Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K_M value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V_max and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Myoinositol hexaphosphate as a potential inhibitor of α-amylases

Myoinositol hexaphosphate (MHP) strongly inhibited α-amylases of different origins. The inhibition of wheat α-amylase is noncompetitive with an apparent K_i value of 1 mM, pH dependent and markedly increased by the preincubation of enzyme with MHP before the addition of substrate. Addition of Ca²⁺ did not reverse the inhibition of α-amylase indicating that its inhibition was not due to the binding of Ca²⁺ by MHP.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Subcellular localization,metal ion requirement and kinetic properties of arginase from the gill tissue of the bivalve Semele solida

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.     

Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain

 We have selectively replaced the catalytic zinc of the catalytic domain of stromelysin-1 (SCD) with other transition metals. Dialysis of the enzyme against 2 mM 1,10-phenanthroline, 20 mM Hepes, pH 7.5 in the presence of 10 mM CaCl₂ removes the catalytic zinc, leaving the structural zinc site intact. Dialysis with metal-free buffer followed by the new metal ion replaces the catalytic zinc forming a metal hybrid enzyme. Full incorporation of 1 mol Co²⁺, Ni²⁺, or Cd²⁺/mol enzyme is confirmed by atomic absorption spectrometry while the weaker binding Mn²⁺ yields a value of 0.4 mol Mn²⁺/mol enzyme after dialysis against 1 μM Mn²⁺. The activity of the monozinc enzyme is <10% while its activity is restored upon the addition of zinc and other transition metals. The k _cat values for the Co²⁺, Mn²⁺, Cd²⁺, and Ni²⁺ enzymes are respectively 99%, 54%, 19%, and 17% of the value for the native enzyme, while the respective k _cat/K _m values are 36%, 29%, 7%, and 16% toward the fluorescent heptapeptide substrate, DnsPLALRAR. The zinc and metal hybrid SCD cleave DnsPLA↓LRAR, and DnsPLE↓LFAR, exclusively at one bond, while DnsPLA↓L↓WAR and DnsPLA↓L↓FAR are cleaved at two positions. The double cleavage of DnsPLALWAR and DnsPLALFAR catalyzed by SCD is in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis is the different cleavage site specificity of the metal hybrids toward the A-L and L-W bonds of the DnsPLALWAR substrate. Thus the k _cat values of the Co/Zn hybrid for the cleavage of the A-L bond in the DnsPLALRAR and DnsPLAWAR substrates are 5- and 8-fold greater than those for the Cd/Zn hybrid compared to a 140-fold difference for the corresponding k _cat values for the L-W bond cleavage. These results imply that the catalytic metal of SCD is not only involved in catalysis but also influences the substrate specificity of the enzyme. Received: 30 December 1997 / Accepted: 23 February 1998     

Thermostable succinyl-Coenzyme A synthetase from Nitrosomonas europaea ATCC 25978: Purification and properties

The ammonia-oxidizing chemoautotrophic bacterium Nitrosomonas europaea possesses prominant succinate-reducing activity of succinyl-Coenzyme A synthetase (SCS, EC 6.2.1.5). SCS was purified as an electrophoretically homogeneous protein from Nitrosomonas europaea strain ATCC 25978 about 275-fold, with a 3.9% activity yield. The molecular mass of the native enzyme was estimated to be about 130 kDa by gel filtration, whereas SDS-PAGE gave two protein bands with M_r values of 29 (α) and 36 kDa (β). The isoelectric point of the enzyme was 5.3. The apparent K_m values of the enzyme for ATP, succinate and CoA were 0.4 mM, 5 mM and 0.1 mM, respectively. The pH and temperature optima of the SCS were about 5.0 and 55°C, respectively. The SCS was stable in the pH range of 8.0–10.0 and up to 70°C. The enzyme was thermostable; 50% of the enzyme activity was retained at 90–100°C for 10 min. The SCS was activated by Mg²⁺ at 1.0–100 mM, but inhibited by Cu²⁺ (0.1 mM) and SDS (1.0 mM). The enzyme utilized ATP as the preferred substrate.     

A d-glucose- and d-xylose-tolerant GH1 β-glucosidase from Cellulosimicrobium funkei HY-13, a fibrolytic gut bacterium of Eisenia fetida

The GluM gene (1491-bp) coding for a β-glucosidase comprising a single catalytic glycoside hydrolase family 1 domain from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium funkei HY-13, was cloned and over-expressed in Escherichia coli BL21. The recombinant histidine-tagged enzyme (rGluM: 56 kDa) displayed the highest cleavage activity toward p-nitrophenyl (pNP)-β-d-glucopyranoside at pH 5.0 and 40 °C. The β-glucosidase activity of rGluM was enhanced over 1.8-fold of its original activity in the presence of 1 mM Ca²⁺, Ni²⁺, Mn²⁺, and Co²⁺ ions, respectively, while it was highly sensitive to 5 mM N-bromosuccinimide and 1 mM Hg²⁺. The susceptibility of some pNP-sugar derivatives and d-cellobiose to rGluM was evaluated to be in the order of pNP-β-d-glucopyranoside > pNP-β-d-galactopyranoside > d-cellobiose > pNP-β-d-cellobioside > pNP-β-d-mannopyranoside. The k_cat/K_m values of rGluM toward pNP-β-d-glucopyranoside, pNP-β-d-galactopyranoside, and d-cellobiose were 302.28, 179.73, and 6.40 mM^-1 s^-1, respectively. At a concentration below 1.0 M, d-galactose was a potent activator of rGluM with β-glucosidase activity enhanced by approximately 160% in a dose-dependent manner. Moreover, the d-glucose (< 400 mM) and d-xylose (≤ 700 mM) stimulation of rGluM suggests that it can be exploited as a potential biocatalyst to generate d-glucose molecules in d-cellobiose degradation.     

Biochemical properties of the alkaline lipase of Bacillus flexus XJU-1 and its detergent compatibility

The possibility of using Bacillus flexus XJU-1 lipase in detergent preparations was studied. The enzyme was monomeric protein as confirmed by liquid chromatography-mass spectrometry and its molecular weight was 15.95 kDa. The lipase showed optimum activity at pH 10.0 and was 100% stable for 24 h at pH 10.0 and 11.0. It exhibited maximum activity at 70°C and retained more than 70% of the initial activity at 60, 70 and 80°C for 24 h. The activity was stimulated by Ca²⁺, Ba²⁺, Mg²⁺ and Co²⁺, whereas 50% of the initial activity was lost with Fe³⁺ and Hg²⁺. The activity was inhibited by 10 mM N-bromosuccinimide and tosyl-L-lysylchloromethylketone, while N-ethylmaleimide, phenylmethylsulphonylfluoride and urea did not show any effect. The enzyme significantly hydrolysed olive, cottonseed, sunflower, groundnut, and gingelly oils. With p-nitrophenyl palmitate, V_max and K_m were 62.5 U/mL and 2.25 mM, respectively. The lipase maintained its stability in Tween-80, Triton-100 and H₂O₂ at 1%, but an activation of 10% and a reduction of 15% in relative activity were observed with NaClO and sodium dodecyl sulphate, respectively. The enzyme retained maximum storage stability for 20 days at ?20, 4 and 30°C. In the presence of 0.7% (w/v) Ariel, Henko, Super wheel, Tide plus and Rin, a retention of more than 84.90% initial activity was recorded after 24 h at 60°C. The supplementation of the lipase to the detergents improved the olive oil stain removal. These properties suggested the present enzyme as a potential additive for detergent preparations.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.     

Red cell Na+,K+-ATPase: a method for estimating the extent of inhibition of an enzyme sample containing an unknown amount of bound cardiac glycoside.

Na⁺, K⁺-ATPase activities of the membranes obtained from intact red cells that are exposed to ouabain, digoxin, and digitoxin are inhibited. The extent of inhibition of each enzyme sample can be found by the following two assays: 1) Activity is measured by the addition of enzyme to a buffered solution containing 2 mM ATP, 3 mM Mg²⁺, 1 mM EDTA, 100 mM Na⁺, and 25 mM K⁺. Since little regeneration of the inhibited enzyme occurs under these conditions, the measured activity is that of the partially inhibited enzyme. 2) Enzyme is preincubated for ten minutes in the same solution from which Mg²⁺ and K⁺ are omitted, and then assayed by the addition of Mg⁺ and K⁺. Since the inhibited enzyme is completely regenerated during the preincubation period, the activity measured here serves as a control for that determined in the first assay.     

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

The conversion of 3-deoxyarabinoheptulosonate 7-phosphate to 3-dehydroquinate by sorghum seedling preparations

Partially purified preparations from etiolated sorghum seedlings catalyzed the conversion of DAHP to DHQ. The reaction catalysed by DHQ synthetase was stimulated by 0.1 μM to 0.1 mM NAD in the presence O-0.5 mM Co²⁺. NADH at 1 μM stimulated the reaction as much as 50% but became inhibitory at 100μM. Co²⁺ at 0.5mM stimulated enzyme activity 3-fold; Mg²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ were not stimulatory. EDTA at 5 mM inhibited the reaction 95% but its effects were reversed by equal concentrations of Co²⁺. Phe, Tyr, Trp, t-cinnamate, several hydroxylated cinnamates, DHS, quinate, and shikimate at 0.3 mM failed to affect enzyme activity but slight inhibition occurred with DHQ and protocatechuic acid at 0.3 mM, inhibition being 14 % and 22 %, respectively. DHQ synthetase activity also was detected in spinach leaves and potato tuber tissue. Synthetase activity appeared to increase in response to injury of potato tuber and sweet potato root tissues.     

Amdxidation and demethylation of N,N-dimethylaniline by rabbit liver and lung microsomes effects of age and metals

Lung N-oxidase enzyme activity was about three times higher than liver N-oxidase at the pH optimum, about pH 8.9, whereas the activities were nearly the same at more physiological ranges of pH. The lung N-oxidase was also stimulated about 2-fold by 100 mM Mg²⁺ and by 0.1 mM Hg²⁺, whereas liver N-oxidase activity was inhibited by these concentrations of ions. The difference in response of liver and lung enzymes to Mg²⁺ and Hg²⁺ was not altered by preparing the microsomes in the presence of 50 mM ethylenediamine tetraacetic acid (EDTA) in 0.1 M Tris (hydroxymethyl) amino methane (Tris) buffer or 50 mM EDTA in 0.1 M KPO₄ buffer, both at pH 7.6, indicating that the differences are probably not due to the presence of endogenous metals. The difference between the liver and lung N-oxidase systems may be due to the tissue environment rather than to the enzyme itself since mercury stimulation of lung N-oxidation began to disappear upon partial purification of the N-oxidase enzymes. In contrast to the effects of Hg²⁺ and Mg²⁺, 1 mM Ni²⁺ enhanced liver N-oxidase activity about 30% and 5 mM Ni²⁺ stimulated lung enzyme activity about 30% whereas concentrations above 10 mM were inhibitory to both N-oxidases. Both liver and lung demethylase activities were inhibited by these concentrations of Mg²⁺, Hg²⁺ and Ni²⁺.Various suifhydryl reagents were also tested for their effects on these enzymes. The mercurials, para-chloromercurybenzoate (pCMB) and phenylmercuryacetate (PMA) at concentrations of 0.1 mM had the same effect as HgCl₂ inhibiting both demethylases and liver N-oxidase, but stimulating lung N-oxidase activity. However, 0.1 mM to 1 mMN-ethylmaleimide (NEM) and iodoacetamide had little if any effect on either liver or lung N-oxidase. It was also shown that Hg²⁺ effects on N-oxidase activity could be overcome by dilution.Changes in N,N-dimethyl aniline (DMA) metabolism with age were followed in rabbits from 4 days old to adult. There was a steady increase in lung demethylase activity and N-oxidase activity in the liver and lung to adult levels. However, the liver demethylase had a sharp increase in activity between 2 weeks and 1 month much like that seen with benzphetamine demethylase in rabbit liver.Activities of N-demethylase in liver and lung, and N-oxidr.se in liver from new-born rabbits were from 10 to 20 % of adult levels. However, in lung, N-oxidase activities in the newborn were about 50 % of adult levels. Microsomal N-oxidation in lungs from 2-day-old rabbits was stimulated by 0.1 mM mercury just as in the adult.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. K_M values were 1.59 and 0.53?mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. V_max values for CDNB and GSH were also determined as 5.58 and 1.88?EU/mL, respectively. In addition, inhibition effects of Ag⁺, Cu²⁺, Cd²⁺, Fe³⁺, Pb²⁺, Cr²⁺, Co²⁺ and Zn²⁺ metal ions were investigated on the enzyme activity and IC₅₀, K_i values were calculated for these metal ions.     

Evidence that Prostaglandin E2 Can Block Calcium-Activated 86Rb Efflux from Rat Brain Synaptosomes via a Protein Kinase C-Dependent Mechanism

Abstract: The effects of prostaglandin E₂ (PGE₂) on ⁸⁶Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K⁺) channel modulation. A selective dose-dependent inhibition of the calcium-activated charybdotoxin-sensitive component of efflux was found upon application of PGE₂. No significant effect was seen on basal and voltage-dependent components over the concentration range of 10–⁸ to 10–⁵M. The protein kinase C (PKC) inhibitors H-7 (10 μM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 40-phorbol 12, 13-dibutyrate, which has been reported to down-regulate PKC, abolished the PGE₂-in- duced inhibition, whereas HA1004 (10 μM) and Rp-3′,5’cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4β-Phorbol 12, 13-dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca²⁺-dependent component of ⁸⁶Rb efflux but also had no effect on the basal and voltage-dependent components. These data suggest that PGE₂ can inhibit rat brain nerve ending calcium-activated ⁸⁶Rb efflux, and this inhibition may involve PKC activation.