The reaction of citraconic anhydride with bovine alpha-crystallin lysine residues. Surface probing and dissociation-reassociation studies

Citraconic anhydride reacts readily with alpha-crystallin's lysine residues at pH 7.4. Upon addition of 2 equivalents of citraconic anhydride per equivalent lysine, 24% of the lysine residues were modified without disrupting the native quaternary structure. Further citraconylation led to dissociation into 10 S aggregates. Complete dissociation into subunits (1.4 S) occurred after adding 100 equivalents of citraconic anhydride, resulting in 98% modification. Decitraconylation did not lead to reaggregates identical with the native ones. The unmodified and the once and twice citraconylated alpha-crystallin subunits were discerned by isoelectric focusing according to their theoretical isoelectric points. In the native alpha-crystallin aggregates, nearly all B chains and approx. 60% of the A chains were found to possess at least one surface-exposed lysine residue. No differences between the susceptibilities to citraconylation of the in vivo deamidated (A1 and B1) and the de novo synthesized (A2 and B2) subunits were found. These results support the three-layer spherical assembly model for the alpha-crystallin quaternary structure.     

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH: In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of disassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles:Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups: The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride: Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides: With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride: Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride:     

Studies on the multi-enzyme complex of yeast fatty-acid synthetase. Reversible dissociation and isolation of two polypeptide chains.

1. The multi-enzyme complex of fatty acid synthetase, Mr 2300,000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the epsilon amino groups of lysine. The complete dissociation into the subunits alpha and beta is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis. 2. This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity. 3. The subunits were isolated by sucrose-density-gradient centrifugation and studied for the different partial enzyme activities involved in long-chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the beta chains of the complex, confirming a pentafunctional role of this subunit.     

Protein-deficient ribosomal particles from yeast 60S subunits obtained by modification with dimethylmaleic anhydride and by treatment with NH4Cl

The protein-deficient particles prepared from yeast 60S subunits by modification of lysine residues with the reversible reagent dimethylmaleic anhydride are compared with those obtained by treatment with NH4Cl. The two procedures cause selective dissociation of certain proteins. With a few exceptions, the dissociation pattern is similar in both cases. When using dimethylmaleic anhydride, a variation in the protein composition of the ribosomal cores is obtained by modification of the ribosomal subunits in the presence of any of the following ligands: elongation factor-2, ricin A, verrucarine A and puromycine.     

Modification of 40S ribosomal subunits from yeast with dimethylmaleic anhydride

Modification of 40S ribosomal subunits from Saccharomyces cerevisiae with dimethylmaleic anhydride (DMMA), a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity and by dissociation of proteins from the particles. The protein-deficient ribosomal particles, originated from 40S subunits by treatment with dimethylmaleic anhydride at a molar ratio of reagent to particle of 250, can partially reconstitute active subunits upon addition of the corresponding released proteins, and regeneration of the modified amino groups.

---

     

Effects of different amino-group reagents on ribosomal integrity: structural role of lysine residues

Treatment of 60S subunits from yeast ribosomes with dicarboxylic acid anhydrides (maleic, dimethylmaleic and tetrahydrophtalic), which introduces negatively-charged residues, is accompanied by substantial dissociation of protein components (35–55%). In contrast, acetic anhydride or cyanate, which introduce uncharged groups, cause practically no protein release, even after extensive modification. Therefore, in addition to blocking lysine-RNA interactions, a large change in the electric charge of the proteins appears to be necessary to obtain dissociation. These results seem to indicate that lysine residues are not essential to ribosome integrity, while arginine-RNA interactions should play an important role in the maintenance of ribosomal structure.     

Primary structure of the cytoplasmic aspartate aminotransferases from the swine myocardium. Isolation, purification and characteristics of the peptides from cyanogen bromide cleavage]

Cytoplasmic aspartate aminotransferase from pig heart muscle was cleaved with cyanogen bromide and 8 peptide fragments were isolated. The high tendency of the large peptides for aggregation was overcome only by the utilization of special procedures of the denaturation and acylation of the lysine residues of peptide with citraconic anhydride. Peptides were separated by gel chromatography on sephadex G-50 and G-75 and by ion exchange chromatography on cellulose DE-22 and DE-32 with use of concentrated urea solutions. Amino acid composition and N-terminal residues of isolated peptides were determined.     

Mitochondrial ATP synthase complex: interaction of its F1 adenosinetriphosphatase moiety with the heavy atom iodine

Studies were carried out to determine whether a simple electron-dense "heavy atom" like iodine could be introduced selectively into one or more of the subunits of the mitochondrial ATP synthase complex of rat liver. Surprisingly, very low amounts of iodine are incorporated into the isolated F1 moiety of this complex under conditions which result in a marked loss of catalytic activity. ATPase activity is inactivated in a concentration-dependent manner at pH 7.5 with half-maximal inactivation occurring at about 40 microM iodine. A maximum of only 10 atoms of iodine are incorporated per F1 molecule under conditions where inhibition of ATPase activity is linearly related to iodine incorporation. The molecular size of F1 after iodination is unchanged, indicating that inactivation is due to modification of essential amino acid residues rather than subunit dissociation. Treatment of F1, with 20-50 microM [125I]iodine followed sequentially by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that the beta subunit is preferentially labeled. Significantly, about two atoms of iodine per beta subunit are incorporated. Some iodine amounting to less than 23% of the total radioactivity placed on the gels is recovered in the alpha and gamma subunits whereas no radioactivity is detected in the delta and epsilon subunits. Iodination of F1 appears to modify essential residues other than those involved in substrate or product binding per se. Thus, nucleotide binding to F1 is unaltered by iodine, and neither phosphate, MgADP, nor MgATP protects F1 against inhibition by this agent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of aspartate aminotransferase with mercurochrome. Relationship of an exposed thiol group of the enzyme to the active centre.

Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 x 10(4) M-1 - min-1 and 2.15 x 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 x 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule.     

Partial reconstitution of 60S ribosomal subunits from yeast

Summary Ribosomal 60S subunits active in polyphenylalanine synthesis can be reconstituted from core particles lacking 20–40% of the total protein. These core particles were obtained by treatment of yeast 60S subunits with dimethylmaleic anhydride, a reagent for protein amino groups. Upon reconstitution a complementary amount of split proteins is incorporated into the ribosomal particles, which have the sedimentation coefficient of the original subunits. Ribosomal protein fractions obtained by extraction with 1.25 M NH₄Cl, 4 M LiCl, 7 M LiCl, or 67% acetic acid, are much less efficient in the reconstitution of active subunits from these core particles than the corresponding released fraction prepared with dimethylmaleic anhydride. Attempts to reconstitute active subunits from protein-deficient particles obtained with 1.25 M NH₄Cl plus different preparations of ribosomal proteins, including the fraction released with dimethylmaleic anhydride, were unsuccessful. Therefore, under our conditions, of the disassembly procedures assayed only dimethylmaleic anhydride allows partial reconstitution of active 60S subunits.Abbreviation DMMA dimethylmaleic anhydride     

Dimethylmaleic anhydride, a specific reagent for protein amino groups

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.     

Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium.

Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef pancreas dissociates into subunits. At pH7, the rate of the dissociation is close to both the reaction rate of the buried--SH groups and the rate of inactivation (Iborra, F., Mourgeon, G., Labouesse B., and Labouesse, J. (1973) Eur. J. Biochem. 39, 547-556). The pH and enzyme concnetration dependences of the reaction rate of the 16 cysteinyl residues of the enzyme as well as that of its inactivation support the idea that inactivation by alkylation of the--SH groups is due essentially to the dissociation of the protein into inactive subunits and not to the chemical blocking of a catalytic residue. This is confirmed by the independence on N-ethylmaleimide concentration of the reaction of the buried--SH groups and of the inactivation of the enzyme at high N-ethylmaleimide concentration. The dissociation becomes in this case the rate-limiting step of the chemical reaction. The monomeric structure is stabilized by the blocking of the--SH groups exposed during the dissociation. The dissociation constant of the dimeric enzyme is progressively increased during the alkylation. The tightness of the associated structure depends on the protonation of groups titrating between pH 7 and pH 9.     

Reaction of ovine interstitial cell stimulating hormone with citraeonic and maleic anhydrides

The free amino groups of ovine interstitial cell stimulating hormone and its subunits are modified with citraconic and maleic anhydrides. Three lysine residues in the native hormone are not available for reaction. Introduction of negatively charged groups does not cause dissociation of the hormone into its subunits. The completely modified interstitial cell stimulating hormone-β combines with the native α subunit to give a recombinant that has biological activity, while the modified interstitial cell stimulating hormone-α is unable to form an active product with native interstitial cell stimulating hormone-β. The results suggest that the ?-NH₂ groups of the α subunit play an important role in determining biological activity.     

Conditions of stability for the quaternary structure and antigenic activity of adenovirus hexon

The procedure of SDS-PAGE was modified by lowering the temperature of protein sample dissociation to allow the separation of denaturated adenoviral hexon chains and native hexon capsomers (trimers) in the same gel. By combining the modified SDS-PAGE with dot and blot radioimmunoassays, the range of stability of the simian adenovirus SA7 hexon quaternary structure and its antigenicity was studied against a number of physical and chemical agents known to dissociate and denaturate proteins. A perfect correlation was found between the hexon native quaternary structure (trimer) and its immunoreactivity with anti-hexon immunoglobulins. The pattern of hexon trimer stability to a wide spectrum of denaturants suggests that its subunits are held together, mainly by hydrophobic interactions, in such a way that the innersubunit contact regions make up the "hydrophobic core" of the hexon molecule.     

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride.

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.     

Protein-deficient ribosomal particles obtained by reversible modification with dimethylmaleic anhydride

Preparation of protein-deficient ribosomal particles from Escherichia coli ribosomes by reversible modification of protein amino groups with dimethylmaleic anhydride (J. A. Pintor-Toro, D. Vázquez, and E. Palacián, 1979, Biochemistry18, 3219) is accompanied by degradation of r-RNA and reagent-independent inactivation. Alternative conditions to regenerate the modified amino groups have been found, which reduce the time needed to prepare the ribosomal “cores” from 9 to 3 days, and prevent RNA degradation and inactivation. The ribosomal particles obtained from 70 S ribosomes and 50 S subunits by this modified procedure show no extensive degradation of RNA and very little reagent-independent inactivation, which allow good recovery of the polypeptide synthesizing activity when incubated with the corresponding split proteins.     

Calculation of the energy difference between the quaternary structures of deoxy- and oxyhemoglobin.

The energy difference between the quaternary structures of deoxy- and oxyhemoglobin is evaluated on the basis of the atomic coordinates determined by X-ray diffraction analysis. Calculation of the van der Waals interaction between subunits shows that in a hemoglobin molecule as a whole, the interaction is more attractive in the oxy form than in the deoxy form by about 8 kcal/mol, and that in each pair of two subunits except the pair alpha1alpha2, the interaction energy varies by about 15 kcal/mol. The electrostatic interactions originating in the partial charges on all constituent atoms of hemoglobin and in the polar residues on the surface of hemoglobin make only a small contribution to the energy difference between the quaternary structures of deoxy- and oxyhemoglobin. Thus, the contribution of the clusters of the polar residues in the internal cavity between like subunits and also of the freedom of rotation of the C-terminal of each subunit in oxyhemoglobin may be important energetically in the transition from deoxy to oxy quaternary structure. In this point, the present calculation supports Perutz' model, but suggests necessity of further investigations on the transitional characteristics of the quaternary structure in the intermediate steps of oxygenation. The discussion on the transitional characteristics is given in the last section.     

Novel dissociation mechanism of a polychaetous annelid extracellular haemoglobin

The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of approximately 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or 'linker' subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS/PAGE, UV-visible spectrophotometry, electrospray-ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

Identification of "buried" lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors.

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or "buried" amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is "buried" in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.