Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204?kDa and 160?kDa. Each was a tetramer or pentamer of 44?kDa and 33?kDa, respectively.     

Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.

Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.     

Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (_•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against _•OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H₂O₂), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a _•OH-trapping agent after UVB exposure (305 nm) to H₂O₂ for 1 min in the presence of rebamipide. The signal intensity of _•OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 10₁₀, 8.16 × 10₉ and 1.65 × 10₁₀ M_-1 s_-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the _•OH generated by UVB/H₂O₂. Specific formation of this product explained the molecular characteristics of rebamipide as a potential _•OH scavenger.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

Hydrolysis of PET and bis-(benzoyloxyethyl) terephthalate with a new polyesterase from Penicillium citrinum

A polyethylene terephthalate (PET) model substrate, bis-(benzoyloxyethyl)terephthalate (3PET), was used to screen for micro-organisms producing enzymes hydrolyzing PET. From this screen, a strain growing on 3PET was isolated and identified as Penicillium citrinum. The polyesterase responsible for 3PET and PET hydrolysis was purified to electrophoretic homogeneity. The polyesterase had a molecular weight of 14.1 kDa, and the K_m and K_cat values on 4-nitrophenyl butyrate were 0.57 mM and 0.21 s^-1, respectively. Highest enzyme activities were obtained when P. citrinum was grown on a medium containing cutin, which was hydrolyzed by the polyesterase. Surface hydrolysis of PET with the enzyme lead to an increase in hydrophilicity based on rising height (+5.1 cm) and drop dissipation measurements (55 s). Both from PET and 3PET bis-(2-hydroxyethyl)terephthalate and mono-(2-hydroxyethyl)terephthalate were released, while only low amounts of terephthalic acid were liberated.     

Kinetics and mechanisms of ligand substitution reactions of molybdenum SO2 complexes. Synthesis and X-ray structure of trans-Mo(CO)2 (dmpe) (PPh3) (SO2)

Kinetic results are reported for intramolecular PPh₃ substitution reactions of Mo(CO)₂(η¹-L)(PPh₃)₂(SO₂) to form Mo(CO)₂(η²-L)(PPh₃)(SO₂) (L = DMPE = (Me)₂PC₂H₄P(Me)₂ and dppe=Ph₂PC₂H₄PPh₂) in THF solvent, and for intermolecular SO₂ substitutions in Mo(CO)₃(η²-L)(η²-SO₂) (L = 2,2′-bipyridine, dppe) with phosphorus ligands in CH₂Cl₂ solvent. Activation parameters for intramolecular PPh₃ substitution reactions: ΔH^≠ values are 12.3 kcal/mol for dmpe and 16.7 kcal/mol for dppe; ΔS^≠ values are −30.3 cal/mol K for dmpe and −16.4 cal/mol K for dppe. These results are consistent with an intramolecular associative mechanism. Substitutions of SO₂ in MO(CO)₃(η²-L)(η²-SO₂) complexes proceed by both dissociative and associative mechanisms. The facile associative pathways for the reactions are discussed in terms of the ability of SO₂ to accept a pair of electrons from the metal, with its bonding transformations of η²-SO₂ to η¹-pyramidal SO₂, maintaining a stable 18-e count for the complex in its reaction transition state. The structure of Mo(CO)₂(dmpe)(PPh₃)(SO₂) was determined crystallographically: P2₁/c, A=9.311(1), B = 16.344(2), C = 18.830(2) Å, ß=91.04(1)°, V=2865.1(7) ų, Z=4, R(F)=3.49%.     

Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227.

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO(2) and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K(m) of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4, 6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

The nitrilases of Rhodococcus rhodochrous NCIMB 11216

Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed -cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the -terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The -terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30°C and 8.0, respectively. The propionitrile-induced nitrilase had a K_m for benzonitrile of 20.7 m and a V_max of 12.4 μmol min⁻¹ mg⁻¹ protein whereas the benzonitrile-induced nitrilase had a K_m for benzonitrile of 8.83 m and a V_max of 0.57 μmol min⁻¹ mg⁻¹ protein.     

Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris

The xynHB gene, encoding alkaline xylanase was cloned from Bacillus pumilus by a shot-gun method. The gene was cloned into vector pHBM905A, and expressed in Pichia pastoris GS115. Xylanase-secreting transformants were selected on plates containing RBB-xylan. Enzymatic activity in the culture supernatants was up to 644 U mL^-1 and the optimal secretion time was 4 days at 25°C. SDS-PAGE showed two bands, of 32.2 kDa and 29.6 kDa, both larger than the predicted mass of 22.4 kDa based on its amino acid sequence. Zymogram analysis demonstrated that the enzyme in both bands could hydrolyze xylan. Deglycosylation by endoglycosidase H revealed that both were derived from the same protein but contain different extents of glycosylation (30 and 25%). The optimal pH and temperature of the enzyme was pH6-9 and 50°C, respectively.     

Gene Sequence and Properties of an s-Triazine Ring-Cleavage Enzyme from Pseudomonas sp. Strain NRRLB-12227

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO₂ and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K_m of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

Cloning and sequencing of the leucine dehydrogenase gene from Bacillus sphaericus IFO 3525 and importance of the C-terminal region for the enzyme activity

The structural gene (leudh) coding for leucine dehydrogenase from Bacillus sphaericus IFO 3525 was cloned into Escherichia coli cells and sequenced. The open reading frame coded for a protein of 39.8 kDa. The deduced amino acid sequence of the leucine dehydrogenase from B. sphaericus showed 76–79% identity with those of leucine dehydrogenases from other sources. About 16% of the amino acid residues of the deduced amino acid sequence were different from the sequence obtained by X-ray analysis of the B. sphaericus enzyme. The recombinant enzyme was purified to homogeneity with a 79% yield. The enzyme was a homooctamer (340 kDa) and showed the activity of 71.7 μmol·min⁻¹·mg⁻¹) of protein. The mutant enzymes, in which more than six amino acid residues were deleted from the C-terminal of the enzyme, showed no activity. The mutant enzyme with deletion of four amino acid residues from the C-terminal of the enzyme was a dimer and showed 4.5% of the activity of the native enzyme. The dimeric enzyme was more unstable than the native enzyme, and the K_m values for -leucine and NAD⁺ increased. These results suggest that the Asn-Ile-Leu-Asn residues of the C-terminal region of the enzyme play an important role in the subunit interaction of the enzyme.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Isolation of an oxygen-evolving Photosystem II preparation containing only one tightly bound calcium atom from a chlorophyll b-deficient mutant of rice

Oxygen-evolving Photosystem II (PS II) particles were prepared from the thylakoid membranes of a chlorophyll b-less rice mutant, which totally lacks light-harvesting chlorophyll a/b proteins, after solubilization with β-octylglucoside. The preparation was essentially free of Photosystem I as judged from its low-temperature fluorescence spectrum and polypeptide composition. The PS II particles contained all the major subunit polypeptides of the PS II reaction center core complexes and the three extrinsic proteins related to oxygen evolution. The relative abundances of the 33, 21 and 15 kDa proteins were 100, 64 and 20%, respectively, of the corresponding proteins in the mutant thylakoids. The chlorophyll-to-Q_A ratio was 53 and there was only one bound Ca²⁺ per Q_A. Thus, one of the two bound Ca²⁺ present in the oxygen-evolving PS II membrane preparations from wild-type rice (Shen J.-R., Satoh, K. and Katoh, S. (1988) Biochim. Biophys. Acta 933, 358–364) is missing. The mutant PS II particles were highly active in oxygen evolution in the absence of exogenously added Ca²⁺, although addition of 5 mM Ca²⁺ enhanced the activity by 30%. When the 21 and 15 kDa proteins were supplemented to the particles, the Ca²⁺-effect disappeared and the rate of oxygen evolution increased to a level exceeding 1000 μmol O₂ per mg chlorophyll per h. The results indicate that the number of Ca²⁺ needed to promote a high rate of oxygen evolution is one per PS II in higher plants.     

Purification and characterization of an enantioselective carbonyl reductase from Candida viswanathii MTCC 5158

A highly enantioselective carbonyl reductase produced by a new yeast strain Candida viswanathii MTCC 5158, which was isolated using an acetophenone enriched medium, has been purified and characterized. The enzyme has been purified to near homogeneity using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The molecular properties of the carbonyl reductase suggested the native enzyme to be tetrameric, with an apparent molecular weight of 120 kDa, the monomer being about 29 kDa. Acetyl aryl ketones were found to be the preferred substrates for the enzyme and the best reaction was the enantioselective reduction of acetophenone. The enzyme yielded (S)-alcohol in preference to (R)-alcohol and utilized NADH, but not NADPH as the cofactor. The purified enzyme exhibited maximum enzyme activity at pH 7.0 and 60 °C. The enzyme retained about 80% of its activity after 7 h incubation at 25 °C in sodium phosphate buffer (50 mM, pH 7.0). The addition of reducing agents like dithiothreitol and β-mercaptoethanol enhanced the enzyme activity while organic solvents, detergents and chaotropic agents had deleterious effect on enzyme activity. Metal chelating agents like hydroxyquinoline and o-phenanthroline have significant effect on enzyme activity suggesting that the carbonyl reductase required the presence of a tightly bound metal ion for activity or stability. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for acetophenone and NADH were 59.21 μmol/(min mg) protein and 0.153 mM and 82.64 μmol/(min mg) protein and 0.157 mM at a concentration range of 0.2–2 mM acetophenone (NADH fixed at 0.5 mM) and 0.1–0.5 mM NADH (acetophenone fixed at 2 mM), respectively.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

Catalytic properties of the endoxylanase I from Thermoascus aurantiacus

Endo-β-1,4-xylanase I previously purified from Thermoascus aurantiacus solid state culture was further characterized. The enzyme had a molecular weight of 33 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 31 kDa by gel filtration. Thin layer chromatography (TLC) analysis showed that endoxylanase liberates aldotetrauronic acid MeGlcA-1,2-Xylβ-1,4-Xylβ-1,4-Xyl as the shortest acidic fragment from glucuronoxylan and an isomeric xylotriose (Xyl₃) of the structure Xylβ1-3Xylβ1-4Xyl from rhodymenan. The enzyme performed ideally on O-acetyl-4-O-methylglucuronoxylan, liberating large amounts of short acetylated and non-acetylated fragments. Also, the enzyme was capable to hydrolyse arabinoxylan to arabinose (Arab), xylose (Xyl) and xylobiose (Xyl₂). The enzyme degraded pNPX (4-nitrophenyl β- -xylopyranoside) by a complex reaction pathway that involved both hydrolysis and glycosyl transfer reactions. The enzyme tolerates the replacement of β-xylopyranosyl units in several artificial substrates by β-glucopyranosyl, - -arabinopyranosyl and - -arabinofuranosyl units and was active on pNPC (4-nitrophenyl β- -cellobioside), pNP-Arap (4-nitrophenyl - -arabinopyranoside) and pNPAraf (4-nitrophenyl - -arabinofuranoside). The enzyme also hydrolysed the 4-methylumbelliferyl glycosides of β- -xylobiose and β- -xylotriose at the agluconic linkage. The results suggested that the xylanase I from T. aurantiacus has catalytic properties similar to those belonging to family 10.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

Dopamine oxidation products inhibit Na+, K+-ATPase activity in crude synaptosomal-mitochondrial fraction from rat brain

The diverse damaging effects of dopamine (DA) oxidation products on brain subcellular components including mitochondrial electron transport chain have been implicated in dopaminergic neuronal death in Parkinson's disease. It has been shown in this study that DA (50-200 μM) causes dose-dependent inhibition of Na₊, K₊-ATPase activity of rat brain crude synaptosomal-mitochondrial fraction during in vitro incubation up to 2 h. The enzyme inactivation is prevented by catalase and the metal-chelator (diethylenetriamine penta-acetic acid) but not by superoxide dismutase or hydroxyl-radical scavengers like mannitol and dimethylsulphoxide (DMSO). Further, reduced glutathione and cysteine, markedly prevent DA-mediated inactivation of Na₊, K₊-ATPase. Under similar conditions of incubation, DA (200 μM) leads to the formation of quinoprotein adducts (protein-cysteinyl catechol) with synaptosomal-mitochondrial proteins and the phenomenon is also prevented by glutathione (5 mM) or cysteine (5 mM).

The available data imply that the inactivation of Na₊, K₊-ATPase in this system involves both H₂O₂ and metal ions. The reactive quinones by forming adducts with protein thiols also probably contribute to the process, since reduced glutathione and cysteine which scavenge quinones from the system protect Na₊, K₊-ATPase from DA-mediated damage. The inactivation of neuronal Na₊, K₊-ATPase by DA may give rise to various toxic sequelae with potential implications for dopaminergic cell death in Parkinson's disease.     

High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

Vasoactive intestinal peptide (VIP) bound with high affinity (K_d 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His¹]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound ¹²⁵I-VIP whereas VIP(16–28) and VIP(1–22-NH₂) were inactive. The binding of ¹²⁵I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, [pCl-D-Phe⁶,Leu¹⁷]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated ¹²⁵I-VIP was bound to receptors on the cell surface. The internalized ¹²⁵I-VIP was completely degraded to ¹²⁵I-tyrosine which was released from the cells. Degradation of internalized ¹²⁵I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of ¹²⁵I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound ¹²⁵I-VIP, but caused retention of internalized ¹²⁵I-VIP.