Effects of reductive methylation on microtubule assembly. Evidence for an essential amino group in the alpha-chain.

Szasz, J.; Burns, Robert; Sternlicht, H.

doi:10.1016/s0021-9258(18)34837-3

Cited by 43 publications

(19 citation statements)

References 42 publications

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“…The extent of methylation measured by H14CHO incorporation showed that the assembly-incompetent tubulin (Figure 3, lanes 1 and 1') was labeled to the extent of 5 methyl groups per dimer or, if there were dimethylation, a minimum of 2.5 amino sites per tubulin dimer (Table I). This degree of modification associated with complete inhibition of assembly is distinctly less than the 13 methyl groups incorporated per dimer found by Szasz et al (1982) using formaldehyde at 2.0 mM. However, in agreement with Szasz et al, we found a consistent preponderance of labeling in a-versus /3-tubulin in a ratio of approximately 1.7 to 1.…”

Section: Resultssupporting

confidence: 92%

“…HCOf/C02 Antagonizes the Inhibition of Microtubule Assembly by Reductive Methylation. As reported by Szasz et al (1982), reductive methylation at relatively low concentrations of formaldehyde inhibited microtubule assembly (Figure la, curve A versus curve C). Under our assembly conditions, a 26% inhibition was attained with 0.1 mM formaldehyde, 80% at 0.25 mM, and complete inhibition at 0.5 mM formaldehyde.…”

Section: Resultssupporting

confidence: 78%

“…In general, our measurements of label incorporation in the absence of C02 were in agreement with Szasz et al (1982) and Mellado et al (1982) with some minor exceptions. Thus, we found that the number of methyl groups introduced that were required for complete inhibition was 5 as compared with 13 found by Szasz et al However, like them, we consistently found preferential labeling of a-as opposed to /3-tubulin.…”

Section: Discussionsupporting

confidence: 82%

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Carbamate formation on tubulin: carbon dioxide/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH

Clark¹,

Volpi²,

Berlin³

1988

Biochemistry

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Carbamoylation and reductive methylation of tubulin have been shown previously to inhibit microtubule assembly, probably by attack on essential internal lysine residues [Mellado, W., Slebe, J., & Maccioni, R.B. (1982) Biochem. J. 203, 675-681; Szasz, J., Burns, R., & Sternlicht, H. (1982) J. Biol. Chem. 257, 3697-3704]. We show first that this inhibition is blocked by the presence of HCO3-/CO2 buffer at physiological concentrations during the carbamoylation or reductive methylation. Under conditions that block assembly, the amount of radiolabeled cyanate or formaldehyde incorporated by these reactions in the absence of HCO3-/CO2 was approximately four carbamoyl or five methyl groups in a ratio of approximately 1.7 alpha chain/beta chain. In the presence of HCO3-/CO2, the formaldehyde incorporation is decreased roughly 0.5 mol in each of the alpha and beta chains, and cyanate incorporation, roughly 1.0 mol/mol of alpha or beta monomer. These results are consistent with the hypothesis that CO2 competed with formaldehyde or cyanate for uncharged amino groups and led to the reversible formation of carbamates. The complete antagonism of the inhibition of microtubule assembly by reductive methylation by CO2, even though the number of methyl groups incorporated was reduced by only 0.5 mol/tubulin monomer, was consistent with the possibility that reductive methylation opened up additional residues for attack. Indeed, using an adaptation of the method of Gros et al. for measurement of carbamates [Gros, G., Forster, R.E., & Lin, L. (1976) J. Biol. Chem. 251, 4398-4407], we found that reductive methylation with 2 mM formaldehyde (assembly blocked) did not decrease carbamate formation (carbamate formation was inhibited at higher formaldehyde concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 78%

Section: Discussionsupporting

confidence: 82%

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Carbamate formation on tubulin: carbon dioxide/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH

Clark¹,

Volpi²,

Berlin³

1988

Biochemistry

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“…lysyl residues. Methylation of these lysyl residues has little effect on microtubule assembly (Szasz et al, 1982(Szasz et al, , 1986. It is consequently difficult to understand why ethoxyformylation of these residues should significantly affect assembly.…”

Section: Discussionmentioning

confidence: 99%

“…We have had a longstanding interest in the -subunit. This subunit contains a highly reactive lysyl residue, Lys 394, whose methylation renders tubulin assembly incompetent (Szasz et al, 1982. Chemical modifications of this reactive residue have been implicated in the pathophysiology of diabetes (Williams et al, 1982) and in alcohol-induced hepatic necrosis (Jennett et al, 1987(Jennett et al, , 1989, two disorders that result in impaired microtubule function.…”

mentioning

confidence: 99%

Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition

Levison¹,

Szasz²,

Sternlicht³

1989

Biochemistry

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In this study we reexamined the basis for the profound inhibitory effects of low concentrations of diethyl pyrocarbonate (DEP) on tubulin's ability to assemble into microtubules [cf. Lee, Y. C., Houston, L. I., & Himes, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 50-56]. Assembly inhibition at low DEP concentrations can be resolved into two components: a component reversible with hydroxylamine (attributed to monoethoxyformylation of histidyl residues) that contributes approximately 40% of the inhibition and a hydroxylamine-resistant component (attributed to ethoxyformylation of non-histidyl residues) that contributes approximately 60% of the inhibition. Comparisons between the extent of assembly inhibition associated with each component and the degree of residue modification argue for the involvement of a small number of highly reactive residues in the inhibition process. To identify these residues, tubulin was reacted with limiting concentrations of [3H]DEP and subjected to tryptic digestion and HPLC analysis. Only one moderately reactive histidyl residue was detected. This residue (approximately 2-3-fold more reactive than the bulk histidyl residues) eluted in an apparently large, hydrophobic fragment. We failed to detect any non-histidyl residues that were exceptionally reactive to [3H]DEP. However, we did observe that the N-terminal methionyl residues in native protein were ethoxyformylated at rates comparable to that of the bulk histidyl residues. In denatured protein these methionyl residues were ethoxyformylated to a much larger extent (approximately 3-4-fold) than the bulk histidyl residues. We suggest that the N-terminal methionyl residues in tubulin are partly buried or are in a salt-bridge interaction in native protein and that ethoxyformylation of these residues disrupts tubulin structure and interferes with microtubule assembly.

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Preferential covalent binding of acetaldehyde to the α-chain of purified rat liver tubulin

et al. 1989

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Hepatic ethanol oxidation generates the reactive intermediate acetaldehyde, which binds to proteins. Previous work, using bovine brain tubulin as a model protein, has shown that acetaldehyde preferentially formed stable adducts on the alpha-chain of the heterodimeric molecule. This binding resulted in functional impairment of the tubulin/microtubule system as evidenced by a decreased ability of adducted tubulin to form microtubules. Since tubulin/microtubules are believed to be very important cytoskeletal components of the hepatocyte and results with brain tubulin were interesting, our goal was to extend these studies to liver tubulin. We purified tubulin from rat liver by a polymerization-based cycle method followed by phosphocellulose chromatography. We then characterized the covalent binding of [14C]acetaldehyde to liver tubulin. Naturally forming and cyanoborohydride-stimulated stable adducts formed linearly with liver tubulin in a manner almost identical to that with brain tubulin. We also found that the alpha-chain of the native heterodimeric liver tubulin molecule was the preferred site of adduct formation at low acetaldehyde to protein ratios. These results confirm and extend our previous findings with the brain tubulin model and further suggest that the alpha-chain of tubulin may be a preferential site for acetaldehyde-adduct formation during ethanol oxidation in the liver.

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Effects of reductive methylation on microtubule assembly. Evidence for an essential amino group in the alpha-chain.

Cited by 43 publications

References 42 publications

Carbamate formation on tubulin: carbon dioxide/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH

Carbamate formation on tubulin: carbon dioxide/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH

Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition

Preferential covalent binding of acetaldehyde to the α-chain of purified rat liver tubulin

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