A sensitive method for determination of 5-fluorouracil and 5-fluoro-2'-deoxyuridine in human plasma by high-pressure liquid chromatography

Polymerization-competent extracts of suspension-cultured HeLa cells and porcine brain tissue were assayed for tubulin content. Five different methods were used to assay identically prepared extracts: two types of sodium dodecyl sulfate-containing acrylamide gels, a DEAE retention assay, a colchicine-binding assay, and a radioimmunoassay. The colchicine-binding and radioimmunoassay results were in close agreement and are therefore considered reliable assays for tubulin content in cell extracts. The DEAE retention assay gave slight overestimates, but the gel methods seriously overestimated tubulin content. Based on data from colchicine binding and radioimmunoassay, the proportion of soluble cell protein which is tubulin is 4.3% for HeLa cell extracts and 12.1% for brain tissue extracts.     

Membrane-bound tubulin in brain and thyroid tissue.

Brain and thyroid tissue contain membrane-bound colchicine-binding activity that is not due to contamination by loosely bound cytoplasmic tubulin. This activity can be solubilized to the extent of 80 to 90% by treatment with 0.2% Nonidet P-40 with retention of colchicine binding. Extracts so obtained contain a prominent protein band in disc gel electrophoresis that co-migrates with tubulin. Membranes, and the solubilized protein therefrom, exhibit ligand binding properties like tubulin; for colchicine the KA is approximately 1 X 10(6) M-1 in brain and approximately 0.6 X 10(6) M-1 in thyroid; for vinblastine the KA is approximately 8 X 10(6) M-1 for both tissues; and for podophyllotoxin the Ki is approximately 2 X 10(-6) M for both tissues. Displacement by analogues of colchicine is of the same order as for soluble tubulin. Although membrane-bound colchicine-binding activity shows greater thermal stability and a higher optimum binding temperature (54 degrees versus 37 degrees) than soluble tubulin, this appears to be the result of the membrane environment since the solubilized binding activity behaves like the soluble tubulin. Antibody against soluble brain tubulin reacts with membranes and solubulized colchicine-binding activity from both brain and thyroid gland. We conclude that brain and thyroid membrane preparations contain firmly bound tubulin or a very similar protein.     

Tyrosination state of free tubulin subunits and tubulin disassembled from microtubules of rat brain tissue.

The content of endogenous COOH-terminal tyrosine in unpolymerized tubulin subunits from the brain extracts of new-born rats was twice as high as that of tubulin isolated from disassembled native microtubules. It was also observed that the proportion of free sites in tubulin from $\text{in}\text{vitro}$ incorporation of tyrosine at the COOH-terminus in unpolymerized protein was about one-half of that obtained for the protein from disassembled microtubules. In the extracts of young adult rats the value for the endogenous COOH-terminal tyrosine content in unpolymerized subunits decreased and almost equaled the value for the polymerized tubulin which remained virtually constant.     

Tubulin and microtubules from bovine kidney: purification, properties, and characterization of ligand binding.

Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or τ-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 m glycerol are similar morphologically as detected by electron microscopy. Molecular weights of α- and β-tubulin from bovine renal medulla are 54,000 ± 700 and 52,000 ± 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 μm vinblastine sulfate increases the half-time of decay to 10.9 h at 37 °C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5-fold both in the absence and presence of vinblastine sulfate at 37 °C. Values of the apparent colchicine-binding constant, K_A, of bovine renal medullary tubulin are 5.9 × 10⁶ and 7.8 × 10⁶m^?1 at 37 °C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent K_i for podophyllotoxin is 4.0 × 10^?7m at 37 °C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are required for the function of vasopressin in mammalian kidneys.     

A modified colchicine-binding assay for the measurement of total and microtubule-derived tubulin in rat liver

The usual measurement of liver tubulin by the colchicine-binding assay does not take into account the accelerated decay of the colchicine-binding capacity of tubulin when liver supernatants, especially those containing microtubule-derived tubulin, are incubated at 37°C. This results in marked underestimations. Our findings indicate that this alteration is due to an inhibitor of colchicine-tubulin binding in liver supernatants that is probably extracted from particulate fractions. The inhibitory activity is decreased by dilution of the supernatants and by increasing the concentration of colchicine. However, the former modification decreases the sensitivity of the assay and the latter increases the nonspecific binding of colchicine to liver proteins other than tubulin. Assessment of the decay and correction for it by calculating the initial binding capacity results in complete recovery of brain tubulin from liver supernatants and values for microtubule-derived tubulin that closely correspond to those expected from simultaneous morphometric assessment of liver microtubules by electron microscopy. The modified method also indicates that the fraction of liver tubulin assembled in microtubules is greater than previously reported.     

Stabilization of hepatic colchicine-binding activity by organic acids

Previous work has shown that the total hepatic tubulin pool and the hepatic microtubule-derived tubulin pool do not have identical [3H]colchicine binding properties. Rapid loss of colchicine-binding activity was noted in the microtubule-derived fractions of liver tubulin. Furthermore, quantitative determination of the total and polymerized tubulin in the liver by the [3H]colchicine-binding assay was hampered by rapid and unequal loss of binding sites under assay conditions. The organic acids, glutamate and glucose 1-phosphate, have been shown to stabilize calf brain tubulin against loss of colchicine-binding sites. Therefore, these compounds were tested as possible protecting agents against loss of colchicine binding activity of liver tubulin. It was found that these agents stabilized liver tubulin under [3H]colchicine-binding conditions. Additional experiments showed that these agents also prevented the rapid loss of colchicine-binding activity that occurred when purified brain tubulin was exposed to liver supernates. These results suggest that the inclusion of the organic acids, glutamate and glucose 1-phosphate, may modify the time decay properties of liver tubulin in solution. Further, these data suggest that these protecting agents may be of analytical value in [3H]colchicine-binding assay systems for liver tubulin.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

A sensitive method for measuring polymerized and depolymerized forms of tubulin in tissues

A rapid method for measuring polymerized and depolymerized forms of tubulin in tissues has been developed. The procedure consists of homogenization and centrifugation of the tissue in a microtubule- stabilizing solution and depolymerization of the precipitated microtubules; polymerized and depolymerized forms of tubulin are quantitated by a colchicine-binding assay. The validity of the technique was assessed by electron microscopy and recovery studies with labeled and unlabeled preparations of polymerized and depolymerized forms of rat brain tubulin. The sensitivity of this technique allows quantitation of tubulin in 150 micrograms of tissue, wet weight. The method demonstrated that both the polymerized and depolymerized forms of tubulin were present in rat liver cells, and that in the fed state 31.3 +/-0.7% of the total tubulin pool was in the polymerized form.     

Immunoperoxidase staining of glial fibrillary acidic (GFA) protein polymerized in vitro: An ultramicroscopic study

The unlabeled antibody peroxidase-antiperoxidase (PAP) method of Sternberger et al. has been employed at the ultramicroscopic level to stain filaments polymerized in vitro from aqueous extracts of multiple sclerosis (MS) plaques. The filaments were heavily decorated with antiserum to the glial fibrillary acidic (GFA) protein but not stained with serum absorbed with GFA protein, preimmunization serum, or anti-rat brain tubulin antiserum. Reassembled rat brain tubulin was heavily stained with antiserum to tubulin but was not stained with antiserum to the GFA protein. The present study provides direct morphological evidence that filaments polymerized in vitro from extracts of MS plaques contain the GFA protein.     

The assembly of tubulin into membranes

Tubulin in high-speed supernatants of brain undergoes an alternate form of polymerization into structures that resemble membranes rather than microtubules. The reaction required elevated temperature (37°C) and was prevented by 1mM CaCl₂ or 10^?4 M maytansine. The membraneous material was composed of tubulin (80%) and microtubule-associated proteins (8%) and contained phospholipids. The tubulin was identified on the basis of comigration in two-dimensional gel electrophoresis and colchicine-binding activity.     

Tubulin constancy during morphological differentiation of mouse neuroblastoma cells

Clonal cell lines N18 and N103 of the mouse neuroblastoma C1300 possess an undifferentiated neuroblast morphology under optimal growth conditions; however, when deprived of serum, N18 can be induced to extend long neurites. Although initial neurite outgrowth is rapid, very long fibers are found only after several days. Both initial outgrowths and established neurites contain microtubules; however, the number and density of these polymerized tubules increase markedly during this time. Optimum conditions have been established for assessing the colchicine-binding activity of neuroblastoma sonicates. A time-decay colchicine-binding assay was used to make a comparative study of the tubulin content of both undifferentiated and differentiated N18 as well as the nondifferentiating N103 and the rat glioma C6. Both morphologies of clone N18 possessed similar concentrations of tubulin (130-140 pmol/10(6) cells). Although cells of clone N103 contain 20% less tubulin than N18 cells, this is considerably more tubulin than is present in the glioma C6 (30 pmol/10(6) cells) which has a similar generation time. Quantitative densitometry of neuroblastoma extracts electrophoresed on SDS-polyacrylamide gels confirmed the constancy of tubulin. Radiolabeled proteins from neuroblastoma cells subjected to both growth conditions show that neurite outgrowth does not create a disproportionate demand for tubulin synthesis. Thus, the morphological differentiation of neuroblastoma cells probably reflects the regulation of tubulin storage and microtubule polymerization.     

Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro

A post-translational modification of tubulin with potential regulatory significance has been revealed by the discovery of an enzyme (tubulin-tyrosine ligase) in brain extracts which can add a tyrosine residue to the alpha chain, apparently through peptide bond linkage to a C-terminal glutamate. We have investigated whether this modification also occurs in vivo, and whether it alters the extent to which tubulin can assemble in vitro. Cytoplasmic tubulin purified from bovine brain by cycles of assembly was shown to be partially tyrosylated. Carboxypeptidase A digestion of isolated alpha chains liberated about 0.3 equivalent of tyrosine. Brief digestion of native tubulin increased the proportion of alpha chains which could be tyrosylated by ligase, from 25 to 45%. The tubulin assembled to the same extent before and after carboxypeptidase treatment. When tubulin was purified after introducing labeled tyrosine with ligase, the labeled species assembled in the same proportion as unlabeled. Thus tubulin can be incorporated into microbubules in vitro with or without C-terminal tyrosine. An apparent resolution of alpha chain into two components by hydroxylapatite chromatography was shown not to be due to the presence or absence of C-terminal tyrosine. Tubulin-tyrosine ligase was found in extracts of every rat tissue examined, but was not detected in sea urchin eggs before or after fertilization, in Tetrahymena cells or cilia, or in yeast. Cultured neuroblastoma cells fixed tyrosine into tubulin alpha chains under conditions where protein synthesis was inhibited; this in vivo fixation appeared to be into an insoluble moiety of tubulin. Incidental to these studies, a new assay utilizing an enamine substrate for carboxypeptidase was investigated.     

Developmental changes in the endogenous Ca2+-stimulated proteolysis of mouse lung cAMP-dependent protein kinases

Regulatory subunits (R subunits) of mouse lung cAMP-dependent protein kinases undergo age-dependent changes in endogenous proteolysis, with the greatest amount of the major M_r = 37,000 proteolytic fragment detectable during fetal and neonatal development. Homogenization of lung in the presence of various protease inhibitors does not affect this age-related difference, suggesting that the observed quantitative change in R subunit proteolysis occurs in vivo. Mechanisms were sought to account for this age-dependent change. The production of a M_r = 37,000 proteolytic fragment can be stimulated in lung extracts by the addition of exogenous calcium and is due to the action of an endogenous Ca²⁺-stimulated protease. Neonatal lung extracts show more Ca²⁺-stimulated proteolysis of R subunits than adult extracts, although only slight agerelated differences in either the Ca²⁺-stimulated protease or its specific endogenous inhibitor were observed. Age-dependent differences in R subunits which may affect sensitivity to proteases were also examined. Analysis of the two-dimensional patterns of adult and neonatal 8-N₃-[³²P]cAMP-labeled R subunits before or after limited proteolysis with trypsin suggests that the R subunits are structurally similar. Differences are found, however, in the relative proportions of adult and neonatal Type I R subunits (R_I) in the holoenzyme or dissociated forms. An increased proportion of neonatal R subunits exist in the dissociated state, whereas adult R subunits exist primarily in the holoenzyme form. Dissociated R subunits from mouse lung are more susceptible than the holoenzyme to limited proteolysis by the partially purified lung Ca²⁺-stimulated protease. Dissociation of the holoenzyme in vivo may be a major factor in the age-dependent proteolytic changes observed in mouse lung protein kinases.     

THE EFFECT OF TRIALKYLTIN COMPOUNDS ON TUBULIN POLYMERISATION

The effects of trialkyltin compounds on the colchicine-binding activity and the in vitro polymerisation of rat brain tubulin have been investigated. Trialkyltins at concentrations between 10 and 100 μM inhibited the colchicine-binding activity of purified tubulin preparations derived from rat brain. Viscometric studies have shown that the same concentrations of trialkyltins also inhibited the in vitro polymerisation of tubulin. Both the effects were found to be concentration dependent. However, trialkyltins did not interfere with the in vitro preassembled microtubules. The reasons which are believed to explain the inhibitory effects of these compounds are discussed.     

In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules.

About 10--20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5 mM MES, 0.5 mM MgSO4, 1.0 mM EGTA, 1.0 mM GTP, and 0 or 50 mM KC1 at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103,000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35degree. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules. Unlike other fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.     

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

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

Increase in polymerized liver tubulin during stimulation of hepatic plasma protein secretion in the rat

Involvement of hepatic microtubules in plasma protein secretion by the liver was investigated by stimulating protein secretion in rat liver and then measuring the different forms of tubulin. Total and free tubulin were estimated in liver supernatants by the [³H] colchicine-binding assay. Polymerized tubulin, assumed to reflect the presence of microtubules, was calculated from the difference between total and free tubulin. To enhance liver plasma protein secretion, an acute inflammatory reaction was induced in one group of rats and a nephrotic syndrome in another. In both cases, total liver tubulin increased significantly compared to normal animals, but free tubulin was unchanged. Accordingly, polymerized tubulin rose by 50% during the inflammatory reaction and by 90% during the nephrotic syndrome. These results support the hypothesis that hepatic microtubules are involved in plasma protein secretion by the liver and also suggest that enhanced secretion requires additional microtubules.     

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

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

The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain.

Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3 +/- 0.11 and 0.8 +/- 0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.     

Comparative analysis of translation accuracy in an Escherichia coli and a mammalian cell-free system

The effect of environmental stress on the accuracy of protein synthesis in an Escherichia coli and a rat brain cell-free system was investigated. Poly-U was translated in a rat brain and an E. coli cell-free extract under identical ionic conditions. The fidelity of translation, both in the E. coli and the rat brain extracts, was commensurate with what is known about the accuracy of translation in vivo. The incorporation of phenylalanine (code: UUU) and leucine (code: CUU, UUG or A) was measured at various Mg²⁺ concentrations (3 to 22 mm), various pH's (6.6 to 8.6), various temperatures (23 to 42 °C), and in the presence or absence of 2.4% (v/v) ethanol. It was observed that (i) the accuracy of translation was generally higher in extracts from E. coli than from rat brain, and (ii) relative to that in E. coli, the translation fidelity in rat brain extracts was about 2 times more sensitive to ethanol, at least 5 times more sensitive to temperature, and at least 50 times more sensitive to pH. It was found that this differential sensitivity was not due to a differential behavior of the bacterial and the mammalian aminoacyl-tRNA synthetases under stress, but rather to the process of chain elongation itself. It is concluded that the accuracy of protein synthesis is more resistant to environmental stress in E. coli extracts than in extracts from at least one mammalian tissue.