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PubMed1984

The effect of myosin sulphydryl modification on the mechanics of fibre contraction.

Glycerinated rabbit psoas fibres have been modified with paramagnetic probes ( IASL and MSL) which react selectively with the reactive sulphydryl on the myosin head. The extent of SH-1 modification was monitored by extracting myosin and measuring its ATPase activity in the presence of EDTA and of Ca2+. The isometric tension, stiffness, maximum velocity of contraction (slack test), and the force-velocity relation was measured as a function of the degree of SH-1 modification. Reaction of up to 50% of SH-1, i.e. 50% reduction in the K+-EDTA ATPase activity of extracted myosin, produced little change (less than 10%) in any of the fibre parameters. Modification of 75% of the SH-1 sites produced small decreases (15-30%) in the magnitude of all parameters, while reaction of more than 90% of SH-1 required long reaction times and produced decreases of 40-75%. In all cases the velocities of contraction decreased in parallel with the decrease in tension, while the decrease in stiffness was less pronounced. We conclude that a large fraction of muscle fibre SH-1 groups can be modified without greatly affecting the mechanical performance of the fibre. At least a portion of the decrease in fibre parameters that is observed at high levels of SH-1 modification can be attributed to modification of other sulphydryls by the probes. The reaction of both SH-1 and nonspecific sulphydryls abolishes myosin ATPase activity, and can account for approximately one half of the decrease in fibre parameters that is observed at high degrees of sulphydryl modification. We conclude that the modification of SH-1 does not greatly affect the function of a myosin head in the filament array of a fibre. This is in contrast to results obtained in vitro where SH-1 modification alters several rates in the interaction of myosin with ATP and decreases the actin-activated ATPase activity of myosin subfragments.

Adenosine Triphosphatases↗
PubMed1988

3,4,5,6-Tetrahydrophthalic anhydride modification of glutamate dehydrogenase: the construction and activity of heterohexamers.

Modification of glutamate dehydrogenase with 3,4,5,6-tetrahydrophthalic anhydride at pH 8.0 results in the progressive loss of enzymatic activity and a concomitant increase in the negative charge of the protein. Although the rate of inactivation at room temperature is too rapid to allow accurate rate constant determination, modification at 4 degrees C shows that the pseudo-first-order rate constant for inactivation appears to show a saturation effect with increasing reagent concentration, with a maximum of approximately 1 min-1. Control experiments showed that tetrahydrophthalic anhydride was hydrolyzed at a much slower rate, with a pseudo-first-order rate constant of 0.041 min-1. Protection studies indicated that inactivation was decreased by the active site ligands, NADP and 2-oxoglutarate. The extents of inactivation, whether assayed with glutamate at pH 7.0 or norvaline at pH 8.0, were the same. Changes in mobility on native gels and isoelectric point were used to follow the incorporated negative charge resulting from modification. Enzyme modified in the presence of protecting ligands (where activity is maintained) showed mobility changes which suggested that a single site of modification was protected. Modified enzyme incorporated 0.78 mol pyridoxal 5-phosphate less than native enzyme, consistent with modification of lysine-126. Enzyme modified under limiting conditions was shown to have a quaternary structure similar to that of the native enzyme, as judged by crosslinking patterns obtained with dimethylpimelimidate. The modified protein is readily resolved from unmodified protein using an NaCl double gradient elution from DEAE-Sephacel. The modification is reversed with regain of activity by incubation of the modified enzyme at low pH. We have made use of the recently demonstrated ability of guanidine hydrochloride to dissociate the hexamer of glutamate dehydrogenase into trimers that can then be reassociated to construct heterohexamers of glutamate dehydrogenase, in which one trimer of the heterohexamer contains native subunits while the other has been inactivated by the 3,4,5,6-tetrahydrophthalic anhydride modification. The heterohexamer is separated from either native or fully modified hexamers by DEAE-Sephacel chromatography. Significantly, the heterohexamer has little detectable catalytic activity, although activity is regained by reversal of the modification of the one modified trimer in the hexamer. This demonstrates that catalytic site cooperation between trimers in the hexamer of glutamate dehydrogenase is an essential component of the enzymatic activity of this enzyme.

Enzyme Activation↗
PubMed2004

Peroxynitrite-mediated oxidative modification of low-density lipoprotein by aqueous extracts of cigarette smoke and the preventive effect of fluvastatin.

Cigarette smoking is known to promote atherosclerosis, possibly through enhanced oxidative stress. The aim of the present study was to elucidate the possible involvement of peroxynitrite in oxidative modification of low-density lipoprotein (LDL) induced by aqueous extract of cigarette smoke (CSE) and the preventive effect of fluvastain, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor with antioxidative activity, in vitro and in vivo. Modification of LDL was monitored by LDL subfraction analysis using anion-exchange HPLC, TBARS formation and 3-nitrotyrosine production. Incubation of LDL with CSE caused a marked increase in oxidative modification of LDL and nitration of tyrosine residues in the apolipoprotein B. These modifications were prevented by treatment with fluvastatin as well as Vitamin E in a concentration-related manner. Fluvastatin was equal to or more effective than Vitamin E for preventing protein nitration, but weaker for preventing oxidative modification. When CSE was injected daily into the ear vein of Watanabe heritable hyperlipidemic rabbits for 5 months, both oxidative modification and nitration of the plasma LDL noticeably occurred. These changes induced by CSE could be effectively prevented by the simultaneous oral administration of fluvastatin (10 and 30 mg/kg) or Vitamin E (150 mg/kg). Fluvastatin prevented the LDL nitration more effectively than Vitamin E. These results suggest that peroxynitrite in CSE is involved in oxidative modification of LDL and that fluvastatin can efficiently prevent LDL modification by scavenging peroxynitrite. Fluvastatin may be potentially beneficial to hypercholesterolemic patients with oxidative stress such as smoking.

Animals↗
PubMed2004

Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions.

Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.

Animals↗
PubMed2001

Modifications of the Cox-Maze III procedure.

BACKGROUND: The extended operative time needed for surgery with complicated atrial incisions may preclude application of the Cox-Maze III procedure (CM-III) as a concomitant operation. And after the CM-III, left atrial (LA) contraction has been reported to recover in reduced magnitude compared with right atrial (RA) contraction. METHODS: To decrease operative time, we have modified the CM-III (modification I) by: obliterating the LA appendage instead of excising it; cryoablating the bridge between the LA appendage and margin of the pulmonary vein encircling incision; extending the lateral incision of RA onto the RA appendage without excising it, and extending the incision more inferiorly toward the inferior vena cava; and omitting the T-incision of RA. We compared the clinical results of the conventional CM-III (group 1, n = 18) with those of the modified CM-III group (group 2, n = 23) performed in patients with rheumatic mitral valve (MV) disease. To enlarge the contractile area of the LA, we modified the CM-III to encircle the right and left pulmonary veins separately (modification II), and compared the LA contractilities of the conventional CM-III (group A, n = 15) with those of the second modification (group B, n = 9). RESULTS: Modification I: Mean aortic cross-clamp (ACC) times (135 +/- 29 versus 104 +/- 18 minutes, p < 0.005) and cardiopulmonary bypass (CPB) times (240 +/- 33 versus 185 +/- 42 minutes, p < 0.001) were significantly decreased in group 2 compared with those in group 1. In group 1, sinus rhythm was restored in 16 patients (88.9%). RA contractility was demonstrated in 100% of patients with sinus rhythm (16 of 16) and LA contractility in 75% (12 of 16) in the latest follow-up echocardiography. In group 2, sinus rhythm was restored in 21 patients (91.3%). RA contractility was demonstrated in 100% of patients with sinus rhythm (21 of 22) and LA contractility in 76.2% (16 of 21). Modification II: Mean ACC times were increased in group B compared with group A (133 +/- 32 versus 172 +/- 39 minutes, p = 0.02). The A velocities at LA contraction and the ratio of atrial contraction to peak early diastolic filling velocity (A/E ratio) of the trans-mitral flow were 0.14 +/- 0.20 m/sec and 0.23 +/- 0.11 in group A, and 0.58 +/- 0.33 m/sec and 0.47 +/- 0.19 in group B, respectively, both showing a significant increase in group B compared with group A (p < 0.05). CONCLUSIONS: Our first modification of the CM-III showed comparable sinus conversion rates and incidence of atrial contractility restoration with significantly shorter ACC and CPB times than the conventional CM-III. The second modification of the CM-III significantly increased the LA contractility when compared with the conventional CM-III, although the second modification required a longer ACC time.

Adult↗
PubMed1997

Nongenetic variation, genetic-environmental interactions and altered gene expression. III. Posttranslational modifications.

The use of protein electrophoretic data for determining the relationships among species or populations is widespread and generally accepted. However, posttranslational modifications have been discovered in many of the commonly analyzed proteins and enzymes. Posttranslational modifications often alter the electrophoretic mobility of the modified enzyme or protein. Because posttranslational modifications may affect only a fraction of the total enzyme or protein, an additional staining band often appears on gels as a result, and this may confound interpretations. Deamidation, acteylation, proteolytic modification, and oxidation of sulfhydryl groups are modifications that often result in an electrophoretic mobility shift. Sialic acid-induced heterogeneity has been documented for many enzymes, but neuraminidase treatment can often remove sialic acids and produce gel patterns that are easier to interpret. In some cases, ontogenetic and tissue-specific expression may be due to posttranslational modifications rather than gene control and restricted expression, respectively. Methods of preventing, detecting and eliminating posttranslational modifications are discussed. Some posttranslational modifications may be useful for detecting cryptic genetic polymorphisms.

Acclimatization↗
PubMed2001

Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities.

Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).

Amino Acid Sequence↗
PubMedDate not supplied

Molecular characterization of a conformational epitope of hen egg white lysozyme by differential chemical modification of immune complexes and mass spectrometric peptide mapping.

A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.

Animals↗
PubMed1992

Protein folding: assignment of the energetic changes of reversible chemical modifications to the folded or unfolded states.

Reversible chemical modifications of a series of single cysteine-containing variants of T4 lysozyme combined with thermal denaturation studies have been used to study the effects of these modifications on the stability of the protein. This allows dissection of the energetic effects of the modification on both the native and denatured states of this protein. At some sites modifications with various chemical reagents have essentially no effect on the stability of the protein, while at others, substantial changes in stability are observed. For example, chemical modification of cysteine at site 146 by cystamine (+NH3CH2CH2SSCH2-CH2NH3+) to form the mixed disulfide lowers the stability of the protein by about 1.1 kcal/mol. The reduction in the free energy of folding caused by the chemical modification is attributed to the destabilization of native state (0.9 kcal/mol), with only a relatively small effect from stabilization of the denatured state (0.2 kcal/mol). Chemical modifications of T4 lysozyme at site 146 with various chemical reagents show that the stability of the protein is lowered by a positively charged group and is relatively independent of the size of the side chains. This approach allows the investigation of the thermodynamic consequences of the reversible insertion of a wide variety of chemical entities at specific sites in proteins and, most importantly, allows dissection of the contribution of the chemical modifications to both the folded and unfolding states. It can be applied to almost any suitable macromolecular system.

Buffers↗
PubMed2001

Enhanced potency of human Sonic hedgehog by hydrophobic modification.

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.

Acyl Coenzyme A↗
PubMed1976

Specific modification of an effector binding site of phosphofructokinase by pyridoxal phasphate.

Conditions are described for the covalent modification of rabbit skeletal muscle phosphofructokinase by pyridoxal phosphate plus sodium borohydride to produce an enzyme that appears by a number of criteria to be modified at the citrate binding site. Evidence that modification occurs at this site is as follows. (1) Protection against activity loss due to modification is provided by the combination of MgATP and citrate, whereas neither low concentrations of citrate nor MgATP alone is effective. This is consistent with the increased affinity for citrate that is observed in the presence of MgATP. (2) The extensive changes in activity and equilibrium binding result from the incorporation of only 1 mol of pyridoxal phosphate per mol of protomer. (3) Modification greatly increases sensitivity to MgATP inhibition, an effect consistent with the known synergism between MgATP and citrate. (4) The affinity of the enzyme for both MgATP and MgIPT at the catalytic site is increased by the modification. (5) The sensitivity of the enzyme to citrate inhibition is greatly diminished following covalent modification. (6) Modification abolishes the equilibrium binding of citrate. (7) Enhanced binding of MgATP is observed following modification, a result consistent with the enhancement of MgATP binding by citrate. Phosphofructokinase protected by citrate plus MgATP can also be modified by the incorporation of 1 or more mol of pyridoxal phosphate, but the enzyme so produced is capable of interacting with citrate and shows none of the properties herein described for the enzyme modified in the absence of citrate.

Adenosine Triphosphate↗
PubMed1975

Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.

Arginine↗
PubMed2005

Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications.

The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.

Base Sequence↗
PubMed2006

Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal.

Methylglyoxal (MG) is an important glycating agent produced under physiological conditions. MG could react with DNA and proteins to generate advanced glycation end products. Human hemoglobin, the most abundant protein in blood cells, has not been systematically investigated as the target protein for methylglyoxal modification. Here we examined carefully, by using HPLC coupled with tandem mass spectrometry (LC-MS/MS), the covalent modifications of human hemoglobin induced by methylglyoxal. Our results revealed that hemoglobin could be modified by methylglyoxal, and the major form of modification was found to be the hydroimidazolone derivative of arginine residues. In addition, Arg-92 and Arg-141 in the alpha chain as well as Arg-40 and Arg-104 in the beta chain were modified, whereas two other arginine residues, that is, Arg-31 in the alpha chain and Arg-30 in the beta chain, were not modified. Semiquantitative measurement for adduct formation, together with the analysis of the X-ray structure of hemoglobin, showed that the extents of arginine modification were highly correlated with the solvent accessibilities of these residues. The facile formation of hydroimidazolone derivatives of arginine residues in hemoglobin by methylglyoxal at physiologically relevant concentrations suggested that this type of modification might occur in vivo. The unambiguous determination of the sites and extents of methylglyoxal modifications of arginines in hemoglobin provided a basis for understanding the implications of these modifications and for employing this type of hemoglobin modification as molecular biomarkers for clinical applications.

Amino Acid Sequence↗
PubMedDate not supplied

VEMS 3.0: algorithms and computational tools for tandem mass spectrometry based identification of post-translational modifications in proteins.

Protein and peptide mass analysis and amino acid sequencing by mass spectrometry is widely used for identification and annotation of post-translational modifications (PTMs) in proteins. Modification-specific mass increments, neutral losses or diagnostic fragment ions in peptide mass spectra provide direct evidence for the presence of post-translational modifications, such as phosphorylation, acetylation, methylation or glycosylation. However, the commonly used database search engines are not always practical for exhaustive searches for multiple modifications and concomitant missed proteolytic cleavage sites in large-scale proteomic datasets, since the search space is dramatically expanded. We present a formal definition of the problem of searching databases with tandem mass spectra of peptides that are partially (sub-stoichiometrically) modified. In addition, an improved search algorithm and peptide scoring scheme that includes modification specific ion information from MS/MS spectra was implemented and tested using the Virtual Expert Mass Spectrometrist (VEMS) software. A set of 2825 peptide MS/MS spectra were searched with 16 variable modifications and 6 missed cleavages. The scoring scheme returned a large set of post-translationally modified peptides including precise information on modification type and position. The scoring scheme was able to extract and distinguish the near-isobaric modifications of trimethylation and acetylation of lysine residues based on the presence and absence of diagnostic neutral losses and immonium ions. In addition, the VEMS software contains a range of new features for analysis of mass spectrometry data obtained in large-scale proteomic experiments. Windows binaries are available at http://www.yass.sdu.dk/.

Algorithms↗
PubMed2006

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.

Age Factors↗
PubMed2005

O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes.

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 microM for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.

Acetylglucosamine↗
PubMed1984

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

Arginine↗