Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Immobilization of catalase via adsorption onto metal-chelated affinity cryogels

A poly (acrylamide-allylglycidyl ether) [p(AAm-AGE)] cryogel was prepared by radical polymerization of acrylamide (AAm) and allylglycidyl ether (AGE). Cibacron Blue F3GA (CB) was covalently attached to the p(AAm-AGE) cryogel via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. The CB-attached p(AAm-AGE) cryogel was chelated with Fe³⁺ ions. This immobilized metal ion affinity chromatography (IMAC) cryogel carrying 25.8 ± 2.0 μmol Fe³⁺ ions was used in adsorption studies to interrogate the effects of pH, protein initial concentration, flow rate, temperature and ionic strength on enzyme activity. Maximum adsorption capacities were found to be 75.7 ± 1.2 mg/g for p(AAm-AGE)-CB-Fe³⁺ cryogels and 60.6 ± 1.0 mg/g for p(AAm-AGE)-CB cryogels, respectively. The adsorbed amounts of catalase per unit mass of cryogel reached a plateau value at about 1.5 mg/mL at pH 6.0. The K_m values were found to be 0.73 ± 0.02 g/L for the free catalase and 0.18 ± 0.02 g/L for the immobilized catalase. The V_max value of free catalase (2.0 × 10³ U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5 × 10³ U/mg enzyme). It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the p(AAm-AGE)-CB-Fe³⁺ cryogel.     

Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane

Poly(itaconic acid) grafted and/or Fe(III) ions incorporated chitosan membranes were used for reversible immobilization of catalase (from bovine liver) via adsorption. The influences of pH and initial catalase concentration on the immobilization capacities of the CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membranes have been investigated in a batch system. Maximum catalase adsorption onto CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membrane were found to be 6.3 and 37.8 mg/g polymer at pH 5.0 and 6.5, respectively. The CH-g-poly(IA)-Fe(III) membrane with high catalase adsorption capacity was used in the rest of the study. The K_m value for immobilized catalase on CH-g-poly(IA)-Fe(III) (25.8 mM) was higher about 1.6-fold than that of free enzyme (13.5 mM). Optimum operational temperature was observed at 40 °C, a 5 °C higher than that of the free enzyme and was significantly broader. The optimum operational pH was same for both free and immobilized catalase (pH 7.0). Thermal stability was found to increase with immobilization. Free catalase lost all its activity within 20 days whereas immobilized catalase lost 23% of its activity during the same incubation period. It was observed that the same support enzyme can be repeatedly used for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. In addition, the CH-g-poly(IA)-Fe(III) membrane prepared in this work showed promising potential for various biotechnological applications.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Development of pH-sensitive tamarind seed polysaccharide-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology

The present study deals with the development of novel pH-sensitive tamarind seed polysaccharide (TSP)-alginate composite beads for controlled diclofenac sodium delivery using response surface methodology by full 3² factorial design. The effect of polymer-blend ratio (sodium alginate:TSP) and cross-linker (CaCl₂) concentration on the drug encapsulation efficiency (DEE, %) and drug release from diclofenac sodium loaded TSP-alginate composite beads prepared by ionotropic gelation was optimized. The observed responses were coincided well with the predicted values by the experimental design. The DEE (%) of these beads containing diclofenac sodium was within the range between 72.23 ± 2.14 and 97.32 ± 4.03% with sustained in vitro drug release (69.08 ± 2.36-96.07 ± 3.54% in 10 h). The in vitro drug release from TSP-alginate composite beads containing diclofenac sodium was followed by controlled-release pattern (zero-order kinetics) with case-II transport mechanism. Particle size range of these beads was 0.71 ± 0.03-1.33 ± 0.04 mm. The swelling and degradation of the developed beads were influenced by different pH of the test medium. The FTIR and NMR analyses confirmed the compatibility of the diclofenac sodium with TSP and sodium alginate used to prepare the diclofenac sodium loaded TSP-alginate composite beads. The newly developed TSP-alginate composite beads are suitable for controlled delivery of diclofenac sodium for prolonged period.     

Development of cloxacillin loaded multiple-unit alginate-based floating system by emulsion-gelation method

This work investigates the development, optimization and in vitro evaluation of liquid paraffin-entrapped multiple-unit alginate-based floating system containing cloxacillin by emulsion-gelation method for gastro retentive delivery. The effect of process variables like drug to polymer ratio by weight, and liquid paraffin to water ratio by volume on various physicochemical properties in case of liquid paraffin-entrapped calcium alginate beads containing cloxacillin applicable to drug entrapment efficiency, density and drug release was optimized using 3² factorial design and analyzed using response surface methodology. The observed (actual values) responses were coincided well with the predicted values, given by the optimization technique. The optimized beads showed drug entrapment efficiency of 64.63 ± 0.78%, density of 0.90 ± 0.05 g/cm³, and drug release of 56.72 ± 0.85% in simulated gastric fluid (pH 1.2) after 8 h with floating lag time of 8.45 min and floated well over 12 h in simulated gastric fluid (pH 1.2). The average size of all dried beads ranged from 1.73 ± 0.04 to 1.97 ± 0.08 mm. The beads were characterized by SEM and FTIR for surface morphology and excipients-drug interaction analysis, respectively. All these beads showed prolonged sustained release of cloxacillin over 8 h in simulated gastric fluid (pH 1.2). The cloxacillin release profile from liquid paraffin beads followed Korsmeyer-Peppas model over a period of 8 h with anomalous (non-Fickian) diffusion mechanism for drug release.     

Catalytic scavenging of peroxynitrite by catalase

Peroxynitrite (ONOO⁻/ONOOH), the product of the diffusion controlled reaction between nitric oxide (NO) and superoxide anion (), is a strong oxidizing and nitrating agent. Several heme proteins react rapidly with peroxynitrite, some of them catalyze its decomposition. In this work we found, contrary to previous reports, that catalase, a ferriheme enzyme, catalytically scavenges peroxynitrite. The second-order reaction rate constants of peroxynitrite decay catalyzed by catalase increase with decreasing pH and are equal to (2.7 ± 0.2) × 10⁶, (1.7 ± 0.1) × 10⁶ and (0.8 ± 0.1) × 10⁶ M⁻¹ s⁻¹ at pH 6.1, 7.1 and 8.0, respectively. This dependence suggests that peroxynitrous acid, ONOOH, is the species that reacts with heme center of catalase. The possible reaction mechanisms of the decay of peroxynitrite catalyzed by catalase and physiological relevance of this reaction are discussed.     

A novel core fractionation process of human plasma by expanded bed adsorption chromatography

Current plasma fractionation technology combines ethanol precipitation with packed bed chromatography. We have developed a novel core fractionation process comprising five expanded bed adsorption (EBA) chromatographic steps on high-density modified agarose/tungsten carbide beads. Plasma was first chromatographed on two diethyl amino-ethyl (DEAE)-tungsten carbide agarose adsorbents (respective mean particle diameters of d_v(0.5) = 190 and 37 μm) to isolate at 50 to 80% recovery a fraction containing 4 to 7 IU/ml factor II (FII), factor IX (FIX), and factor X (FX) (specific activity >1 IU/mg) and another enriched in FVIII and von Willebrand factor (vWF) (∼1 IU/ml and 0.6 IU/mg, respectively). The flow-through was adsorbed on 4% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand to isolate an 80% pure immunoglobulin G (IgG) at a 93% step recovery. A highly purified α1-antitrypsin was isolated at 95% step recovery by adsorbing the flow-through on 4% epoxy-crosslinked agarose-10% tungsten carbide adsorbent material coupled with a cationic ligand. Isolation of 98% pure albumin was achieved at a 99% step recovery by pH 4.5 adsorption of the flow-through on 6% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand. EBA may represent a feasible alternative core plasma fractionation tool.     

Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Impact of hapten presentation on antibody binding at lipid membrane interfaces

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K_D) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, K_D(bulk) = 1.7 ± 0.2 nM vs. 2.9 ± 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, K_D = 3.6 ± 1.1 nM vs. 2.0 ± 0.2 μM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands’ relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the K_D value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 ± 1.1 μM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (K_D = 21 ± 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.     

Metformin induces suppression of NAD(P)H oxidase activity in podocytes

Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms.The experiments were performed in normal (NG, 5.6 mM) and high (HG, 30 mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-³H]-glucose.ROS production increased by about 27% (187 ± 8 vs. 238 ± 9 arbitrary units AU, P < 0.01) in HG. Metformin (2 mM, 2 h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58 ± 0.07 to. 0.99 ± 0.06, and from 0.53 ± 0.03 to 0.64 ± 0.03; P < 0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C.We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2 ± 0.7 mg/g spores, corresponding to a calculated activity of 63,960 U/g spores; however, the measured activity was 41,120 ± 990.1 U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t_1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90 °C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1 M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety.     

A novel alkaline lipase from Ralstonia with potential application in biodiesel production

With the aim of isolating a biocatalyst able to catalyze biodiesel production from microbial source, Ralstonia sp. CS274 was isolated and a lipase from the strain (RL74) was purified. Molecular weight of RL74 was estimated to be 28,000 Da by SDS-PAGE. The activity was highest at 50-55 °C and pH 8.0-9.5 and was stable at pH 7.0-12.0 and up to 45 °C. It was resistant to oxidizing and reducing agents and the activity was enhanced by detergents. RL74 was 1,3 specific and K_m and V_max for p-nitrophenyl palmitate were 2.73 ± 0.6 mM and 101.4 ± 1.9 mM/min mg, respectively. N-terminal amino acid sequence showed partial homology with that of Penicillium lipases. RL74 produced biodiesel more efficiently in palm oil than in soybean oil; and the production was highest at pH 8.0, at 5% methanol and at 20% water content.     

Synthesis and characterization of a stable paramagnetic hexacoordinated oxochromium(IV) complex with dianionic tetradentate Schiff base ligand salen

A paramagnetic octahedral oxochromium(IV) complex with dianionic tetradentate ligand salen (where H₂salen is N,N′-bis(salicylidene)-1,2-ethylenediamine) has been synthesized. This compound [CrO(OH₂)(salen)] (1) is characterized by elemental analysis, magnetic moment measurement, IR, UV-Vis and EPR spectroscopic studies. Measured room temperature (RT) magnetic moment value is 2.96 BM for 1 indicates a d² system with a triplet ground state. The magnetic moment value rules out a large spin-orbit coupling. The RT and LNT powder EPR spectra of 1 in X-band clearly shows two lines, one around g = 1.965 and the other with larger intensity at g = 4.26 ± 0.10. The first line at g = 1.965 corresponds to the |0> ↔ |±1> transition from the Kramers doublet |±1>, while the broad and intense line at low field with the g-value of 4.26 ± 0.10 is due to the forbidden transition |−1> ↔ |+1>. Compound 1 displays two successive reductions at −0.76 and −1.63 V (versus Ag/AgCl), respectively, while it undergoes only one irreversible oxidation as evident from the well-defined anodic wave at +1.48 V in its cyclic voltammogram.     

Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n = 12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300 mM Tris, 28 mM glucose, 95 mM citric acid 5% glycerol to a concentration of 200 × 10⁶ sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100 mg/mL skimmed milk powder and 27.75 mM glucose (without glycerol) to a concentration of 400 × 10⁶ sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200 × 10⁶ sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25 mL straws, held for 2 h at 4 °C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean ± SEM) were significantly lower (P < 0.05) in P1 as compared to P2 (47.50 ± 1.23% vs. 55.63 ± 1.72%; 80.04 ± 1.29% vs. 84.04 ± 1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P > 0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.     

Comparative effects of cationic triarylmethane, phenoxazine and phenothiazine dyes on horse serum butyrylcholinesterase

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25 ^°C in 50 mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K_i values of 4.5 ± 0.50 μM, 0.41 ± 0.007 μM, 0.44 ± 0.086 μM and 0.050 ± 0.0074 μM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K′ values (≡[I]²_0.5 extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20 ± 0.096 μM, 0.0018 ± 0.0015 μM, 0.92 ± 0.23 μM and 0.23 ± 0.08 μM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.     

Regulation of respiration controlled by mitochondrial creatine kinase in permeabilized cardiac cells in situ: Importance of system level properties

The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, K_ia and K_a (1.94 ± 0.86 mM and 2.04 ± 0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44 ± 0.08 mM and 0.016 ± 0.01 mM, respectively). Apparent dissociation constants of creatine, K_ib and K_b (2.12 ± 0.21 mM 2.17 ± 0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28 ± 7 mM and 5 ± 1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.     

pH dependent copper binding properties of a CuA azurin variant with both bridging cysteines replaced with serines

A double mutant of Cu_A azurin was prepared in which both bridging cysteine thiolate ligands of the binuclear Cu_A center were replaced by serine. The copper binding properties of this protein were investigated, and shown to be pH dependent. At lower pH (5.2 ± 0.1), the protein binds one copper per protein molecule as demonstrated by electrospray ionization mass spectrometry. Copper titrations resulted in electronic absorptions at 730 nm (peak) and ca. 330 nm (shoulder) in the UV-Vis spectrum. EPR data show a four line pattern with hyperfine A_∥ = 150 G and g_∥ and g_⊥ values 2.32 and 2.03, characteristic of a type II (T2) copper. Superhyperfines to two nitrogen atoms were also observed. At higher pH (8.5 ± 0.1), the protein binds upto two copper atoms per protein molecule, and copper titrations exhibit a blue transition at 595 nm in the UV-Vis spectrum. The EPR data are consistent with two monomeric sites very similar to one another having hyperfines A_∥ = 182 and 150 G, g_∥ = 2.24 and 2.22 and a similar g_⊥ value of 2.01. These results indicate that both bridging cysteines play a critical role in the Cu_A center, and replacing them with serines is not enough to maintain the symmetrical diamond core structure or the characteristic electronic and functional properties of the Cu_A center.