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1.
  • 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
  • 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
  • 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
  • 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower Mr, Stokes-radius and diffusion coefficient.
  • 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the Mr of the subunits were unaffected.
  • 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
  • 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
  • 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.
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2.
  • 1.1. The number of tyrosine and tryptophan residue-equivalents on the surfaces of the α-amylase and of two of its protein inhibitors has been determined.
  • 2.2. The solvent-exposed tyrosine and tryptophan residue-equivalents in the dimeric inhibitor are respectively four and two times as much as those ones of the monomeric inhibitor.
  • 3.3. On the basis of the homology among their polypeptide chains, it is suggested that the outer tryptophan residues in either inhibitors are located at the positions 4 and 51 of the primary structure.
  • 4.4. On the interaction of the monomeric inhibitor with the amylase, two tryptophan residue-equivalents became no more accessible to the solvent.
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3.
  • 1.1. The inorganic pyrophosphatase from Escherichia coli was almost completely inactivated on chemical modification of Trp-149 with N-bromosuccinimide (NBS).
  • 2.2. The presence of a complex of Mg2+ and a substrate analogue, iminodiphosphate (PNP), provided considerable protection against the inactivation, whereas Mg2+ or PNP alone afforded only slight protection.
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4.
  • 1.1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone.
  • 2.2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved.
  • 3.3. Methionine 4 is the most reactive group, followed by methionines 72 and 178—methionine 123 being the less reactive residue.
  • 4.4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues.
  • 5.5. Results agree with data previously obtained with bovine growth hormone.
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5.
  • 1.1. The reactivities of lysine residues of recombinant rat guanidinoacetate methyltransferase were determined by trace labeling with acetic anhydride.
  • 2.2. Lys-113 and -160 were weakly reactive and Lys-178 and -234 were unreactive toward the reagent. The six lysines (Lys-38, -83, -104, -108, -152 and -180) showed moderate reactivities. The N-terminal amino group was very reactive.
  • 3.3. S-Adenosylmethionine did not alter the reactivities of lysines significantly, but the reactivity of Lys-38 was substantially reduced in the presence of S-adenosylmethionine and guanidinoacetate.
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6.
  • 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
  • 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
  • 3.3. The inhibitor is a small protein (Mr = 23,000) which did not show proteolytic activity.
  • 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
  • 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
  • 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.
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7.
  • 1.1. The role of histidine on the decarboxylation of porphyrinogens of 7-, 6-, and 5-COOH III brought about by porphyrinogen carboxy-lyase (PCL) was studied.
  • 2.2. For this purpose hepatic PCL from normal and hexachlorobenzene (HCB) treated rats were modified with diethylpyrocarbonate.
  • 3.3. The results indicated that the enzyme from both normal and porphyric animals had histidine at the binding sites of all the porphyrinogens assayed.
  • 4.4. Comparative studies between the enzyme from normal and porphyric rats suggested that in vivo HCB treatment affected the active site for the decarboxylation of 7-, 6- and 5-COOH porphyrinogens III at histidine residues.
  • 5.5. On the other hand arginine modification by 2,3-butanedione treatment altered 5-COOH porphyrinogen III decarboxylation for both enzymes. However this amino acid was not involved at the binding site of this substrate.
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8.
  • 1.1. Purified thyroidal NAD+ glycohydrolase has been subjected to the action of a number of group specific reagents in order to gain information concerning its mode of action.
  • 2.2. Modification of histidyl residues with diethylpyrocarbonate strongly suppresses the NAD+ glycohydrolase activity. Inactivation with this reagent can be reversed to some extent by subsequent treatment with hydroxylamine.
  • 3.3. NAD+ and ADP-ribose partially protect against inactivation with similar efficiencies.
  • 4.4. The incomplete reactivation with hydroxylamine after diethylpyrocarbonate treatment and the selective inactivation by 2,4-pentanedione indicates that apart from one or more essential histidyl residue(s) also lysyl residues are important for activity. NAD+ and to a smaller extent ADP-ribose again protect against inactivation by 2,4-pentanedione.
  • 5.5. The sensitivity of the enzyme towards N-ethyl-5-phenyl-isooxazolium-3'-sulfonate further points to the importance of carboxylate containing side chains.
  • 6.6. The mechanistic implications of these results are discussed.
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9.
  • 1.1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin: cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions.
  • 2.2. In the absence of lipids no fluorescence energy transfer was measurable.
  • 3.3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface.
  • 4.4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.
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10.
  • 1.1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [3H]palmitic acid and [35S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells.
  • 2.2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes.
  • 3.3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [3H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da.
  • 4.4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30–43 amino acids and those of 18–25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10–15 amino acids.
  • 5.5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occuring in an immediate vicinity of the signal peptide fragment.
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11.
  • 1.1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident Mr and pI values of both ATPase-ADPase activities.
  • 2.2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher.
  • 3.3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DBS and slightly by DCCD.
  • 4.4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A.
  • 5.5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of −SH, COO, −OH, and probably also Tyr and Trp.
  • 6.6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes.
  • 7.7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.
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12.
  • 1.1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered.
  • 2.2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered.
  • 3.3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated.
  • 4.4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation.
  • 5.5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.
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13.
  • 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
  • 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
  • 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
  • 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
  • 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent Km of 1 mM and Vmax of 0.84 μmol diene/min/mg protein.
  • 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
  • 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.
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14.
  • 1.1. A fluorescent derivative of the Griffonia simplicifolia I-B4 isolectin was used to examine historically the distribution of terminal methyl-α-galactosyl residues in the microcirculatory vessels of the gastrocnemius muscle and the ventricles from four families of anuran amphibians.
  • 2.2. The isolectin preferentially bound to capillaries in the gastrocnemius muscles from members of the families Ranidae, Bufonidae and Pipidae, but not from the family Hylidae.
  • 3.3. Histological and ultrastructural analyses revealed a primitive sinusoidal endothelial system in the anuran heart, with a less extensive expression of the GSI-B4 receptors than in skeletal muscle.
  • 4.4. These results suggest phylogenetic differences among families of anuran amphibians with regard to the distribution of GSI-B4 receptors in skeletal and cardiac muscle.
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15.
  • 1.1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri).
  • 2.2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots.
  • 3.3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions.
  • 4.4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.
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16.
  • 1.1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity.
  • 2.2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially.
  • 3.3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity.
  • 4.4. Polyamine content was increased. The maximum modification was observed for putrescine and N1-acetylspermidine.
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17.
  • 1.1. The inhibitory effect of N,N,N′,N′-tetramethylethylene diamine (TEMED) on water soluble (WSAChE) and membrane bound (MBAChE) acetylcholinesterase was investigated.
  • 2.2. TEMED (0.5–4.0 mM) reversibly inhibited WSAChE activity (18–62%) and MBAChE (20–61%) in a concentration dependent manner.
  • 3.3. The IC50 being about 2.8 mM for WSAChE and 2.6 mM for MBAChE.
  • 4.4. Lineweaver-Burk plots indicated that the nature of inhibition is noncompetitive for both water soluble and membrane bound acetylcholinesterase, with Km values 68 μM and 123 μM respectively.
  • 5.5. An Arrhenius plot showed that the transition temperature (TT) is unaffected in the presence of TEMED.
  • 6.6. The activation energy was increased below and above TT in the case of WSAChE only.
  • 7.7. On the basis of this behaviour of TEMED with AChE. it can be proposed that it can be used as an eluting agent for the bounded AChE to affinity ligand and may have beneficial action on the reactivatability of irreversibly-inhibited AChE due to its structure.
  • 8.8. Moreover there is a possibility that it can be used as a therapeutic agent for the treatment of Alzheimer's disease, myasthenia gravia and glaucoma like some other inhibitors of AChE.
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18.
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Highlights
  • •Dimethyl fumarate covalently modifies cysteine residues in neurons and astrocytes.
  • •Cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2) are targets.
  • •DMF-modified cofilin-1 reduces actin-severing ability, preserving filamentous actin.
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19.
  • 1.1. Intermediates in the process of melanin synthesis formed through oxidation of catechols by tyrosinase produced the inactivation of ornithine decarboxylase (ODC), a key enzyme in the polyamine biosynthesis pathway.
  • 2.2. The inactivation was dependent on the substrate used (dihydroxybenzylamine ⪢ l-3,4-dihydroxy-phenylalanine ⪢ l-tyrosine) and on the concentration of intermediate produced rather than on the rate of formation.
  • 3.3. Sulfhydryl compounds (dithiothreitol and glutathione) or quinone-reducing agents (ascorbic acid) prevented the inactivation of ODC; l-ornithine, but not other aminoacids, also protected partially ODC. The results suggest that different cysteine residues in ODC molecule are implicated in the inactivatory event.
  • 4.4. When 14C-labeled catechols were used, numerous polypeptides resulted labeled, showing that the reactive quinones formed as intermediates in the process of melanin biosynthesis bind covalently to many cellular proteins.
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20.
  • 1.1. The phosphorylation of Escherichia coli proteins was analyzed comparatively before and after induction of the SOS response in a temperature-sensitive mutant strain.
  • 2.2. The presence of phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after labelling with radioactive orthophosphate in vivo or radioactive adenosine triphosphate in vitro.
  • 3.3. Significant changes in the intensity of protein labelling were observed upon induction of the SOS functions: six proteins were found to be more phosphorylated while two others were less phosphorylated. Moreover, five additional proteins appeared to become phosphorylated exclusively during the SOS response. The molecular mass and isoelectric point of these various proteins were determined.
  • 4.4. For most proteins, the changes in the pattern of protein phosphorylation were concomitant with variations in the amount of protein synthesized.
  • 5.5. The changes in the pattern of phosphoproteins observed during the SOS response were not due to the temperature shift required experimentally for expressing the SOS phenotype.
  • 6.6. Phosphorylation was found to be catalyzed by protein kinases that modify amino acid residues at hydroxyl groups in protein substrates.
  • 7.7. Both in vivo and in vitro studies brought evidence that neither RecA nor LexA, the two key regulatory proteins of the SOS functions, were capable of undergoing phosphorylation.
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