Alginate esters via chemoselective carboxyl group modification

Alginates are (1 → 4) linked linear copolysaccharides composed of β-d-mannuronic acid (M) and its C-5 epimer, α-l-guluronic acid (G). Several strategies for synthesis of carboxyl modified alginate derivatives exist in the literature. Most of these however employ aqueous chemistries, such as carbodiimide coupling reactions. Based on our recently discovered method for homogeneous dissolution of tetrabutylammonium (TBA)-alginate, we now describe use of tetrabutylammonium fluoride (TBAF)-based two component solvent systems as media for synthesis of carboxyl-modified alginate esters. Partially and fully esterified benzyl, butyl, ethyl, and methyl alginates were synthesized via reaction with the corresponding alkyl halides. The newly synthesized derivatives were soluble in polar aprotic solvents without the addition of TBAF. Saponification was performed to demonstrate that alkylation was completely regioselective for carboxylate groups in preference to hydroxyl groups to form esters. We demonstrate the utility of these alginate esters to enhance aqueous solubility of the flavonoid naringenin by formation of solid dispersions.     

Assembly properties of tubulin after carboxyl group modification

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.     

Antibodies specific for 1-methylguanosine as a probe of tRNA conformation

Antibodies specific for 1-methylguanosine (m1G) were produced by immunization of rabbits with a bovine serum albumin conjugate of m1G. Antibodies specificity was determined by measuring the inhibition of binding of 3H-m1G trialcohol by various nucleosides or related derivatives. The relative affinities of the unpurified antibodies for various nucleosides showed that m1G trialcohol had an 8-fold higher affinity than m1G; further, guanosine and 2'-O-methylguanosine had at least a 500-fold lower affinity than m1G. The antibodies were purified on m1G-AH-Sepharose column and subsequently immobilized to Sepharose. Immobilized m1G antibodies quantitatively and exclusively retained m1G-containing oligonucleotides derived from ribonuclease A digests of 32P-labeled phage T4 tRNAPro. On the other hand, intact 32P-labeled T4 tRNAPro or its precursor RNA(s) did not bind to the same column. These findings indicate that at least a portion of m1G adjacent to the 3' end of the anticodon in intact T4 tRNAPro is not accessible for antibody binding.     

Thermostabilization of carboxymethylcellulase from Aspergillus niger by carboxyl group modification

The carboxyl groups of purified carboxymethylcellulase (CMCase) from Aspergillus niger NIAB280 were modified by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) in the presence of glycinamide for 15 min (GAM15) and glycinamide plus cellobiose for 75 min (GAM75). The half-lives of GAM15 at different temperatures were significantly enhanced whereas those of GAM75 were reduced as compared with the native CMCase. The activation energies of denaturation of native, GAM15 and GAM75 were 40, 35 and 59kJ mol respectively. Native CMCase and GAM15 showed no compensation effect, whereas native and GAM75 gave temperature of compensation of 44¡C. Gibb's free energy of activation for denaturation (DG*) of GAM15 was increased as compared with native CMCase. Surprisingly the entropies (DS*) of activation for denaturation were negative for native and GAM75 and decreased further for GAM15 between the temperature range of 45 to 65¡C. A possible explanation for the thermal inactivation of native and increased thermal stability of GAM15 is also discussed.     

Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme

The tRNA(Gm18) methyltransferase (TrmH) catalyzes the 2'-O methylation of guanosine 18 (Gua18) of tRNA. We solved the crystal structure of Thermus thermophilus TrmH complexed with S-adenosyl-L-methionine at 1.85 A resolution. The catalytic domain contains a deep trefoil knot, which mutational analyses revealed to be crucial for the formation of the catalytic site and the cofactor binding pocket. The tRNA dihydrouridine(D)-arm can be docked onto the dimeric TrmH, so that the tRNA D-stem is clamped by the N- and C-terminal helices from one subunit while the Gua18 is modified by the other subunit. Arg41 from the other subunit enters the catalytic site and forms a hydrogen bond with a bound sulfate ion, an RNA main chain phosphate analog, thus activating its nucleophilic state. Based on Gua18 modeling onto the active site, we propose that once Gua18 binds, the phosphate group activates Arg41, which then deprotonates the 2'-OH group for methylation.     

Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group

The role of carboxyl group in the catalytic action of xylanase (M_r 35 000) from an alkalothermophilic Bacillus sp. was delineated through iinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M⁻¹ min⁻¹. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar K_m and lower k_cat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Dehspande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172–177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp.     

Mitochondrial tRNA valine as a recurrent target for mutations involved in mitochondrial cardiomyopathies

The aim of this study was to identify the genetic defect in two patients having cardiac dysfunction accompanied by neurological symptoms, and in one case MRI evidence of cortical and cerebellar atrophy with hyperintensities in the basal ganglia. Muscle biopsies from each patient revealed single and combined mitochondrial respiratory chain deficiency. The complete mtDNA sequencing of both patients revealed two transitions in the mitochondrial tRNA(Val) gene (MT-TV) (m.1628C>T in Patient 1, and m.1644G>A in Patient 2). The functional and molecular analyses reported here suggest that the MT-TV gene should be routinely considered in the diagnosis of mitochondrial cardiomyopathies.     

A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K

Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.     

The nucleus as the site of tRNA methylation

Mouse L-cells were enucleated by exposure to cytochalasin B followed by centrifugation. The resulting karyoplasts, nuclei surrounded by a thin shell of cytoplasm and an outer cell membrane, and cytoplasts, the enucleated cell cytoplasm, were assayed for tRNA methyltransferase activity. The bulk of the enzyme activity was found to be localized in the nuclei. Analysis of the methylated nucleosides produced by the enzyme from the two sources showed that all the base-specific enzyme activities which are found in whole cell extracts were present in the nuclear extracts. The cytoplast extracts retained a low but detectable enzyme activity, which was composed predominantly of only two base-specific activities. This may represent tRNA methyltransferases of the mitochondria or may be cytoplasmic enzymes for late modification reactions.     

Identification of a site for carboxyl methylation in human alpha-globin

The human erythrocyte protein carboxyl methyltransferase modifies unusual protein D-aspartyl and L-isoaspartyl residues which arise spontaneously from internal rearrangements accompanying asparaginyl deamidation and aspartyl isomerization. A site of methylation associated with alpha-globin in intact cells has been identified by peptide mapping of radiolabeled globin isolated from human erythrocytes previously incubated with L-[methyl-3H]methionine. The site is located in a Staphylococcus V8 peptide containing residues 1-30 of alpha-globin. Two potential sources of methylation sites are present in this sequence at Asp-t and Asn-9.     

Triplex mediated delivery of a platinum complex to a specific DNA target site

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

Spectrophotometric titration of a single carboxyl group at the active site of ribonuclease T1.

Low-pH-induced difference spectra for ribonuclease T1, which were determined using a reference solution at pH 6, consisted of a shorter wavelength component from 270 to 285 nm that relfected an ionization having a pKa of 3.54 and a longer wavelength component above 285 nm that reflected an ionization having a pKa of 4.29. The temperature dependence of the pKa value for data at 300 nm is consistent with its representing the dissociation of a carboxyl group. In addition, the pKa determined at this wavelength significantly decreased at lower ionic strength. Similar experiments which were conducted using catalytically inactive gamma-carboxymethyl-Glu-58-ribonuclease T1 gave difference spectra having only the shorter wavelength component and were characterized by a single pKa of 3.53. It is concluded that the longer wavelength component of the difference spectra is due to the ionization of Glu-58. The pKa determined for this residue in the present study agrees with one found previously from kinetic studies which supports a role for Glu-58 in catalysis. Furthermore, the results suggest a model for the interaction of Glu-58 with histidine and tryptophan residues at the active site.