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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↗
PubMed1991

Design and statistical issues of the Modification of Diet in Renal Disease Trial. The Modification of Diet in Renal Disease Study Group.

The Modification of Diet in Renal Disease Trial is a multicenter randomized clinical trial for men and women aged 18-70 years with chronic renal disease who are not on dialysis and who have not had a kidney transplant. Study participants are randomized in a 2 x 2 factorial design to diets containing different amounts of protein and phosphorus and to two levels of blood pressure control. The prescribed modifications differ depending on the level of a patient's kidney function. The primary outcome variable to compare diet or blood pressure groups is each patient's slope (or the change) in glomerular filtration rate with time. This paper describes the study design with particular emphasis on sample size determination. Special statistical analysis issues that arise with slope as the outcome are also discussed.

Adolescent↗
PubMed1985

Investigation of restriction-modification enzymes from M. varians RFL19 with a new type of specificity toward modification of substrate.

The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported. Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence. The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine. This type of modification protects the substrate from R.MvaI cleavage. 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage. R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine. N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure.

5-Methylcytosine↗
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↗
PubMed1988

UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification.

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.

Adenosine Diphosphate↗
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↗
PubMed1990

Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification.

The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of Leu129 or Asp119.

In the cross-linking reaction of lysozyme between Leu129 (alpha-COO-) and Lys13 (epsilon-NH3+) using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a side reaction of the peptide bond inversion from alpha to beta between Asp101 and Gly102 was greatly reduced by addition of beta-(1,4)-linked trimer of N-acetyl-D-glucosamine [(NAG)3]. When methylamine or 2-hydroxyethylamine was further added, the extent of the cross-link formation was decreased and the derivative where the alpha-carboxyl group of Leu129 was modified with the amine was newly obtained. On the other hand, when ammonia was added, the beta-carboxyl group of Asp119 instead of the alpha-carboxyl group was mainly amidated. From these results, the presence of a salt bridge between Asp119 and Arg125 besides that between Lys13 and Leu129, is proposed. Enzymatic activities of the derivatives prepared here indicated that the modification of the alpha-carboxyl group reduced the activity to approximately 90% of that of native lysozyme. Des-Leu129 lysozyme, which lacks Leu129, also showed approximately 90% of the activity of native lysozyme. Therefore, the salt bridge between Lys13 and Leu129 may play some role in maintaining the active conformation of lysozyme.

Amino Acid Sequence↗
PubMed1990

Inactivation of bacteriophage T7 DNA-dependent RNA polymerase by 5'-p-fluorosulfonylbenzoyladenosine. Identification of the modification site and the effect of the modification on enzyme action.

Bacteriophage T7 RNA polymerase was covalently modified by 5'-[4-fluorosulfonyl)benzoyl]adenosine (4-FSO2BzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the phi 10 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4-FSO2BzAdo within the covalent enzyme-inhibitor complex restores the RNA synthesis at a lower rate. Sequence studies show that Lys172 is the target of modification by 4-FSO2BzAdo. This residue, which is situated in the polypeptide region connecting two domains of RNA polymerase, was shown to be the primary site of the limited proteolysis occurring in vivo [Ikeda, R. A. & Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799]. We propose that Lys172 is located outside the active site. Once this residue has reacted with 4-FSO2BzAdo, the nucleoside moiety of the analog is fixed in the NTP-binding site of the active centre and prevents binding of the substrates. Here, Lys172 per se is not important for the activity but serves as an 'anchor' for binding of the inhibitor.

Adenosine↗
PubMed1990

Covalent modification and metabolic control analysis. Modification to the theorems and their application to metabolic systems containing covalently modifiable enzymes.

A study of the sensitivity properties of metabolic systems containing covalently modifiable enzymes and cascades has been carried out with the aid of metabolic control analysis. We have considered how the theorems of metabolic control analysis must be modified to take into account covalently modifiable enzymes, and have used these results to investigate the effects of increasing the total amount of modifiable enzyme. The sensitivity of system variables to an effector acting through a covalent-modification cycle has also been investigated.

Energy Metabolism↗
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↗
PubMed1990

Towards an artificial cornea: surface modifications of optically clear, oxygen permeable soft contact lens materials by ammonia plasma modification technique for the enhanced attachment and growth of corneal epithelial cells.

The advent of high water content, oxygen permeable contact lens materials has made the intracorneal implants more feasible. A major obstacle encountered is the regrowth of a stable epithelium over the implant. Therefore, ammonia gaseous plasma modification technique was used to modify the surface chemical properties of soft contact lens material such as poly(2-hydroxyethyl methacrylate and methacrylic acid), PHEMA-MAA copolymer, in an attempt to enhance the cell attachment and growth of rabbit corneal epithelial cells.

Ammonia↗
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↗
PubMed1987

[Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. IX. Cleavage of substrates with point modifications in the recognition site and flanking sequences].

Ability of the EcoRII restriction endonuclease to cleave 14-base-pair DNA duplexes with nucleotide substitutions in the recognition site CCA/TGG and in the adjacent base pair has been studied. Modifications leading to a local change in the substrate conformation (rU residue in and outside the recognition site, A.A- or A.C-pairs in the flanking sequence) reduce the rate of hydrolysis, the effect being maximal when the modified base pair is outside the recognition site. No digestion occurs when the internal dC-residue of the recognition site is 5-methylated in one or both strands. Replacement of dT residue in the EcoRII recognition site by dfl5U residue results in a dramatic inhibition of hydrolysis. Km and kcat for the cleavage of 14-base-pair DNA duplex have been determined. The cleavage rate of the dT-containing strand of the recognition site in 1.5 fold higher comparing with the dA-containing strand. The cleavage of both strands of the substrate by EcoRII endonuclease is confirmed to proceed in one enzyme-substrate complex.

Base Sequence↗