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At least 19 recordsLinked to original sources
PubMed1992

Non-specific influence of chemical modification upon the properties of antithrombin III:modification of carboxyl groups.

The ability of antithrombin III to inhibit thrombin was observed to be rapidly inactivated upon specific modification of carboxyl groups. The loss of activity, upon treatment with nitrotyrosyl ester in the presence of 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, was concomitant to the incorporation of 2 moles of nitrotyrosine per mole of inhibitor indicating the modification of only two carboxyl groups. Moreover, the modification occurred with loss, also, of the ability of the native protein to bind tightly to heparin. The modified antithrombin III retained a reduced affinity for heparin (eluting at 0.3M NaCl from heparin Agarose) and was observed to be a competitive inhibitor of the heparin-dependent rate of inhibition of thrombin by native antithrombin III. However, FAB-MS (fast atom bombardment mass spectroscopy) analysis of digests of modified material gave no indication that modification was localized to specific Asp or Glu residues. It is concluded that the loss of activity is due to deleterious change in conformation during modification. These findings, together with our previous report upon tryptophan modification of antithrombin III [1] suggest that the nature of the molecule is such that considerable care must be taken in interpretation of results when investigating the structure/function relationships of this protein by chemical modification.

Antithrombin III↗
PubMed1975

Restriction and modification in B. subtilis. The biochemical basis of modification against endo R. Bsu R restriction.

The content of 5-methylcytosine (5MC) and 6-methyladenine (6MA) in modified and nonmodified DNAs from B. subtilis and B. subtilis phage SPP1 were determined. Non-modified SPP1-O DNA contains about 15 5MC residues/molecule. Each modified SPP1-R DNA molecule carries 190 modification specific methyl groups. This number is sufficient to account for modification of the 80 restriction sites in SPP1 DNA (Bron and Murray, 1975) against endo R-Bsu R, assuming each modified site contains two 5MC residues. Resistance of SPO1 DNA against endo R-Bsu R restriction both in vivo and in vitro is probably not due to methylation of endo R-Bsu R recognition sites.

Bacillus subtilis↗
PubMed1975

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.

Cell-Free System↗
PubMed1975

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.

Amino Acids↗
PubMed1976

Selective chemical modification of Escherichia coli elongation factor G. N-Ethylmaleimide modification of a cysteine essential for nucleotide binding.

Escherichia coli Elongation Factor G is inhibited ireversibly by the chemical modification of 1 cysteine residue with N-ethylmaleimide. At pH 5.2, this cysteine is approximately 130 times more reactive than beta-mercaptoethanol toward N-ethylmaleimide. Inhibition is not prevented by either the ribosome or GTP alone at concentrations approximately equal to that of Elongation Factor G, but in combination they reduce the inhibition by 50%. Increasing the stability of the Elongation Factor G-ribosome-GDP complex by the addition of fusidec acid, completely protects against N-ethylmaleimide inhibition. The modified protein cannot form either the Elongation Factor G-ribosome-GMP-P(CH2)P or the Elongation Factor G-ribosome-GDP-fusidic acidcomplex. However, the modification had no effect on its ability to form the Elongation Factor G-ribosome complex. These results suggest that the cysteine residue modified by N-ethylmaleimide is at or near the nucleotide binding site.

Binding Sites↗
PubMed1979

[Chemical modification of proteins. 5. Modification of the 11-S-globulin from sunflower seed by reaction with dialdehyde starch].

Dialdehyde starch (DAS) reacts unspecifically with the amino acid residues of the 11 S globulin from sunflower seed. The modification of the protein causes a decrease of the content of each amino acid. Their blocking reaches maximum values at high pH levels (9,5) and high concentration of protein (5%). Especially high reactivity is shown by arginine as well as by the hydrophobic amino acids isoleucine, valine, and proline, and furthermore by histidine, lysine, asparagine (aspartic acid), and glutamine (glutamic acid). By reaction with DAS at pH 8.0 70% of the amino groups are blocked within 6 h; on the contrary, glyoxale blocks only 30% of the amino groups. Owing to the blockage of charged amino acid groups, a shift of the isoelectric point of the protein to a lower pH (4,3-4,4) takes place; this effect can be followed for 2 days. As a result of the reaction with DAS, only small amounts (10-15%) of intermolecular crosslinkage products with sedimentation coefficients of 17 S and greater than 17 S were formed. But by means of SDS-gel electrophoresis, dimers and trimers of the polypeptide chains in the protein were detected.

Aldehydes↗
PubMed1992

C-terminal modifications of nonpeptide renin inhibitors: improved oral bioavailability via modification of physicochemical properties.

We describe the development of a series of soluble, potent, and bioavailable nonpeptide renin inhibitors. These inhibitors derived from a series of novel nonpeptide renin inhibitors which were recently identified in our laboratories, by alteration of the nature of the C-terminus (P2') of the molecules. Introduction of basic substituents into modified hydroxyethylene dipeptide isosteres gave inhibitors with improved solubility as well as improved potency against human plasma renin. In addition, these modifications produced inhibitors which displayed markedly improved intraduodenal bioavailability in both the ferret and cynomolgus monkey. We also present data which demonstrate excellent efficacy in the monkey for A-74273 (65), with an intraduodenal bioavailability of 16 +/- 4% in the monkey, compared to 1.7 +/- 0.5% for the dipeptide renin inhibitor enalkiren (A-64662, 75). A-74273 is an example of a nonpeptide inhibitor which possesses a good balance of the desirable properties of potency, solubility, and lipophilicity and which is well absorbed into the intestine.

Administration, Oral↗
PubMed1976

Chemical modification of crude timothy grass pollen extract. I. Antigenicity and immunogenicity changes following amino group modification.

Glutaraldehyde modification was found to reduce the allergenic potency of crude timothy pollen extracts yet even highly substituted materials retained the capability of inducing the formation of allergen-specific antibody in animals. Experiments showed that these antibodies were capable of blocking skin test reactions to native timothy allergens in pollen-sensitive human volunteers. The value of glutaraldehyde-modified allergen extracts for use in desensitization vaccine therapy is discussed.

Aldehydes↗
PubMed1979

[Modelling modification of chemical mutagenesis in human cells. III. Linear index of protection as the standardized criterion of modification].

The effect of substances with radioprotective activity, APAETP 2,3 (aminopropylaminoethylthiophosphoric acid 2,3), APAETP 3,3 and cystaphos, on chromosome aberrations, induced by thioTEPA in the culture of human lymphocytes was investigated. It is shown that the obtained curves "concentration -- effect" for thioTEPA can be described by equations rho = 1 -- e-(KC + alpha)2 and X = E -(KC + alpha)2 --1 for aberrant cells and for chromosome breaks in the presence of the investigated substances. On the basis of comparison of angle coefficients of regression the unificated characteristic of the efficiency of chemical mutagenesis is proposed: the linear protection index (LPI), with generalizes the effect of modificators in chemical mutagenesis.

Cells, Cultured↗
PubMed1977

Selective chemical modification of Escherichia coli elongation factor G: butanedione modification of an arginine essential for nucleotide binding.

Treatment of Escherichia coli elongation factor G with the arginine reagent, 2,3-butanedione, leads to the inactivation of the enzyme when performed in sodium borate buffers. The inhibition follows pseudo-first-order kinetics until 95% of the activity has been lost and further incubation results in complete inhibiton. Removal of the borate by exhaustive dialysis results in the restoration of approximately 85% of the original activity. The pH dependence of the reaction suggests that the ionization of a group in the protein with a pKa of approximately 8.8 facilitates the reaction with butanedione. A reaction order of 1.01 +/- 0.13 was calculated for the inhibition reaction, indicating that the incorporation of one butanedione per elongation factor G results in the inactivation of the enzyme. The kinetics of inhibition in the presence of GTP indicate that the elongation factor G-GTP complex is refractory to butanedione inhibiton. Elongation factor G which has been partially inactivated by butanedione has the same apparent Km for GTP as does the native enzyme. These results indicate that elongation factor G contains only one essential arginine residue which is reactive with butanedione and that this residue is located at its nucleotide binding site.

Butanones↗
PubMed1992

Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.

Amino Acid Sequence↗
PubMed1976

Studies of enzyme-catalyzed modification of proteins. I. Tyrosinase-catalyzed modification of asparaginase.

Asparaginase [EC 3.5.1.1.] of Escherichia coli, an anti-tumor enzyme, was inactivated in a time-dependent fashion by mushroom tyrosinase [EC1.14.18.1.]. The inactivation did not proceed, however, when heat-inactivated tyrosinase was used. Exculusion of the atmospheric oxygen or addition of diethyldithiocarbamate, a copper selective chelating agent, prevented the inactivation. The difference absorption spectrum of tyrosinase-inactivated asparaginase versus intact asparaginase exhibited the appearance of marked absorption peaks at 300 and 350 nm. These results indicate that the tyrosyl residue(s) of asparaginase, which is essential for the activity is enzymatically modified by tyrosianes.

Asparaginase↗
PubMed1992

Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.

In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.

Amino Acid Sequence↗