of the tubulin from both an intermediate and a completely unfolded state. All these data therefore suggest that tubulin has intrachain disulfide bonds in the alpha- and beta-subunits and that these disulfides assist in correct refolding of tubulin from the intermediate unfolded state or help to recover the hydrophobic domains from the completely unfolded state. These disulfides also regulate microtubule assembly and the stability of tubulin in vitro. Our results suggest that tubulin disulfides may play a role in tubulin folding and that thiol-disulfide exchange in tubulin could be a key regulator in microtubule assembly and dynamics of tubulin in vivo. 相似文献
7.
NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5''-deiodinase. Demonstration by alkylation with iodoacetamide. 总被引:1,自引:1,他引:0
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We have assessed a previously proposed mechanism mediating 5'-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5'-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5'-deiodinase correlated with the amount of available (unalkylated) Fraction B. 相似文献
8.
C3-translational product, which was synthesized with rabbit liver mRNA in a reticulocyte lysate protein-synthesizing system, did not react with [14C]methylamine, indicating the lack of an internal thioester bond. Instead, the C3-translational product reacted with iodo[1-14C]acetamide, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate after immunoprecipitation of the product, indicating the presence of a reactive thiol group. When the C3-translational product was treated with rabbit liver homogenate, the product acquired reactivity with [14C]methylamine and lost the reactivity with iodo[1-14C]acetamide. Thus, the liver homogenate seemed to contain a factor (or factors) required for the formation of an internal thioester bond. The factor was partially purified from the liver homogenate by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose. 相似文献
9.
The bullfrog (Rana catesbeiana) major hemoglobin dissociates into its constituent globin chains (alpha and beta) which are separated by Sulfopropyl-Sephadex C-25 column chromatography after alkylation with iodo[14C]acetamide. Each globin chain has two cysteine residues and those of the beta-globin chain in the tetramer are preferentially alkylated with iodoacetamide. 相似文献
10.
Rapid and Reversible High-Affinity Binding of the Dinitroaniline Herbicide Oryzalin to Tubulin from Zea mays L 总被引:7,自引:1,他引:6
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Oryzalin, a dinitroaniline herbicide, was previously reported to bind to plant tubulin with a moderate strengthe interaction (dissociation constant [Kd] = 8.4 [mu]M) that appeared inconsistent with the nanomolar concentrations of drug that cause the loss of microtubules, inhibit mitosis, and produce herbicidal effects in plants (L.C. Morejohn, T.E. Bureau, J. Mole-Bajer, A.S. Bajer, D.E. Fosket [1987] Planta 172: 252-264). To characterize further the mechanism of action of oryzalin, both kinetic and quasi-equilibrium ligand-binding methods were used to examine the interaction of [14C]-oryzalin with tubulin from cultured cells of maize (Zea mays L. cv Black Mexican Sweet). Oryzalin binds to maize tubulin dimer via a rapid and pH-dependent interaction to form a tubulin-oryzalin complex. Both the tubulin-oryzalin binding strength and stoichiometry are underestimated substantially when measured by kinetic binding methods, because the tubulin-oryzalin complex dissociates rapidly into unliganded tubulin and free oryzalin. Also, an uncharacterized factor(s) that is co-isolated with maize tubulin was found to noncompetitively inhibit oryzalin binding to the dimer. Quasi-equilibrium binding measurements of the tubulin-oryzalin complex using purified maize dimer afforded a Kd of 95 nM (pH 6.9; 23[deg]C) and an estimated maximum molar binding stoichiometry of 0.5. No binding of oryzalin to pure bovine brain tubulin was detected by equilibrium dialysis, and oryzalin has no discernible effect on microtubules in mouse 3T3 fibroblasts, indicating an absence of the oryzalin-binding site on mammalian tubulin. Oryzalin binds to pure taxol-stabilized maize microtubules in a polymer mass- and number-dependent manner, although polymerized tubulin has a much lower oryzalin-binding capacity than unpolymerized tubulin. Much more oryzalin is incorporated into polyment during taxol-induced assembly of pure maize tubulin, and half-maximal inhibition of the rapid phase of taxol-induced polymerization of 5 [mu]M tubulin is obtained with 700 [mu]M oryzalin. The data are consistent with a molecular mechanism whereby oryzalin binds rapidly, reversibly, and with high affinity to the plant tubulin dimer to form a tubulin-oryzalin complex that, at concentrations substoichiometric to tubulin, copolymerizes with unliganded tubulin and slows further assembly. Because half-maximal inhibition of maize callus growth is produced by 37 nM oryzalin, the herbicidal effects of oryzalin appear to result from a substoichiometric poisoning of microtubules. 相似文献
11.
Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl 总被引:5,自引:1,他引:4
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Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a substoichiometric endwise mechanism. 相似文献
12.
J F Biellmann G Branlant J C Nicolas M Pons B Descomps A Crastes de Paulet 《European journal of biochemistry》1976,63(2):477-481
3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label. 相似文献
13.
The mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate (DCBT) has been examined in detail. Shown in previous studies to inhibit tubulin polymerization [Abraham, I., Dion, R. L., Duanmu, C., Gottesman, M. M., & Hamel, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6839-6843] and to form a covalent bond preferentially with beta-tubulin [Bai, R., Duanmu, C., & Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20], DCBT has now been documented to interact at low concentrations with a high degree of specificity at cysteine residue 239 of beta-tubulin. These low DCBT concentrations also result in the partial inhibition of tubulin polymerization. Such findings strongly indicate that cysteine-239 of beta-tubulin is essential for microtubule assembly. Although alpha-tubulin is alkylated almost as well as beta-tubulin when the drug:tubulin ratio = 5:1 (Bai et al., 1989), beta-tubulin is alkylated about 25 times as extensively as alpha-tubulin, almost exclusively at Cys-239, when the drug:tubulin ratio = 1:5. In addition, we find that low concentrations of DCBT do not affect the binding of colchicine to tubulin but that colchicine and related compounds do reduce the alkylation of tubulin by DCBT. This suggests that Cys-239 of beta-tubulin is not involved in the binding of colchicine to tubulin but that this amino acid residue is at least partially masked by the drug when it is bound to the protein. We also describe a column chromatography procedure (hydrophobic chromatography on decylagarose) useful for the preparative resolution of unalkylated, although denatured, alpha- and beta-tubulin. 相似文献
14.
The product of the incorporation of [14C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [3H]colchicine-binding method (10.5% of the total protein). The incorporated [14C]-tyrosine was found in the alpha-subunit of tubulin. Protein labelled with [3H]colchicine and [14C]tyrosine was precipatated with vinblastine sulphate and the radioactivity of 3H and that of 14C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the 14C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Bineing of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit. 相似文献
15.
An examination of the inhibitory effects of N-iodoacetylglucosamine on Escherichia coli and isolation of resistant mutants 总被引:2,自引:2,他引:0
1. After incubation of Escherichia coli with N-iodo[1,2-(14)C(2)]acetylglucosamine, 95-99% of the (14)C taken up by whole cells is located in a cold-trichloroacetic acid-soluble fraction. Two major components of this fraction are S-carboxymethylcysteine and S-carboxymethylglutathione. The same compounds accumulate during incubation with iodo[(14)C]acetate but not with iodo[(14)C]acetamide. The amount of (14)C associated with a cold-trichloroacetic acid-insoluble fraction are comparable for all three alkylating agents. After incubation with iodo[(14)C]acetamide, 50% of the label bound to whole cells is recoverable in a cold-trichloroacetic acid-insoluble fraction. 2. Uptake and incorporation of (14)C from [U-(14)C]glycerol is blocked at an early stage by N-iodoacetylglucosamine. No specific inhibition of macromolecular synthesis could be demonstrated. 3. Mutants selected for resistance to iodoacetate are partially resistant to iodoacetate and N-iodoacetylglucosamine, but show no resistance to iodoacetamide. 4. Mutants selected for resistance to N-iodoacetylglucosamine are not resistant to iodoacetate or iodoacetamide, and are defective in their ability to grow on N-acetylglucosamine. Resistance to N-iodoacetylglucosamine is not absolute, and depends on the presence of glucose or certain other sugars; there is no resistance during growth on maltose, glycerol or succinate. 5. Absolute resistance can be achieved by selecting for a second mutation conferring resistance during growth on maltose; double mutants isolated by this procedure are unable to grow on N-acetylglucosamine and grow poorly on glucosamine. Resistant single mutants have a slightly diminished uptake of N-acetyl[1-(14)C]glucosamine, but in resistant double mutants the uptake of both [1-(14)C]glucosamine and N-acetyl[1-(14)C]glucosamine is severely diminished. 6. These observations are consistent with the presence of two permeases for N-acetylglucosamine, one that also permits uptake of glucosamine and another that allows entry of methyl 2-acetamido-2-deoxy-alpha-d-glucoside. N-Iodoacetylglucosamine can gain entry to the cell by both permeases. 相似文献
16.
A sex difference in hepatic glutathione S-transferase B and the effect of hypophysectomy. 总被引:4,自引:4,他引:0
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[2-14C]Methyl cyanide (acetonitrile) is metabolized to citrate, succinate, fumarate, malate, glutamate, pyrrolidonecarboxylic acid and aspartate. Non-radioactive acetamide and acetate compete with 14C from methyl cyanide, and [2-14C]acetate and [2-14C]methyl cyanide are metabolized at similar rates, giving identical products. This evidence, combined with the inhibitory effect of fluoroacetate and arsenite on methyl cyanide metabolism, indicates that the pathway is: methyl cyanide leads to acetamide leads to acetate leads to tricarboxylic acid-cycle intermediates. The pathway was investigated in a species of Pseudomonas (group III; N.C.I.B. 10477), but comparison of labelling patterns suggests that it also exists in several higher plants. 相似文献
17.
S G Cohen E Salih M Solomon S Howard S B Chishti J B Cohen 《Biochimica et biophysica acta》1989,997(3):167-175
1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl. 相似文献
18.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1995,1254(2):180-186
Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-14C]oleate resulting in esterification of [1-14C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-14C]oleate and esterification of [1-14C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 μM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 μM 2′,5′ dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM chloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal. 相似文献
19.
Release of C-terminal tyrosine from tubulin and microtubules at steady state. 总被引:3,自引:0,他引:3
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Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase. 相似文献
20.
Fast non-covalent interactions of 16S rRNA Escherichia coli with 14C labeled 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivatives of deoxyribooligonucleotides d(pACCTTGTT)rA, d[pTTACGATC)rU, d(pTTTGCTCCCC)rA (less than[14C]CHRCl-reagents) observed at 0 degrees C were investigated. It was shown, that 16S rRNA and [14C]CHRCl-reagents at 0 degrees C form stable complexes which can not be disrupted under mild acidic conditions (pH 4, 40 degrees C) and under denaturing conditions (7 M urea, 50 degrees C), but are completely disrupted in the course of centrifugation in sucrose density gradient in the presence of SDS. Formation of such complexes of 16S rRNA with greater than[14C]CHRCl-reagents at 0 degrees C was observed due to the presence in the reagent preparation of a number of unidentified products, formed in the course of the synthesis of benzylidene derivatives, and having a hydrophobicity larger, than those for greater than CHRCl-derivatives of deoxyribooligonucleotide. Preparation of [14C]CHRCl-reagents, subjected for purification by reverse-phase chromatography, were unable to form such a complex with 16S rRNA at 0 degrees C. Studies on the complementary addressed modification at 0 degrees C (or incubation at 0 degrees C) with the use of the oligonucleotide benzylidene derivatives not purified from hydrophobic contaminations may lead to alkylation within these complexes during subsequent treatments and in such a way give incorrect information about the level of alkylation within the complex under investigation. 相似文献