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PubMed1978

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.

Adenine↗
PubMed2004

The 2004 Canadian recommendations for the management of hypertension: Part III--Lifestyle modifications to prevent and control hypertension.

OBJECTIVE: To provide updated, evidence-based recommendations regarding the role of lifestyle modification in the treatment and prevention of hypertension. OUTCOMES: Lifestyle modification interventions including exercise, weight reduction, alcohol consumption, dietary modification, intake of dietary cations and stress management are reviewed. Antioxidants and fish oil supplements are also reviewed, although specific recommendations cannot be made at present. EVIDENCE: MEDLINE searches were conducted from January 2002 to September 2003 to update the 2001 recommendations for the management of hypertension. Supplemental searches in the Cochrane Collaboration databases were also performed. Reference lists were scanned, experts were contacted, and the personal files of the subgroup members and authors were used to identify additional published studies. All relevant articles were reviewed and appraised independently using prespecified levels of evidence by content and methodology experts. RECOMMENDATIONS: Key recommendations include the following: lifestyle modification should be extended to nonhypertensive individuals who are at risk for developing high blood pressure; 30 min to 45 min of aerobic exercise should be performed on most days (four to five days) of the week; an ideal body weight (body mass index 18.5 kg/m2 to 24.9 kg/m2) should be maintained and weight loss strategies should use a multidisciplinary approach; alcohol consumption should be limited to two drinks or fewer per day, and weekly intake should not exceed 14 standard drinks for men and nine standard drinks for women; a reduced fat, low cholesterol diet that emphasizes fruits, vegetables and low fat dairy products, and maintains an adequate intake of potassium, magnesium and calcium, should be followed; salt intake should be restricted to 65 mmol/day to 100 mmol/day in hypertensive individuals and less than 100 mmol/day in normotensive individuals at high risk for developing hypertension; and stress management should be considered as an intervention in selected individuals. VALIDATION: All recommendations were graded according to the strength of the evidence and voted on by the Canadian Hypertension Education Program Evidence-Based Recommendations Task Force. Individuals with irreconcilable competing interests (declared by all members, compiled and circulated before the meeting) relative to any specific recommendation were excluded from voting on that recommendation. Only those recommendations achieving at least 70% consensus are reported here. These guidelines will continue to be updated annually.

Adult↗
PubMed1992

Malondialdehyde modification of lipoprotein(a) produces avid uptake by human monocyte-macrophages.

Increased plasma levels of the apoB-100-containing lipoprotein(a) (Lp(a)) are associated with an increased risk for atherosclerosis and myocardial infarction, but the mechanisms by which lipoprotein(a) may accelerate these processes remain obscure. In this study we have investigated the impact of the association of apoprotein(a) with the low density lipoprotein (LDL)-like Lp(a) particle upon specificity of receptor recognition after lipoprotein modification by malondialdehyde or transition metal-induced oxidation. We have determined that radioiodination labels both apoprotein components of Lp(a), that malondialdehyde modification produces an anionic lipoprotein comparable to native Lp(a) in Stokes' radius, and that N,N'-disubstituted 1-amino-3-iminopropene derivatives preferentially cross-link apoprotein(a) to apoB-100 protein. Like LDL, native Lp(a) is recognized in human monocyte-macrophages by the LDL receptor. Like LDL, progressive modification of Lp(a) by malondialdehyde abolishes lipoprotein recognition by the LDL receptor and produces uptake and hydrolysis by the scavenger receptor of human monocyte-macrophages. We propose that intimal retention of Lp(a) by extracellular components of the atherosclerotic reaction places the lipoprotein in a microenvironment favoring subsequent peroxidative modification. The chronic production of lipid peroxide-modified Lp(a) together with unmitigated cellular clearance by scavenger receptors may contribute to the accumulation of lipoprotein-derived lipid in macrophage-derived foam cells of the atherosclerotic reaction.

Cells, Cultured↗
PubMed2005

[Preliminary study on graft versus leukemia effect of camouflage of mice bone marrow transplantation with methoxy polyethylene glycol modification].

OBJECTIVE: To study if methoxy polyethylene glycol modification (mPEG) affects grafts versus leukemia (GVL) when donor bone marrow mononuclear cells are camouflaged with mPEG in murine bone marrow transplantation (BMT). METHODS: Sixty (BALB/c(H-2d) x 615(H-2k))F(1) mice were divided into four groups randomly. Mice in group A were only irradiated with 8.0 Gy (60)Cogamma, and mice in the other groups were inoculated intraperitoneally with 1 x 10(6) L615 cells 3 days before irradiation with the same dose (60)Cogamma. BALB/c(H-2d) mice were sacrificed and bone marrow cells and spleen cells were collected. The bone marrow cells (1 x 10(7)) were mixed with the spleen cells (1 x 10(7)), which were camouflaged or not camouflaged with mPEG, were transplanted into irradiated leukemia mice in C and D groups. GVL effects were assessed by L615 cells proportion in peripheral blood, histopathological changes and survival time. RESULTS: Severe GVHD was observed in group C (without mPEG modification), and the mice rapidly died, the mean survival time was 6.9 days. The mice in irradiated group (group B) with leukemia cell died of leukemia. The average survival time of group D (with mPEG modification) was 24.2 days, which was longer than that of the other groups (P < 0.05), and the survival rate of group D (27%) was significantly higher than that of the others (P < 0.05), 11 mice (11/15) died of leukemia and the others were still alive. CONCLUSION: The camouflage with mPEG modification is capable of preserving GVL effect and preventing GVHD in mice BMT.

Animals↗
PubMed2005

Discovery of disease-induced post-translational modifications in cardiac contractile proteins.

Post-translational modifications to myofilament proteins are essential for the regulation of cardiac function in both normal and disease states. Recent developments in the field of proteomics have produced a variety of useful tools to study protein modifications. Current applications of proteomic technologies in the study of modifications to myofilament proteins are summarized. The separation, identification and characterization of myofilament modifications using gel electrophoresis and mass spectrometry approaches are discussed. Each method is illustrated and evaluated with selected examples, and several powerful emerging technologies are assessed.

Animals↗
PubMed2005

[Modifications of actinomycin D structure as example of actinomycins structure-activity relationship].

For over 60 years, actinomycins, well-known antibacterial and anticancer antibiotics, have been the subject of the scientific research. These compounds exhibit high toxicity and therefore are not widely used in the chemotherapeutic treatment of antibacterial and antifungal diseases. However, actinomycin D, the best-known compound from the actinomycin group, has been introduced into clinical practice as an anticancer drug. Actinomycin D, together with 7-amino-actinomycin D, also became a useful tool in biochemistry and molecular biology. The isolation, production, chemistry, and biological and clinical use of the actinomycins have been thoroughly investigated. Many derivatives of actinomycins, differing in chemical structure as well as biological activity, have been isolated and synthesized and their modifications involved not only the chromphoric phenoxazone ring, but also two cyclic pentapeptide lacton rings. Modifications of the actinomycins' chromophore mainly concerned introducing an amino group in position 2 and a carbon atom in position 7, but also modifications in positions 4, 6, and 8 of the phenoxazone ring. The actinomycin peptide moiety was mainly modified by replacement of amino acids in the pentapeptide rings and also by the synthesis of actinomycin derivatives with open peptide lacton rings. These modifications enabled separating the elements in the actinomycin structure which are responsible for the biological activity of these compounds. That was key information for recognizing the performance of these compounds, and an important way of planning effective new chemotherapeutics.

Animals↗
PubMed2003

[Chemical modification of Streptococcus suis type 2 haemolysin].

The haemolysin of Streptococcus suis type 2 Jiangsu isolate was purified. Effects of protein modification reagents on the haemolysin activity were detected. The haemolysin was not affected by PCMB, succinic anhydride (SA), EDC, and N-AI modification, and it indicated that sulfhudryl groups, amino groups, carboxyl groups and tyrosine residues were not essential to haemolysin activity. The haemolysin activity was significantly decreased after NBS, DEPC, 2, 3-Diacetyl, H2O2 modification and was greatly increased after DTT modification. The results indicated that tryptophane residues, histidine residues, arginine residues and disulfides groups seemed to be essential to the haemolysin activity.

Bacterial Proteins↗
PubMed2005

[Effect of acid-base two steps surface modification on the adsorption of Cr(VI) onto activated carbon].

Effect of HNO3-NaOH two steps surface modification on the adsorption of Cr(VI) from aqueous solution onto activated carbon was evaluated. Activated carbon was oxidized in HNO3 aqueous solution at first (AC1), then treated in the mixture of NaOH and NaCl solution (AC2). Batch equilibrium and continuous adsorption experiments were conducted to determine the adsorption characteristics. Boehm titration method, element analysis were used to characterize the surface properties. N2/77 K adsorption isotherm method was used to characterize the pore structure. The results reveal that adsorption capacity and adsorption rate increase significantly, which in the following order: AC2>AC1>AC0. Surface modification caused BET surface area decreased and the total number of surface oxygen acid groups increased. First oxidation modification in HNO3 solution produced positive acid groups on the surface of activated carbon. Subsequent 2nd modification replaced H+ of carbon surface groups by Na+, the acidity of AC2 was decreased. The main cause of higher Cr(VI) adsorption capacity and rate for AC2 was the more oxygen surface acid groups, and suitable solution pH provide by surface groups.

Adsorption↗
PubMed2005

Screening for transglutaminase-catalyzed modifications by peptide mass finger printing using multipoint recalibration on recognized peaks for high mass accuracy.

Detection of posttranslational modifications is expected to be one of the major future experimental challenges for proteomics. We describe herein a mass spectrometric procedure to screen for protein modifications by peptide mass fingerprinting that is based on post-data acquisition improvement of the mass accuracy by exporting the peptide mass values into analytical software for multipoint recalibration on recognized peaks. Subsequently, the calibrated peak mass data set is used in searching for modified peptides, i.e., peptides possessing specific mass deviations. In order to identify the location of Lys- and Gln-residues available for transglutaminase-catalyzed isopeptide bond formation, mammalian small heat shock proteins (sHsps) were screened for labeling with the two hexapeptide probes GQDPVR and GNDPVK in presence of transglutaminase. Peptide modification due to cross-linking of the GQDPVR hexa-peptide probe was detected for C-terminal Lys residues. Novel transglutaminase-susceptible Gln sites were identified in two sHsps (Q31/Q27 in Hsp20 and HspB2, respectively), by cross-linking of the GNDPVK hexapeptide probe. Deamidation of specific Gln residues was also detected, as well an isopeptide derived from intramolecular Gln-Lys isopeptide bond formation. We conclude that peptide mass fingerprinting can be an efficient way of screening for various posttranslational modifications. Basically any instrumentation for MALDI mass spectrometry can be used, provided that post-data acquisition recalibration is applied.

Amino Acid Sequence↗
PubMed1991

The effects of p-hydroxymercuribenzoic acid modification and heat treatment on the CuA reduction potential of cytochrome c oxidase.

p-Hydroxymercuribenzoic acid modification of cytochrome c oxidase converts the CuA center into a type 2 copper site while heat treatment of the oxidase in lauryl maltoside can transform CuA almost stoichiometrically (greater than 90%) to a blue type 1 copper site. These modifications of the protein have previously been shown to have a profound effect on the dioxygen reduction and proton pumping activities of the enzyme (Li, P. M., Morgan, J. E., Nilsson, T., Ma, M., and Chan, S. I. (1988) Biochemistry 27, 7538; Nilsson, T., Gelles, J., Li, P. M., and Chan, S. I. (1988) Biochemistry 27, 296; Sone, N., and Nicholls, P. (1984) Biochemistry 23, 6550). In this work, the intrinsic reduction potentials and the midpoint reduction potentials of the "CuA" site in both these modified oxidases have been measured under various conditions in order to clarify the intramolecular electron transfer pathways in these systems. The study reveals that the CuA intrinsic reduction potential decreases by almost 150 mV upon p-hydroxymercuribenzoic acid modification whereas it increases by 100 mV upon heat treatment. In addition, the redox interactions between CuA and the remaining metal centers are perturbed upon CuA modification. It is argued that these results bear on the role of CuA in the proton-pumping paradigm of cytochrome c oxidase.

Animals↗
PubMed1975

The carboxylic acid groups of bovine luteinizing hormone. The effects of their modification on receptor site binding and subunit-subunit interaction.

The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies.

Amino Acid Sequence↗
PubMed1990

[Expression of genes controlling modification of amylase in hybrids and original representatives of a cattle subfamily (Bovinae)].

Modifications of similar type were noted in the number of multiple AMY-1 forms of amylase isozyme for various species of Artiodactyla. It is supposed that these modifications are linked to periodical activation during evolution of genes responsible for modification of the molecules. Data obtained on Bovinae hybrids testify to this point of view. Inactivation of active and reinactivation of "silent" genes responsible for modification of AMY-1 molecules are observed in hybrids of bison x cow and bison x aurochs combinations.

Amylases↗
PubMed1991

[Complementary addressed alkylation of 16S rRNA of Escherichia coli by 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligodeoxyribonucleotides. V. Study of the factors affecting selectivity of modification].

We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribonucleotide) stabilizing duplex RNA.reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16s rRNA was determined. The temperature rise from 20 to 40 degrees C brought about a 1.5-fold increase in the relative extent of modification at the target site 771-781. In the presence of oligonucleotide-effector, which is a full complementary copy of the 782-789 fragment of 16S rRNA (reagent concentration is 1 x 10(-6) M), the selectivity of the RNA modification at the target site is doubled and a high level of the modification is retained. When the reagent concentration in the reaction mixture was decreased down to 1 x 10(-7) M, the same level of selectivity was achieved without the oligonucleotide-effector. Under these conditions, however, a drastic (20-fold) drop of the level of the 16S rRNA alkylation was observed.

Alkylation↗
PubMed1992

Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Structural and functional changes.

As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50%. Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.

Glucosephosphate Dehydrogenase↗
PubMed1991

Strategies for identification of covalent xenobiotic modifications in proteins by mass spectrometry.

The structural elucidation of covalent xenobiotic modifications in proteins is a powerful approach to identify compounds that may be responsible for genotoxic damage in humans. Many proteins, such as hemoglobin can be readily obtained in relatively large quantities. Since covalent modifications in proteins are not repaired, xenobiotic damage can accumulate over the lifetime of the protein and reveal exposure over extended time periods. Recently developed mass spectrometric methods are ideally suited to tackling protein structural problems involving covalent modifications. A variety of strategies to identify xenobiotic covalent modifications in proteins using mass spectrometry are discussed.

Amino Acid Sequence↗
PubMed1990

ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro.

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [alpha-32P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [alpha-32P]ATP- and [alpha-32P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [alpha-32P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes (Moss, J., Watkins, P.A., Stanley, S.J., Purnell, M.R., and Kidwell, W.R. (1984) J. Biol. Chem. 259, 5100-5104). A 32P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H3 32PO4-grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification.

Adenosine Diphosphate Ribose↗
PubMed1991

Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification.

Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1991

Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis.

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.

Adenylosuccinate Synthase↗