Reaction of aspirin with cysteinyl residues of lens γ-crystallins: a mechanism for the proposed anti-cataract effect of aspirin

Incubation of lens crystallins with aspirin inhibits the development of opacities caused by cyanate. Cyanate-induced opacities are thought to be due to carbamylation of the lysyl residues which causes a decrease in the protein charge and subsequent conformational changes that permit disulfide bonding. Because aspirin can also react with lysyl residues, it has been proposed that the aspiring inhibition of cataractogonesis is due to acetylation of the lysyl which would block their reaction with cyanata. However, acetylation oflysyl residues also lowers the protein charge and would be expected to effect changes in protein conformation similar to those caused by carbamylation. Therefore, acetylation of the lysyl residues is not a satisfactory explanation for the inhibitory effect of aspirin on lens opacification. Our investigations of the reactions of cyanate and aspirin with bovine γ_II-crystallins show that the cysteinyl residues are also carbamylated and acetylated at pH 7.4. At this pH, the carbamylation at the cysteinyl residues is reversible, leading to regeneration of the thiol group and disulfide bonding. In contrast, the acetylation at cysteinyl residues is stable at pH 7.4 and can prevent disulfide bonding. This difference in stability explains how cyanate promotes, and aspirin inhibits, cataractogenesis.     

Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides

Chemical modification of proteins is often carried out to generate protein-small molecule conjugates for various applications. The high resolution and mass accuracy of a Fourier transform mass spectrometer is particularly useful for assessing the extent or sites of covalent modifications. As protein-small molecule reactions often produce products with variable numbers of the compound incorporated at different sites, a direct mass analysis of the reaction products at times yields mass spectra hard to interpret. Chromatographic separation at protein level could reduce the complexity of a sample, thus allowing more accurate mass spectrometric analysis. In this report, we demonstrate the utility of reversed-phase protein chromatography and FT-ICR mass spectrometry in analyzing CCNU (lomustine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-urea, MW: 233.7 Da) modification of stathmin. With this combined approach, we determined the stoichiometry as well as sites of CCNU incorporation into the protein, demonstrating differential reactivity of several lysyl residues to CCNU alkylation.     

Capillary isoelectric focusing with laser-induced fluorescence whole column imaging detection as a tool to monitor reactions of proteins

Capillary isoelectric focusing (CIEF) with laser-induced fluorescence (LIF) whole column imaging detection (WCID) has the characteristics of high resolution, high speed and high sensitivity for separation of amphoteric biomolecules. These features enable a CIEF-LIF-WCID system to monitor the dynamic process of a protein reaction. The reaction can be a physical change or a chemical reaction, provided that the kinetics of the reaction is slower than the focusing speed or that the intermediates involved have long enough life-span compared to the analysis time. The processes of denaturation (a physical reaction), reduction and carbamylation (both chemical reactions) were dynamically monitored. The CIEF profiles at successive reaction times clearly displayed the formation of different products at different stages. At incomplete denaturation, intermediates with higher apparent pI values relative to the products at complete denaturation were detected. Carbamylation products of a protein were detected when the protein reacted with a urea solution that had prepared three months earlier, exhibiting gradually decreased pI values. Mechanisms involved in these reactions were rationalized. A combined mechanism of denaturation and reduction was suggested to explain the denaturing process under high concentrations of urea. Potential applications and critical factors to manipulate these reactions were also discussed.     

In vivo carbamylation and acetylation of water-soluble human lens alphaB-crystallin lysine 92

Several post-translational modifications of lysine residues of lens proteins have been implicated in cataractogenesis. In the present study, the molecular weight of an alpha-crystallin isolated from the water-soluble portion of a cataractous human eye lens indicated that it was a modified alphaB-crystallin. Further analysis by mass spectrometry of tryptic digests of this modified protein showed that Lys 92 was modified and that the sample was structurally heterogeneous. Lys 92 was acetylated in one population and carbamylated in another. Although carbamylation of lens crystallins has been predicted, this is the first documentation of in vivo carbamylation of a specific site. These results are also the first documentation of in vivo lysine acetylation of alphaB-crystallin. Both modifications alter the net charge on alphaB-crystallin, a feature that may have significance to cataractogenesis.     

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.     

Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture, neurons were shown to express a high degree of tubulin heterogeneity (8 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.     

Oligonucleotides conjugated to short lysine chains

A new method for synthesizing oligonucleotide peptide conjugates by an in-line approach is presented. A phosphorothioate oligonucleotide with the sequence of bcl-2 targeted Oblimersen by employing a modified 2'-amino-2'-desoxy-uridine nucleotide bearing a succinyl linker at the 2' position was prepared. The carboxyl group was protected for solid-phase synthesis as the benzyl ester. Ester cleavage was afforded by a phase transfer reaction using palladium nanoparticles as catalyst and cyclohexadiene as hydrogen donor. Short tails of up to three lysyl residues were conjugated to the oligonucleotide by an inverse stepwise peptide synthesis. The conjugates were characterized by HPLC, mass spectrometry, and circular dichroism. Influence of lysyl tails on CD spectra were minimal. Melting profiles revealed only minimal destabilizing effects on duplexes by conjugation of peptides.     

Failure of highly purified lysyl hydroxylase to hydroxylate lysyl residues in the non-helical regions of collagen.

The activity of highly purified lysyl hydroxylase towards lysyl residues within both the helical and the N-terminal non-helical telopeptide regions of chick type I collagen has been examined. The peptides alpha 1(I)-CB1 and alpha 2(I)-CB1, isolated from protocollagen following CNBr digestion and containing the N-terminal telopeptidyl lysyl residues, failed themselves to act as substrates. With protocollagen as substrate, analysis of products obtained following bacterial collagenase digestion of the reaction mixture showed that overall 37% hydroxylation of lysyl residues within the helical region of collagen had been obtained, which may be maximal. No hydroxylation, however, of the single lysyl residue in either alpha 1(I)-CB1 or alpha 2(I)-CB1, isolated following CNBr digestion of the reaction mixture, was observed, despite the known susceptibility of these residues to hydroxylation. These findings provide strong circumstantial evidence for the suggestion that a lysyl hydroxylase specific for the telopeptidyl residues and distinct from that active towards lysyl residues in the helical portion of the molecule may exist [Barnes, Constable, Morton & Royce (1974) Biochem. J. 139, 461-468].     

Characterization of a new electrophoretically silent hemoglobin variant. Hb saale OR alpha 2beta 2 84(EF8)Thr --> Ala

A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --> Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A.     

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.     

Antisera to synthetic peptides as probes of structural changes during aging of alpha-crystallin from the bovine lens

Polyclonal antisera have been made to synthetic peptides of 11-15 residues that correspond to nine different regions of the alpha A crystallins. These antisera have been used in a radioimmunoassay to quantitatively probe for structural and/or covalent changes of alpha-crystallins in the nucleus versus cortex of the adult bovine lens. Antisera specific for the C-terminal and N-terminal regions of the alpha-crystallins bind more to alpha-crystallins from cortex. Antisera to three out of the seven internal sequences (residues 75-89, 87-101 and 135-149) bind better to alpha-crystallins from the bovine lens nucleus, suggesting a greater accessibility of these sequences to antisera binding. Together, these studies demonstrate that antisera against synthetic peptide sequences of alpha A crystallins are very specific probes that can detect structural and/or covalent changes in specified regions of the alpha-crystallins during the process of aging in the bovine lens.     

Evidence for the synthesis of thymosin alpha 1 by calf thymocytes and the production of this peptide by natural processing

Thymus and thymocytes from calf were extracted under isotonic conditions in the presence of protease inhibitors or under severe denaturing conditions (after quick freezing and thawing in boiling 0.1 M NaCl). The extracts, as well as the medium in which the thymocytes were obtained from thymus fragments (thymocyte supernatants), were size-fractionated by ultrafiltration. As in whole thymus isotonic extracts, thymosin alpha 1 [A. L. Goldstein, T. L. K. Low, M. McAdoo, J. McClure, G. B. Thurman, J. Rossio, C-Y. Lai, D. Chang, S-S. Wang, C. Harvey, A. H. Ramel, and J. Meienhofer (1977) Proc. Natl. Acad. Sci. USA 74, 725-729] was contained in isotonic extracts from thymocytes and also in thymocyte supernatants, as determined by isoelectric focusing and reverse-phase HPLC analysis. The extraction under denaturing conditions mainly yielded products with molecular masses over 50,000, showing very similar isoelectric focusing patterns in both thymocytes and whole thymus extracts. As deduced by isoelectric focusing analysis of diverse size-fractionated products, a strong association capacity seems to be responsible for an apparently high molecular mass of the components of these extracts. According to the pI, two of these components were prothymosin alpha [A. A. Haritos, G. J. Goodall, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 1008-1011] and thymosin alpha 1. Prothymosin alpha was not detected in any isotonic extracts or thymocyte supernatants. These data suggest that calf thymocytes are capable of producing thymosin alpha 1, which would arise by natural processing of its precursor.     

In vivo acetylation identified at lysine 70 of human lens alphaA-crystallin.

Posttranslational modification of protein lysyl residues that change the net charge of the molecule may alter the protein conformation. Such modifications are of particular significance among lens proteins, because conformational changes are associated with the development of cataract. A previously unidentified acetylated form of alphaA-crystallin has been isolated from the water-soluble portion of human lenses. The alphaA-crystallins were fractionated by anion exchange HPLC into seven peaks, each containing more than one form of alphaA-crystallin. The previously reported deamidated and phosphorylated forms were identified by their molecular masses, determined by electrospray ionization mass spectrometry. In addition to these modifications, approximately 5% of alphaA-crystallin had a modification that decreased the charge by one and increased the molecular mass by 42 u. This modification, identified as acetylation, was located uniquely at Lys 70. Like any modification that alters the surface charge, acetylation may affect protein conformation and intermolecular interactions, thereby altering the solubility or chaperone properties of alphaA-crystallin. Acetylation of lysine 70 is potentially significant since it is located in a region that has been implicated in the chaperone activity of alphaA-crystallin.     

Purification, characterization and gene cloning of a novel phospholipase A2 from the venom of Agkistrodon blomhoffii ussurensis

A new phospholipase A2 with Gln at the site 49, abbreviated as Gln49-PLA2, has been purified from the venom of Agkistrodon blomhoffii ussurensis by using ion-exchange chromatography, gel filtration chromatography and reversed-phase HPLC, and behaves as a single-band on SDS-PAGE. Its molecular weight is 13881.85+/-0.33 Da given by mass spectrometry and pI is about 8.56 given by isoelectric focusing. Gln49-PLA2 does not show phospholipase A2 and hemorrhagic activity, whereas shows weak toxic and apparent anticoagulant activity. Based on the N-terminal sequencing and peptide mass fingerprint analysis, Gln49-PLA2 cDNA has been cloned by means of RT-PCR. Gln49-PLA2 consists of 122 amino acid residues and has the structural features of class II of snake venom phospholipase A2.     

Mass measurements of C-terminally truncated alpha-crystallins from two-dimensional gels identify Lp82 as a major endopeptidase in rat lens

Molecular chaperone activity of lens alpha-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on alpha-crystallins, 2) identify alpha-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mM Ca2+. Resulting fragmented alpha-crystallins were separated by two-dimensional gel electrophoresis. Eluted alpha-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble alpha-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of alphaA-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from alphaA were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of alphaA. Using uniquely truncated alphaA-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of alpha-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications.     

Studies on the amino groups of myosin ATPase. Trinitrophenylation of reactive lysyl residue in ventricular and atrial myosins

Myosin and heavy meromyosin from ventricular, atrial, and skeletal muscle were purified and trinitrophenylated by 2,4,6-trinitrobenzene sulfonate. The trinitrophenylation reaction followed a complex kinetics consisting of a fast and slow reaction in all preparations studied. Reactive lysine residues were trinitrophenylated during the fast reaction with a concomitant decrease in K⁺ (EDTA)-activated ATPase and an increase in Mg²⁺-stimulated ATPase activities of myosin. The extent of increase in Mg²⁺-mediated ATPase was the highest with skeletal and the lowest with atrial myosin. The trinitrophenylation of the less reactive lysyl residues continued during the slow reaction. The rate constants of the reactions and the number of reactive lysine residues were evaluated by computer analyses of the trinitrophenylation curves. Two reactive lysine residues were found in skeletal and ventricular myosins while their number in atrial myosin was somewhat lower. The rate of trinitrophenylation in skeletal muscle myosin or heavy meromyosin was always higher than in the two cardiac myosin isozymes. Addition of KCl increased the trinitrophenylation of both highly reactive and slowly reactive lysyl residues in all of the three heavy meromyosins, however, the effect was more profound with cardiac heavy meromyosins. Addition of MgADP induced spectral changes in trinitrophenylated skeletal but not in cardiac myosins. Similar changes occurred in skeletal and to a lesser degree in ventricular heavy meromyosin, but no definite spectral changes were observed in atrial heavy meromyosin. The findings suggest that structural differences exist around the reactive lysyl residue in the head portion of the three myosins.     

Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates

A quenching technique for the study of rapid protein reactions is described which consists of injecting a small volume of aqueous solution of reactants into a large volume (× 10) of hydro-organic solvent cooled at subzero temperature and mechanically shaken. The protein reaction intermediates, stabilized at subzero temperature and brought into a hydro-organic solution, can then be separated by subzero temperature electrophoretic methods, such as isoelectric focusing, in the same solvent. The alkaline hydrolysis of 2,4-dinitrophenylacetate was studied by the use of this quenching technique in order to compare the quenching and the rate constants of the reaction with those obtained by normal rapid quenching methods. It was found that first-order reactions having rate constants up to about 5 s^?1 can be satisfactorily studied by this technique. The technique is not suitable for the study of faster reactions because of the high value of the quenching time (40–100 ms). The hybridization reaction of carboxyhemoglobins A and C in aqueous solution at 22°C was studied as an example of the application of this quenching technique and of the isoelectric focusing method at subzero temperature to the isolation of unstable intermediates in a protein reaction.     

The role of the 6 lysines and the terminal amine of Escherichia coli single-strand binding protein in its binding of single-stranded DNA

Differential chemical modification of the lysines and amino-terminus of Escherichia coli single-strand binding (SSB) protein was used to determine their roles in the binding of SSB to single-stranded DNA (ssDNA). A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride. First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single-stranded ssDNA. Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride. Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry. The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA. Acetylation rate constants were determined from time course measurements. In the absence of ssDNA, the terminal alpha-amine of SSB was 10-fold more reactive than Lys residues at positions 43, 62, 73, and 87. The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition. In the presence of ssDNA, the reactivities of the amino-terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7-25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA. Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer-monomer interface.     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys 394 region.

Previous studies have implicated at least two regions in alpha-tubulin that are important for the regulation of microtubule assembly. These regions include a cluster of basic residues consisting of Arg 390, His 393, and Lys 394 and the highly acidic carboxyl terminus. Lys 394 is highly reactive to HCHO and NaCNBH3. The reductive methylation of Lys 394 by these reagents is thought to be responsible for the profound inhibitory effects of low concentrations of HCHO on microtubule assembly (cf. Szasz J., M. B. Yaffe, M. Elzinga, G. S. Blank, and H. Sternlicht. 1986. Biochemistry. 25:4572-4582). In this study we reexamined the basis for this inhibition. Lys 394 in a human keratinocyte alpha-tubulin (k alpha 1) was replaced by a glutamic acid residue using site-directed mutagenesis. The mutant K394E was synthesized in vitro using rabbit reticulocyte lysates, and its ability to coassemble with bovine brain microtubule protein (MTP) before and after reaction with HCHO and NaCNBH3 was compared with that of wild-type. No differences in the coassemblies of the unmethylated proteins were detected suggesting that Lys 394 is not essential for microtubule assembly. However, methylated K394E prepared at low HCHO concentrations (< 1 mM) incorporated into microtubules to a greater extent (approximately 30-40%) than methylated wild-type. This result is consistent with the hypothesis that methylation of Lys 394 interferes with microtubule assembly. However, the extent of protection afforded by the replacement of Lys 394 with Glu 394 was less than half as large as that predicted from the earlier studies. We tentatively conclude that another residue(s) besides Lys 394 contributes significantly to the assembly-inhibition observed with low concentrations of HCHO. Since this residue(s) is less reactive than Lys 394, it would have to inhibit assembly substoichiometrically when methylated. Potential candidates for this residue include bulk lysyl residue(s), a lysyl residue(s) with intermediate reactivity toward HCHO, and the NH2-termini. The NH2-termini are especially attractive candidates since they appear to have a structural role in microtubule assembly.