CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Miniaturization and validation of a sensitive multiparametric cell-based assay for the concomitant detection of microtubule-destabilizing and microtubule-stabilizing agents

The authors describe a cell-based assay for anti-microtubule compounds suitable for automation. This assay allows the identification, in a single screening campaign, of both microtubule-destabilizing and microtubule-stabilizing agents. Its rationale is based on the substrate properties of the tubulin-modifying enzymes involved in the tubulin tyrosination cycle. This cycle involves the removal of the C-terminal tyrosine of the tubulin alpha-subunit by an ill-defined tubulin carboxypeptidase and its readdition by tubulin tyrosine ligase. Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin, whereas non-dynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can easily be detected with double-immunofluorescence staining using antibodies specific to tyrosinated and detyrosinated tubulin. The authors have scaled this assay to the 96-well plate format and adapted its process for an automated handling, including a readout using a microplate reader. They describe the different steps of this adaptation. This assay was validated using known compounds. This new cell-based assay represents an alternative to both global cytotoxicity assays and in vitro tubulin assembly assays commonly used for the detection of microtubule poisons.     

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.     

A sensitive radiometric assay for cysteic acid decarboxylase activity in crude enzyme preparations of rat liver and brain.

1. A method is described for the synthesis of L-[U-14C]cysteic acid from L-[U-14C] cysteine hydrochloride and for its subsequent utilisation as a substrate for cysteic acid decarboxylase activity in liver and brain. 2. The enzyme determination relies on the entrapment of radio-labelled carbon dioxide in Hyamine hydroxide. 3. The assay is sensitive, reliable and convenient and is particularly suitable for measuring the activity of the decarboxylase in crude enzyme preparations.     

Tyrosination of microtubules and non-assembled tubulin in brain slices

Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of alpha-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of [14C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non-assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubulin:tyrosine ligase during the in vitro isolation of tubulin was discarded. We also found that tubulin:tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule-preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin:tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules. The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin:tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non-assembled tubulin.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Post-translationally modified tubulins in Artemia: prelarval development in the absence of detyrosinated tubulin

The synthesis of post-translationally modified tubulins was examined during Artemia development. Tubulin, either purified to homogeneity or in cell-free extracts, was blotted to nitrocellulose and probed with a panel of antibodies. When purified tubulin was examined, tyrosinated tubulin underwent a large decrease as development progressed and this was accompanied by the appearance of detyrosinated tubulin in samples from organisms developed 24 hr. The inclusion of carboxypeptidase inhibitors had a small effect on the relative amounts of tyrosinated and detyrosinated tubulins in 24-hr preparations. The amount of alpha- and beta-tubulin in cell-free extracts of Artemia either remained relatively constant during development or increased slightly. The same result was obtained for acetylated and tyrosinated tubulin. Detyrosinated tubulin first appeared in 24-hr cell-free extracts and was only post-translationally modified tubulin to increase, relative to the total amount of tubulin, as the brine shrimp developed. As revealed by immunofluorescence staining, detyrosinated tubulin occurred in many cell types of developing nauplii and was prominently displayed in mitotic figures. Artemia, a complex metazoan animal, is thus able to grow for an extended period of time in the absence of detyrosinated tubulin. This isoform is however, synthesized in early larvae and may be required for the development of elongated cells including those which encircle the gut. Detyrosination remains as the only developmentally related change observed for brine shrimp tubulin.     

Incorporation of 3-nitrotyrosine into the C-terminus of alpha-tubulin is reversible and not detrimental to dividing cells.

The C-terminus of the alpha-chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3-nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells. Nitrotyrosine is formed in cells by nitration of tyrosine by nitric oxide-derived species. We studied properties of tubulin modified by in vitro nitrotyrosination at the C-terminus of the alpha-subunit, and the consequences for cell functioning. Nitrotyrosinated tubulin was a good substrate of tubulin carboxypeptidase, and showed a similar capability to assemble into microtubules in vitro to that of tyrosinated tubulin. Tubulin of C6 cells cultured in F12K medium in the presence of 500 micro m nitrotyrosine became fully nitrotyrosinated. This nitrotyrosination was shown to be reversible. No changes in morphology, proliferation, or viability were observed during cycles of nitrotyrosination, denitrotyrosination, and re-nitrotyrosination. Similar results were obtained with CHO, COS-7, HeLa, NIH-3T3, NIH-3T3(TTL-), and A549 cells. C6 and A549 cells were subjected to several passages during 45 days or more in the continuous presence of 500 micro m nitrotyrosine without noticeable alteration of morphology, viability, or proliferation. The microtubular networks visualized by immunofluorescence with antibodies to nitrotyrosinated and total tubulin were identical. Furthermore, nitrotyrosination of tubulin in COS cells did not alter the association of tubulin carboxypeptidase with microtubules. Our results demonstrate that substitution of C-terminal tyrosine by 3-nitrotyrosine has no detrimental effect on dividing cells.     

Release of C-terminal tyrosine from tubulin and microtubules at steady state.

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.     

Serine Transhydroxymethylase: A Simplified Radioactive Assay; Purification and Stabilization of Enzyme Activity Employing Affi-Gel Blue

An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [¹⁴C]HCH0 which is generated when [3-¹⁴C]-serine is employed as the substrate. The new assay eliminates 14 the need for a solvent extraction of a [C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach.

The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4° indefinitely.     

Enzymatic Detyrosination of Tubulin Tyrosinated in Rat Brain Slices and Extracts

Abstract: Tubulin was tyrosinated in slices and in extracts of brain of rats of 3, 25, and 120 days of age by successive incorporation of [¹⁴C]tyrosine and [³H]-tyrosine, respectively. The release of the incorporated amino acid was measured by using tubulinyl-tyrosine carboxypeptidase, carboxypeptidase A, and tubulin-tyrosine ligase. With the carboxypeptidases no differences in either the rates or the extents of the release of tyrosine between these two differently labeled tubulins were found. Differences were found when the detyrosination was catalyzed by the ligase and these were attributed to a higher inactivation of tubulin labeled in slices than of that labeled in extracts.     

A sensitive radiometric assay to measure D-xylulose kinase activity

A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.     

A new assay and solubilization procedure for steroid 17,20-lyase from rat testes

We report an assay for testicular 17, 20-lyase which depends on the use of [21?¹⁴C]progesterone as a substrate. The method is made possible by a simplified procedure for the synthesis of [21?¹⁴C]progesterone. A chromatographic separation of the unreacted substrate and the 2-carbon by-product on mini silica gel colums permitted a quantitative assay of the lyase activity.The lyase complex from rat testes has been solubilized by treatment with Triton CF-54 detergent. The solubilized enzyme complex catalyzes the formation of androstenedione (4-androstene-3,17-dione) from progesterone without equilibrium with added 17-hydroxyprogesterone and the solubilized enzyme complex responds to the presence of cytosol activator. Both of these characteristics are similar to the properties of the intact microsomes. Thus, solubilization with this detergent preserves the special properties of the microsome bound enzyme complex.     

Tyrosination State of Tubulin and the Activity of Tubulin:Tyrosine Ligase and Tubulin Carboxypeptidase in the Developing Retina of the Chick

The tyrosination state of tubulin and the enzymes involved in the tubulin tyrosination/detyrosination cycle--tubulin:tyrosine ligase and tubulin carboxypeptidase--were determined in chick retina during development. The amount of tyrosinable (tyrosinated plus detyrosinated) tubulin increased approximately 110% from embryonic day 7 to 14. Then it decreased, and by day 19 it was similar to the value on day 7. This result did not change after hatching, at least up to day 20. The proportion of tyrosinated and detyrosinated tubulin significantly changed with the development of the animal. At embryonic day 7, these tubulin species were at a proportion of 70 and 30%, respectively, and after hatching, the values inverted, to 30 and 70%, respectively. This change did not correlate with the activity of the ligase relative to that of the carboxypeptidase, as measured in vitro. This observation suggested that a change in the turnover rate of microtubules, in the proportion of assembled and nonassembled tubulin pools, or in both had occurred. Coincident with the last possibility, the proportion of assembled tubulin was found to increase during the development of the animal. This finding suggests that the tyrosination state of tubulin may be determined, at least in part, by the assembly state.     

Coumarin-Ser-Asp-Lys-Pro-OH, a fluorescent substrate for determination of angiotensin-converting enzyme activity via high-performance liquid chromatography

N-Acetyl-Ser-Asp-Lys-Pro-OH (AcSDKP-OH), a negative regulator of hematopoietic stem cell proliferation, is shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), a zinc-dipeptidyl carboxypeptidase, involved in cardiovascular homeostasis. Recently, a study carried out on captopril-treated volunteers revealed that the kinetics of [3H]AcSDKP-OH hydrolysis in vitro in the plasma of donors correlates closely to the plasmatic ratio angiotensin II/angiotensin I, which characterized the conversion activity of ACE. This prompted us to design a fluorescent substrate, 2-[7-(dimethylamino)-2-oxo-2H-chromen-4-yl]acetyl-SDKP-OH, or coumarin-SDKP-OH, which could be an alternative to the radiolabeled analogue used in that study, allowing an easier and more rapid determination of enzyme activity. We report here the synthesis and the determination of the kinetics constants of this fluorescent derivative compared with those of [3H]AcSDKP-OH with human plasma ACE (133 and 125 microM, respectively), which are in the same range as those of the physiological substrate angiotensin I. Furthermore, the hydrolysis of the fluorescent substrate shows the same sensitivity toward chloride concentration as the natural substrate, demonstrating its specificity for N-domain hydrolysis. This fluorescent derivative was used to develop a sensitive assay for the determination of ACE activity in human plasma.     

A direct radioassay for pyruvate kinase activity

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. A direct radioassay for this enzyme using [14C]PEP as substrate has been developed. The product, [14C]pyruvate, can be separated from the substrate rapidly and easily by applying the mixture to a hydroxyapatite column, and eluting the [14C]pyruvate directly into a scintillation vial. The [14C]PEP is bound to the column which can be regenerated and used indefinitely. The assay is sensitive, rapid, and particularly well suited for the simultaneous assay of large numbers of samples.     

A quantitative method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase activity with lectin affinity chromatography

A quantitative method for the activity of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase has been developed using a well-characterized substrate to which other fucosyltransferases fail to transfer and lentil lectin-Sepharose, which will bind this substrate only after fucosylation of the asparagine-linked N-acetylglucosamine. The enzyme was extracted from human skin fibroblasts and incubated with GDP-[14C]fucose and a specific substrate, asialo-agalactotransferrin glycopeptide. The product of the enzyme reaction, [14C]fucose alpha 1----6 to the asparagine-linked N-acetylglucosamine of the substrate, bound to lentil lectin-Sepharose and was eluted with 0.4 M methyl alpha-D mannopyranoside. The method was shown to be specific after characterization of the lentil lectin-bound glycopeptides by enzyme degradation and affinity chromatography. Quantitation of the method was shown by several parameters, including the linearity of product formed with respect to time, GDP-[14C]fucose concentration and enzyme concentration.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Adenylate cyclase and cyclic AMP phosphodiesterase activity during the mitotic cycle of Physarum polycephalum.

Tyrosine, as well as small amounts of phenylalanine, were removed selectively and quantitatively from purified chick brain tubulin by enzymatic digestion with carboxypeptidase. The fraction of molecules containing hydrolyzable tyrosine changed with the stage of development and had the highest value (～0.5) around days 14–16 of the embryo. The increase in the fraction of tyrosinated molecules was found to be temporally correlated with an increased specific activity of the enzyme catalyzing the incorporation reaction. In addition, it was found that the availability of α-chain C-termini for $\text{in vitro}$ tyrosination also reached a maximum during the same period. Changes in the extent of modification of the C-terminus of tubulin may be relevant for the participation of the resultant microbubules in different developmental events.     

Hydroxylaminolysis of penicillin binding componenets is enzymatically catalyzed.

The hydroxylaminolysis of the penicilloyl moiety from [14C]penicillin G binding component (PBC) complexes of the Bacillus subtilis D-alanine carboxypeptidase and of the mixture of PBC's of Staphylococcus aureus was inhibited by denaturation of the complexes by heat (55 degrees), detergent (1% sodium dodecyl sulfate), or trichloroacetic acid. The kinetics of inhibition by denaturation were comparable to those of the inhibition of [14C]penicillin G binding to the PBC's and of carboxypeptidase activity of the B. subtilis enzyme under identical denaturing conditions. These data establish that the hydroxylaminolysis is an enzymatically catalyzed process suggesting that penicillin G is bound to an enzymatically active site. Treatment of the denatured [14C]penicillin G-carboxypeptidase complex with sodium borohydride or at pH 12 resulted in the release of the penicilloyl moiety. These results are consistent with a carboxylic ester bond for the penicilloyl-PBC instead of a thiolester linkage as was initially presumed.