The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 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 M_r, 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 M_r 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.

     

Inactivation of ornithine decarboxylase by intermediates of tyrosinase-catalyzed reaction

• 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.

     

Identification of essential amino acids in the active center of thyroidal NAD+ glycohydrolase

• 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.

     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

The effects of organic and inorganic pollutants on intracellular phosphate compounds in blue mussels (Mythus edulis)

• 1.1. The effects of sublethal concentrations of organic and inorganic pollutants on intracellular energy-rich phosphates in blue mussels, Mytilus edulis, were investigated by in vivo ³¹P-NMR.
• 2.2. Formaldehyde (30 and 10mg/l), phenol, pyridine, mercury and cadmium gave marked reductions in phosphoarginine and, in some cases, the ATP amounts. The reduction in high-energy phosphate was accompanied by an increase in inorganic phosphate in all groups.
• 3.3. A “phosphorus index”, the product of the ratios between phosphoarginine and inorganic phosphate, and ATP and inorganic phosphate, is suggested, which might serve as an early warning (“alarm”) parameter in environmental monitoring.
• 4.4. Diversity in the responses to different pollutants make phosphorus compounds in M. edulis also an interesting element in a finger print parameter system designed to distinguish between pollutants in the marine environment.

     

Dose-dependent vitamin D3 and 1,25-dihydroxyvitamin D3-induced hypercalcemia and hyperphosphatemia in male cyprinoid Cyprinus carpio

• 1.1. Vitamin D₃ (10IU and 100IU/100 g body weight) and 1,25(OH)₂D₃ (0.5, 5 and 50 U) were administered daily to unfed male carp Cyprinus carpio for 10 days. The serum calcium and inorganic phosphate levels were measured colorimetrically.
• 2.2. The serum calcium level increased significantly in all treated groups; this increase is dose-dependent.
• 3.3. The serum inorganic phosphate was elevated in the treated groups on days 3 and 5.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

Phosphorylation of a 25,000-dalton protein in slow and fast muscles of the chicken

• 1.1. Protein phosphorylation in intact chicken latissimus dorsi muscle, slow anterior (ALD) and fast posterior (PLD), was compared.
• 2.2. A major difference in [³²P]phosphate incorporation was found between the ALD and PLD in a 25,000-dalton heat soluble protein.
• 3.3. The 25,000-dalton protein was purified from both the ALD and PLD.
• 4.4. The two proteins had similar amino acid composition and both contained approximately 1 mole phosphate per mole of protein.
• 5.5. The difference in their content of radioactive phosphate was determined to be due to faster turnover in the ALD.

     

Changes in high-energy phosphate metabolism in the water scorpion,Ranatra chinensis,under cold water-warm water stress

• 1.1. To evaluate changes in high-energy phosphate metabolism in the water scorpion (Ranatra chinensis) under restraint and cold water-warm water stresses, in vivo [³¹P]NMR spectra were obtained.
• 2.2. Under restraint stress, arginine phosphate (Arg-P) decreased by 10% after 1 hr and remained at that level thereafter, while β-ATP showed negligible changes over 6 hr.
• 3.3. As the water temperature gradually increased or decreased, the relative concentration of Arg-P decreased due to enzyme regulation.
• 4.4. Repeated cold water-warm water stress, which consisted of repeated 15 min exposures to cold water (5°C) followed by 15 min exposures to warm water (30°C) caused distinct decreases in Arg-P and β-ATP concentration. These decreases were dependent on the frequency of exposure.
• 5.5. Phosphomonoesters (PME) increased not only with restraint stress but also with cold water-warm water stress.
• 6.6. The effect of cold water-warm water stress on high-energy phosphate metabolism was greater than that of restraint stress.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

A very stable and potent fibrinolytic enzyme found in earthworm Lumbricus rubellus autolysate

• 1.1. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity.
• 2.2. It also showed a clot-forming activity in the fibrinogen- or plasma-added system.
• 3.3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and 15,000 in the autolysate.
• 4.4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N-terminal amino acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

     

Regulation of horse-liver glutathione reductase

• 1.1. The enzyme was rapidly inactivated by NAD(P)H, GSH, dithionite or borohydride, while activity increased in the presence of NAD(P)+ or GSSG. NADH was more efficient for inactivation than NADPH. Redox inactivation required neutral or alkaline pH, was maximal at pH 8.5, and depended on the presence of metal cations.
• 2.2. GSSG and dithiothreitol fully protected the enzyme from inactivation at concentrations stoichiometric with NAD(P)H. Ten-fold higher ferricyanide or GSH concentrations were required to obtain partial protection. NAD+ or NADP+ were quite ineffective.
• 3.3. GSSG fully reactivated the inactive enzyme at 38°C and neutral to acidic pH (5.5–7.5). Reactivation by dithiothreitol was accomplished in short periods of time at pH 8.5 although the activity was progressively lost afterwards. Ferricyanide and GSH also reactivated the enzyme to different extents.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Human placental atp-diphosphohydrolase: Biochemical characterization,regulation and function

• 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 M_r 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.

     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Variation in plasma constituents during the natural vitellogenic cycle of tuatara,Sphenodon Punctatus

• 1.1. The vitellogenic cycle of female tuatara was investigated by monitoring plasma levels of vitellogenin, calcium, total protein, inorganic phosphate (P₁) and cholesterol.
• 2.2. Vitellogenin was not detected in females in the non-reproductive condition, but was found perenially in plasma of reproducing females during vitellogenesis, which normally lasts about 3 years out of the 4 year ovarian cycle.
• 3.3. No large year-to-year variations were found in the plasma constituents measured and there was no correlation between the oestradiol peak at mating and plasma levels of vitellogenin.
• 4.4. The results provide further evidence that tuatara have an extraordinary prolonged and gradual vitellogenic cycle spanning several years for a single clutch of eggs. This type of reproductive cycle is unique among reptiles.

     

Studies on the active centre(s) of rat liver porphyrinogen carboxy-lyase. in vivo effect of hexachlorobenzene on decarboxylation site(s) of porphyrinogens

• 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.

     

Oxidation of methionine residues in equine growth hormone by chloramine-T

• 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.