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PubMed1993

Reversible modification of cysteine residues of NADPH-cytochrome P-450 reductase.

A reversible chemical modification of SH-groups of NADPH-cytochrome P-450 reductase is the subject of the present study. The enzyme was modified using first biradical RS-SR (R being the imidazolidine derivative) and a new affinity reductase inhibitor beta-cystamine adenosine diphosphate (ANSSN). These reagents were shown to be covalently bound to reductase SH-groups via the reaction of thiol-disulfide exchange resulting in the loss of reducing activity for cytochrome c. NADP+ protected reductase from inactivation and decreased the extent of the modification by RS-SR. The modification of reductase was reversible: the modified enzyme was partially reactivated with glutathione and dithiothreitol. The method proposed can be used to study both the reductase structure and the reversible inhibition of microsomal monooxygenase systems.

Adenosine Diphosphate↗
PubMed1994

Stimulation of cell-mediated low-density lipoprotein oxidative modification by oncostatin M.

The effect of oncostatin M on low-density lipoprotein oxidative modification by human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation products content and the electrophoretic mobility of the particle. In the range of concentrations from 10 to 30 ng/ml, oncostatin M induced a dose-dependent increase in LDL oxidation. The LDL degradation by J774 macrophage-like cells was also stimulated. Preincubation of cells with oncostatin M also enhanced the subsequent LDL modification. This effect was accompanied by a parallel increase in superoxide anion release by cells. Since oncostatin M is produced by immune cells, the described effect suggests a relationship between infection, inflammation and LDL oxidative modification.

Cell Line↗
PubMed1996

ADP-ribosylation of wild-type p53 in vitro: binding of p53 protein to specific p53 consensus sequence prevents its modification.

We have recently reported that mutant but not wild-type (wt) p53 protein was ADP-ribosylated in primary rat cells overexpressing the temperature-sensitive murine p53val135 gene. To examine whether the lack of susceptibility to modification is a specific feature of p53val135 adopting wt conformation or rather a general property of this tumor suppressor protein, we have studied ADP-ribosylation of wt p53 of different origin in vitro using semi-purified poly(ADP-ribose) transferase (pADPRT). In vitro pADPRT modified human and mouse wt p53 and p53val135. Under limiting substrate concentration, the molar mass of ADP-ribosylated p53 was only slightly altered. Chase experiments with high NAD concentration resulted in the formation of poly(ADP-ribosyl)ated p53 protein shifted to 64 kD. However, preincubation of wt p53 proteins with a p53 consensus sequence resulting in complex formation abolished the modification of wt p53. This indicates that in the cellular environment the specific DNA binding of wt p53 prevents its covalent modification by poly(ADP-ribose).

Adenosine Diphosphate Ribose↗
PubMed1996

Differential modification of activities of the high-affinity and low-affinity insulin receptors of 3T3-L1 fibroblasts by phosphonolipids in vivo.

The low-affinity and high-affinity forms of the insulin receptor respond differently to modifications of cellular phospholipid content in mouse 3T3-L1 fibroblasts in vivo. When cells are cultured with 2-aminoethylphosphonate the resulting phosphonolipid, which has previously been demonstrated to prevent the insulin-induced differentiation of the fibroblasts into adipocytes [J. D. Smith et al., Biochem. Arch. 8, 339-344 (1992)] results in alterations in both the affinity for insulin and receptor number of the low-affinity receptor while leaving the high-affinity receptor unaffected. That this phospholipid modification induces a specific change in the cellular insulin effect is demonstrated by the lack of alteration in the mobilization of GLUT-4 and glucose transport in the lipid modified cells. The results suggest that this specific cellular phospholipid modification will be useful in dissecting the specific functions of the two forms of the mammalian insulin receptor.

3T3 Cells↗
PubMed2000

Modification of cullin-1 by ubiquitin-like protein Nedd8 enhances the activity of SCF(skp2) toward p27(kip1).

The periodic expression of cell cycle proteins is important for the regulation of cell cycle progression. The amount of CDK inhibitor, p27(kip1), one such protein, seems to be regulated by the ubiquitin-proteasome system. The ubiquitin ligase (E3) toward p27(kip1) is thought to be SCF(skp2). The activity of SCF(skp2) was increased by the addition of Roc1 protein to the complex. Furthermore, the ubiquitination of p27(kip1) seemed to be dependent on the phosphorylation of T187 of p27(kip1) because the mutant T187A was not ubiquitinated at all in an in vitro ubiquitination system. Cullin-1, a component of SCF, is modified by ubiquitin-like protein Nedd8. The modification site of cullin-1 was shown to be K696 because the K696R mutant was not modified. When the effect of the Nedd8 modification on the SCF(skp2) activity toward p27(kip1) was investigated, the activity was markedly decreased by using the Nedd8-unmodified mutant cullin-1 (K696R), indicating that the modification may play an important role on the SCF(skp2) activity toward p27(kip1).

Amino Acid Sequence↗
PubMed2002

Modification of histidine (B10) is the causative agent for a superactive form of insulin.

The site of modification that is responsible for the formation of superactive insulin (ILM) was determined. The insulin derivative was prepared by treatment of insulin-Sepharose with ammonium bicarbonate. It was found that the insulin was bound to the resin through histidine B10, His (B10), and its ammonium bicarbonate-mediated release resulted in an insulin analog in which His (B10) was modified on the imidazole ring. This modification was reversible upon storage, resulting in normal levels of insulin activity. Amino acid analysis of a peptide containing this modified histidine revealed some aspartic acid. Since Asp (B10) insulin is also superactive, the observed superactivity may thus stem from either modification of the histidine or its conversion to aspartic acid.

Amino Acid Substitution↗
PubMed2001

Fine structural modifications of liver, pancreas and brown adipose tissue mitochondria from hibernating, arousing and euthermic dormice.

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia-hibernation-arousal cycle.

Adipose Tissue, Brown↗
PubMed2002

Modification of glycosylation reduces microvilli on rat liver epithelial cells.

Effect of glycosylation modification on the shape of microvillus was investigated on rat liver epithelial cell line WB-F344. Since rat ER alpha-mannosidase has 89% identity to human 6A8 alpha-mannosidase, we used an antisense 6A8 cDNA fragment to inhibit expression in WB-F344 cells. Cells were transfected with antisense 6A8, the relevant sense fragment or the mock plasmid. Genomic PCR for neo(R) demonstrated integration of the transfected gene into host DNA. Enzymatic activity assay on p -nitro-phenyl-alpha-D-mannopyranoside showed suppression of ER alpha-mannosidase expression in the cells transfected with antisense 6A8. Concanavalin A binding to these cells was enhanced, indicating a modification in glycosylation. Number reduction and blunting of microvillus on these cells was observed. Transduction with the sense fragment or the mock had no effect. Cells with suppressed ER alpha-mannosidase expression grew slower in culture. Our results indicate an obvious effect of glycosylation modification on microvilli, which might be related with malfunctioning of cells.

Animals↗
PubMed1993

Chemical modification of alpha crystallin.

Calf lens alpha-crystallin was isolated and the lysine residues were extensively modified with a variety of chemical agents. The effect of these modifications on elastase inhibitor activity, apparent molecular size, antibody reactivity and solubility were determined. The addition of either a methyl group or a threose residue did not alter the charge on the lysine residues and had little or no effect on either inhibitor activity or apparent molecular size. The introduction of a negative charge by either carboxymethylation or citraconylation caused a marked decrease in size and an almost complete loss of inhibitor activity. The introduction of a hydrophobic residue by reaction with either a trinitrobenzenesulfonic acid or Bolton Hunter reagent caused a slight increase in size, but a 70% increase in elastase inhibitor activity. Reaction with fluorescamine resulted in the dissociation of alpha-crystallin in a 200-kDa species, yet caused a two to four-fold increase in elastase inhibitor activity, which was similar to the activity of the water-insoluble fraction isolated from aged human lens and cataract. Several of these modified alpha-crystallins were compared for reactivity with a polyclonal alpha-crystallin antiserum using a quantitative slot blot assay. Charge neutral modifications resulted in a two to three-fold loss of antibody recognition, whereas the other preparations showed an almost complete loss of antigenic activity. None of the modifications caused the alpha-crystallin to precipitate at higher salt concentrations (0.3 M) with the exception of threose which caused a 30% decrease in soluble protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Suppression of phase separation in bovine gamma IV crystallin solutions: effect of modification by charged versus uncharged polar groups.

gamma IVa Crystallin, the ocular lens protein with the highest critical temperature (Tc) for phase separation, has been chemically modified with N-ethylmaleimide (NEM) at neutral pH. NEM, a polar but uncharged modifier reacts with the cysteine residues of the protein. A maximum of 80-85% of the thiol groups are modified and the phase separation temperature is lowered by about 10 degrees C at a protein concentration of 30 mg ml-1. The coexistence curve of the NEM-modified protein with nearly four out of the six cysteines modified was determined and compared with that obtained by modifying the protein with a charged reagent, N-bromoacetylethanolamine phosphate (NBAEP). NBAEP modifies both cysteine and methionine residues of gamma IVa crystallin. The results of these two modification studies indicate that for the same total degree of modification of the sulfur-containing residues, the suppression in Tc due to the charged NBAEP is at least twice as large as that due to the polar but uncharged NEM. In order to obtain some measure of the relative hydrophilicities of the two modifiers, we have estimated the free energies of hydration of the nonionic segments of NEM and NBAEP, using structural additivity schemes. We find that the intrinsic hydrophilicities of the nonionic segments of these two modifiers are nearly equal. Hence the much larger suppression of Tc resulting from NBAEP modification is most probably due to the strongly polar, doubly charged phosphate group.

Amino Acids↗
PubMed1999

Prevention of naphthalene-1,2-dihydrodiol-induced lens protein modifications by structurally diverse aldose reductase inhibitors.

The effects of aldose reductase inhibitors on lens protein modifications induced by naphthalene-1,2-dihydrodiol were investigated in vitro to confirm the role of aldose reductase on naphthalene cataract formation. HPLC analysis of naphthalene-1, 2-dihydrodiol incubated with aldose reductase and NAD+indicated the formation of a metabolite peak corresponding to 1,2-naphthoquinone. Soluble proteins from rat lenses prepared by gel filtration of crude lens extracts through Sephadex PD-10, incubated with naphthalene-1, 2-dihydrodiol in the presence of NAD+displayed an absorbance ca 450 nm and their spectra were essentially identical to those of 1, 2-naphthoquinone-protein adducts. Similar spectra were also obtained from proteins isolated from the intact rat lens after in vitro incubation in medium containing naphthalene-1,2-dihydrodiol. The spectra obtained from lens proteins incubated with 1, 2-dihydroxynaphthalene were distinct from those of either naphthalene-1,2-dihydrodiol or 1,2-naphthoquinone. Aldose reductase inhibitors possessing either hydantoin or carboxylic acid groups prevented protein modification induced by naphthalene-1, 2-dihydrodiol but not protein modification induced by 1, 2-dihydroxynaphthalene or 1,2-naphthoquinone. Therefore, the metabolite formed from naphthalene-1,2-dihydrodiol by aldose reductase is 1,2-naphthoquinone. Lens proteins modified by naphthalene-1,2-dihydrodiol appear essentially identical to protein adducts formed with 1,2-naphthoquinone and their formation can be prevented by both hydantoin and carboxylic acid containing aldose reductase inhibitors.

Aldehyde Reductase↗
PubMed1994

Modification of hepatic immunoglobulin heavy chain binding protein (BiP/Grp78) following exposure to structurally diverse peroxisome proliferators.

This investigation was conducted to determine the comparative effect of structurally diverse peroxisome proliferators (PP) on the two-dimensional protein pattern of rat liver whole homogenates. Perfluoro-n-decanoic acid (PFDA), perfluoro-n-octanoic acid (PFOA), clofibrate, and di(2-ethylhexyl)phthalate (DEHP) are all known to cause the proliferation of hepatic peroxisomes and the induction of peroxisomal beta-oxidative and microsomal omega-oxidative enzymes. To clarify the mechanistic differences between these compounds with regard to the liver, we examined the unique patterns of protein alteration produced by in vivo exposure to them. Following exposure to various doses, whole liver homogenates were prepared and separated by two-dimensional gel electrophoresis (2DE) using the ISO-DALT system. Stained gels were digitized and protein patterns analyzed using the Kepler 2D gel analysis system. Immunoglobulin heavy chain binding protein (BiP), also known as 78-kDa glucose-regulated protein (Grp78), was identified immunologically and by comigration of recombinant Grp78. BiP is a luminal endoplasmic reticular protein that functions in the assembly and folding of nascent proteins as they enter the ER. The present results suggest a selective posttranslational modification of BiP following PFDA exposure. Single-dose exposure to PFDA was associated with a notable charge modification of BiP that persists up to 30 days. PFOA, clofibrate, and DEHP had less effect in this regard. The identity of BiP/Grp78 as the halothane hepatitis-associated trifluoroacetylated protein was also demonstrated. The nature of this PFDA-associated protein modification (reactive metabolite conjugation, abnormal ribosylation, or phosphorylation) is currently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Covalent Modification of Emulsified beta-Casein Resulting from Lipid Peroxidation.

Competitive displacement of adsorbed protein from emulsion droplets by the surfactant Tween 20 has been used to determine the influence of the oil phase and aging on the behavior of beta-casein, the displaced protein being analyzed by reverse-phase high performance liquid chromatography HPLC. Unlike soluble beta-casein or the protein displaced from tetradecane droplets where aging had no effect on the appearance of the HPLC profile, the protein displaced from a soya oil emulsion interface was observed to change. As the soya oil emulsion aged, the retention time of the protein decreased. Mass spectrometry of the modified protein showed that the molecular weight increased, indicating that some form of covalent modification was occurring. Gas chromatography-mass spectrometry of steam distillates of the samples showed the presence of a variety of aldehydes in microfluidized soya oil samples that were not present in either the original oil or the tetradecane emulsions. Aldehydes, particularly alpha,beta-unsaturated aldehydes (enals), which are the major components formed in these soya oil emulsions, are known to react with nucleophilic amino acid side chains such as lysine. This is the probable cause of the observed modification of the emulsified protein. These aldehydes, whose concentration increased with storage time, are formed by peroxidation of the unsaturated fatty acyl chains present in the soya oil as a result of the microfluidization process used in the preparation of the emulsions. The tryptic peptide pattern also changed with age due to modification of the primary structure of the protein. Potential consequences of these chemical changes arising as a result of microfluidization are discussed. Copyright 1999 Academic Press.

Journal Article↗
PubMed2000

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway.

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.

Catalysis↗
PubMed1993

Durability of the in situ bypass following modification of abnormal vein segment.

Modification procedures performed during in situ bypass grafting to correct an injured or inadequate saphenous vein segment result in a significant increase in the incidence of vein graft complications in the follow-up period. Modification procedures were performed in 96 in situ saphenous vein bypasses and consisted of primary closure (n = 28), vein patch angioplasty (n = 31), or resection and/or replacement (n = 37). At 4 years primary patency was 54%, secondary patency was 73%, and limb salvage was 89%. The incidence of subsequent vein graft stenosis and revision or graft failure was similar for grafts requiring vein patch angioplasty (7 of 31, 23%), primary repair (9 of 28, 32%), and resection and/or replacement (16 of 37, 43%) (p not equal to ns). Only 4 bypass revisions were performed for stenosis at the site of the original modification procedure. The type of vein graft repair did not significantly affect the primary patency at 18 months (primary closure, 65%, vein patch angioplasty, 66%, and resection and/or replacement, 58%) or the secondary patency at 30 months (primary closure, 80%, vein patch angioplasty, 90%, and resection and/or replacement, 77%). Modified autogenous conduits maintain patency and limb salvage but are prone to develop graft complications in the follow-up period.

Coronary Artery Bypass↗
PubMed1999

Posttranslational modification of CLN3 protein and its possible functional implication.

The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium. The function of CLN3 protein remains unknown. Computer-based analysis predicted that CLN3 may contain several posttranslational modifications. Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line. Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein. We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein. Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors.

Alkaline Phosphatase↗
PubMedDate not supplied

Queuosine modification of tRNA: a case for convergent evolution.

Queuosine is a hypermodified nucleoside found in position 34, the anticodon wobble position, of four tRNA species. This modification is distributed with near uniformity across all life forms found on this planet. Yet the molecular mechanisms involved with accomplishing this ubiquitous posttranscriptional modification of tRNA are dramatically different between prokaryotic and eukaryotic organisms, which suggests that these were formed by convergent evolution of a fundamental life process essential to nearly all life forms. This minireview describes the differences between these modification systems and points to a new direction for developing research on the molecular function queuosine-modified tRNA in diverse species.

Animals↗
PubMed2002

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).

Animals↗