Time-dependent Structure and Activity Changes of α-Chymotrypsin in Water/Alcohol Mixed Solvents

Secondary structure of α-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. α-Chymotrypsin formed β-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, α-chymotrypsin took on a native-like structure, which gradually changed to β conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, α-chymotrypsin tended to form stable β-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to α-helix structure with substantial loss in catalytic activity was observed when α-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50%. In water/2,2,2-trifluoroethanol 90/10, α-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of β-sheet structure.     

A Study on the Process of Lipase-catalyzed Synthesis of α-pinene Oxide in Organic Solvents

The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L^?1 h^?1 when the amount of H₂O₂ increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H₂O₂ for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.     

Probes for Catalytic Action of α-Chymotrypsin in Plastein Synthesis

A plastein was synthesized with α-chymotrypsin from a dialyzable fraction of a peptic hydrolysate of soybean protein.

The plastein was obtainable also by use of an insoluble preparation of α-chymotrypsin. This may rule out the possibility that the plastein is a product resulting from some chemical peptide-protein (enzyme) aggregation.

No appreciable amount of the plastein was produced when chymotrypsinogen was used instead of α-chymotrypsin.

The plastein synthetic, as well as the protein hydrolytic, activity of α-chymotrypsin was inhibited more or less by a hydrophobic inhibitor (n-hexane), a competitive inhibitor (benzolyl-d,l-phenylalanine), and divalent cations (Zn²⁺, Hg²⁺ and Cu²⁺); the degree of inhibition in each case was approximately similar against both the synthetic and the hydrolytic activities.

Either diisopropylphosphorylation of the β-O of Ser-195 or methylation of the 3-N of His-57 imidazole of α-chymotrypsin repressed the synthetic, as well as the hydrolytic, activity.

Based on these results a possible mechanism was discussed of the plastein synthesis by α-chymotrypsin, especially in relevance to its acylation and deacylation.     

Salt Effects on Plastein Formation by α-Chymotrypsin

The plastein formation by α-chymotrypsin from an ovalbumin hydrolysate was affected in an order of valency of salts when the concentration of each salt was 1 m. Monovalent cations were rather effective at this concentration and enhanced the plastein yield by 10%. In the presence of NaCl, the plastein formation showed two distinct maximal rates at its concentrations of 0.1 m and 0.8 m. The first maximum was considered to be resulted from an increase in enzyme activity, since chymotryptic hydrolysis of both N-acetyl-l-tyrosine ethyl ester and benzyloxycarbonyl-l-phenylalanine p-nitrophenyl ester was activated at an NaCl concentration of 0.1 ~ 0.2 m. The second maximum was ascribed to the salting-out of the product due to the higher concentration of NaCl. A salt-tolerant protease was also used to confirm the above conclusions. It was observed that this enzyme was much effective in producing a plastein at a high NaCl concentration. This may be due to the fact that both the enzyme activation effect and the product salting-out effect participate co-operatively.     

Increase of Catalytic Activity of α-Chymotrypsin by Metal Salts for Transesterification of an Amino Acid Ester in Ethanol

α-Chymotrypsin-catalyzed transesterification of n-acetyl-l-tyro-sine methyl ester in ethanol was markedly accelerated by addition of small amounts of divalent metal salts. The reaction rate depended not only on the nature of metal ions but also on the nature of anionic counter ions. Calcium acetate was the most effective among the metal salts used. The reaction followed Michaelis–Menten kinetics, and it was found that the reaction increase is due to the increase in k_cat.     

Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N′-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of α-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20°C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.Linus Olofsson and Pernilla Söderberg authors have contributed equally to the work.     

Sugar Specificity in the Induction and on the Activity of Streptomyces α-d-Galactosidases

The α-d-galactosidases of six Streptomyces strains were examined on their inducer susceptibility, substate specificity, and inhibitor susceptibility. In all strains examined, α-d-galactosidase was induced by d-galactose, but neither by d-fucose nor by l-arabinose. α-d-Fucosidase activity was always induced accompanying with α-d-galactosedase activity. β-l-Arabinosidase activity, however, was never observed. These α-d-galactosidases were purified to electrophoretically pure degree by successive ammonium sulfate and ethanol precipitation, and ion exchange and gel filtration chromatography. The purified preparations from six strains were different from each other in their chromatographic behaviors and in some physical properties, but they all showed strong α-d-fucosidase activity as well. The α-d-galactosidase activities were strongly inhibited by d-galactose and l-arabinose, but scarcely by d-fucose. On the other hand, their α-d-fucosidase activities were inhibited by d-fucose as well as by d-galactose and l-arabinose.     

Is the Adenosine Receptor Modulation of Histamine-Induced Accumulation of Inositol Phosphates in Cerebral Cortical Slices Mediated by Effects on Calcium Ion Fluxes?

In this report, we show that under conditions designed to provide an initially uniform incorporation of [3H]inositol into mouse and guinea pig cerebral cortical slices prior to agonist stimulation, the accumulation of 3H-inositol phosphates (3H-InsPx, x = 1-4) induced by histamine in mouse and guinea pig cerebral cortical slices increased in a quasilinear manner with increasing added calcium. Raising the ambient calcium ion concentration failed to reduce the adenosine receptor-mediated inhibition of the histamine-induced 3H-InsPx response in mouse cerebral cortical slices. Similarly, the potentiation of the histamine response by adenosine receptor activation in guinea pig cerebral cortical slices was unaffected by lowering the added calcium ion concentration. The presence of the calcium ionophore A23187 (33 microM) produced 3H-InsPx responses in both mouse and guinea pig cerebral cortical slices, which were not affected by the presence of the stable adenosine analogue 2-chloroadenosine. A23187 also potentiated the accumulation of 3HInsPx induced by histamine in both species. Both the inhibitory and potentiatory modulations of the histamine response by 2-chloroadenosine in mouse and guinea pig, respectively, were still apparent in the presence of A23187. These results indicate that the histamine-induced 3H-InsPx accumulations in both mouse and guinea pig cerebral cortical slices are sensitive to variations in calcium ion concentrations. However, the adenosine receptor modulations of the histamine responses are relatively insensitive to fluctuations in either extra- or intracellular calcium ion concentrations, and thus cannot be mediated by effects on calcium ion movements.     

Influence of Organic Solvents on Catalytic Behaviors and Cell Morphology of Whole-Cell Biocatalysts for Synthesis of 5′-Arabinocytosine Laurate

A whole-cell based method was developed for the regioselective synthesis of arabinocytosine laurate. Among the seven kinds of bacteria strains tested in the acylation reaction, Pseudomonas fluorescens gave the highest productivity and a higher 5′-regioselectivity than 99%. Compared with pure organic solvents, the use of organic solvent mixtures greatly promoted the yield of the whole-cell catalyzed reaction, but showed little influence on the 5′-regioselectivity. Of all the tested solvent mixtures, the best reaction result was found in isopropyl ether/pyridine followed by isopentanol/pyridine. However, the whole-cells showed much lower thermostability in isopropyl ether/pyridine than in THF-pyridine. To better understand the toxic effects of the organic solvents on P. fluorescens whole-cells and growing cells were further examined. Significant influences of organic solvents on the biomass of the cells were found, which differed depending on the type of solvents used. SEM analysis visually revealed the changes in the surface morphology of whole-cells and growing cells cultured in media containing various organic solvents, in terms of surface smoothness, bulges and changed cell sizes. Results demonstrated that organic toxicity to cell structure played an important role in whole-cell mediated catalysis.     

Effects of water content on the enantioselectivity of α-chymotrypsin catalysis in organic media

Summary The -chymotrypsin-catalyzed transesterification between N-trifluoroacetyl-DL-phenylalanine trifluoroethyl ester and 1-propanol was carried out in a variety of organic solvents. The addition of small quantities of water enhanced both the rate of reaction and enantioselectivity. A high enantioselectivity was achieved in ethyl acetate (E = 120), diethyl ether (86), or acetonitrile (60). The competing hydrolysis became significant at water content higher than 0.5% (w/w).     

Catalytic Role of the Metal Ion in the Metallo-��-lactamase GOB

Metallo-β-lactamases (MβLs) stand as one of the main mechanisms of bacterial resistance toward carbapenems. The rational design of an inhibitor for MβLs has been limited by an incomplete knowledge of their catalytic mechanism and by the structural diversity of their active sites. Here we show that the MβL GOB from Elizabethkingia meningoseptica is active as a monometallic enzyme by using different divalent transition metal ions as surrogates of the native Zn(II) ion. Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give rise to the most active enzymes and are shown to occupy the same binding site as the native ion. However, Zn(II) is the only metal ion capable of stabilizing an anionic intermediate that accumulates during nitrocefin hydrolysis, in which the C–N bond has already been cleaved. This finding demonstrates that the catalytic role of the metal ion in GOB is to stabilize the formation of this intermediate prior to nitrogen protonation. This role may be general to all MβLs, whereas nucleophile activation by a Zn(II) ion is not a conserved mechanistic feature.     

Effects of Hofmeister ions on the α-helical structure of proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO₄ in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO₄ originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO₄ denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.     

Effects of Dietary Casein and Adrenal Hormone on the Dietary Induction of α-Amino-β-carboxymuconate-ε-semialdehyde Decarboxylase Activity in Rat

The effects of dietary casein and adrenal hormone on the dietary induction of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSDase) activity in rat liver and kidneys were examined. Of three levels of casein in the diet examined (7.5%, 15% and 30%), only the feeding of a 30% casein diet to normal rats after three days on a protein-free diet resulted in a significant increase in the activity of ACMSDase in liver and kidneys on the following morning. The degree of the increase in this activity was higher in the liver than the kidneys. Dietary induction of ACMSDase activity disappeared when rats were adrenalectomized six days prior to the start of the experiment. In sham-operated rats, dietary induction of liver ACMSDase activity was as high as that in normal rats, however, that in kidneys disappeared. Prednisolone (synthetic adrenal hormone with glucocorticoid activity) injection of adrenalectomized rats led to the recovery of the dietary induction of liver ACMSDase activity. Blood serum corticosterone levels in normal rats on the last day of the feeding period remained maximum and then decreased gradually until 04:00, and tended to be higher in rats fed a protein-free diet than in those fed a 30% casein diet. These results suggest that the dietary induction of ACMSDase activity occurs only when a sufficient amount of dietary casein is ingested in the presence of a physiologically significant concentration of blood serum glucocorticoid in rats.     

Effects of phospholipids on the antioxidant activity of α-tocopherol in the singlet oxygen oxidation of canola oil

This study evaluated the effects of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the antioxidative activity of α-tocopherol during oxidation of canola oil by singlet oxygen at 10°C for seven hours. Singlet oxygen was produced by chlorophyll b (4 ppm) under 1,700 lux. The oxidation of oil was evaluated by headspace oxygen consumption by gas chromatography and peroxide values (POVs). Concentrations of PC, PE, chlorophyll, and α-tocopherol were determined by HPLC. PC and PE protected chlorophyll from degradation, but they accelerated the degradation of α-tocopherol under singlet oxygen. Contents of PC and PE did not change for seven hours under singlet oxygen. α-Tocopherol significantly lowered POV and headspace oxygen consumption of canola oil under singlet oxygen, and its antioxidant activity was increased by the co-presence of PC and PE. PC and PE increased chemical quenching of singlet oxygen by tocopherol in decreasing the oil oxidation.     

γ-Irradiation Effects on the Antioxidative Activity Developing in the Amino Acid-Sugar Reaction

The effects of γ-irradiation on the antioxidative activity developing in the amino acid-sugar reaction were investigated. The antioxidative activity of the nondialyzable melanoidin prepared from glycine and d-glucose was not much affected by γ-irradiation. However, the development of the antioxidative activity of an l-leucine-d-glucose solution on heating was markedly accelerated when the mixture had been preirradiated with γ-rays, and the development of the activity was more prominent than that of the brown color. The irradiation of a glucose solution alone accelerated the antioxidative activity development when heated with leucine, but the irradiation of a leucine solution alone did not cause a similar effect when heated with glucose. Except an l-cysteine-glucose combination, all combinations of amino acids and sugars tested gave rise to almost similar antioxidative effects.     

Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases

summary. A thermostable -amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca²⁺. The stability of the native enzyme was found to increase upon modification as observed from the increase in t_1/2 in the absence of Ca²⁺ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with C_m and T_m values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.This article is dedicated to Dr. P.V. Sundaram.     

Effects of macromolecular crowding on the structural stability of human α-lactalbumin

The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments. α-Lactalbumin, as a model system for protein-folding studies, has been used extensively because it can form stable molten globule states under a range of conditions. Here we report that the crowding agents Ficoll 70, dextran 70, and polyethylene glycol (PEG) 2000 have different effects on the structural stability of human α-lactalbumin (HLA) represented by the transition to a molten globule state: dextran 70 dramatically enhances the thermal stability of Ca(2+)-depleted HLA (apo-HLA) and Ficoll 70 enhances the thermal stability of apo-HLA to some extent, while PEG 2000 significantly decreases the thermal stability of apo-HLA. Ficoll 70 and dextran 70 have no obvious effects on trypsin degradation of apo-HLA but PEG 2000 accelerates apo-HLA degradation by trypsin and destabilizes the native conformation of apo-HLA. Furthermore, no interaction is observed between apo-HLA and Ficoll 70 or dextran 70, but a weak, non-specific interaction between the apo form of the protein and PEG 2000 is detected, and such a weak, non-specific interaction could overcome the excluded-volume effect of PEG 2000. Our data are consistent with the results of protein stability studies in cells and suggest that stabilizing excluded-volume effects of crowding agents can be ameliorated by non-specific interactions between proteins and crowders.     

Effect of Environmental Conditions on the α-Glucosidase Inhibitory Activity of Mulberry Leaves

Mulberry leaves have been used as the sole food for silkworms in sericulture, and also as a traditional medicine for diabetes prevention. Mulberry leaf components, for example 1-deoxynojirimycin (1-DNJ), inhibit the activity of α-glucosidase and prevent increased blood glucose levels, and they are highly toxic to caterpillars other than silkworms. The α-glucosidase inhibitory activity of mulberry leaves changes with the season, but it is unknown which environmental conditions influence the α-glucosidase inhibitory activity. We investigated in this study the relationship between the α-glucosidase inhibitory activity and environmental conditions of temperature and photoperiod. The results demonstrate that low temperatures induced decreasing α-glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing α-glucosidase inhibitory activity. These results suggest that the α-glucosidase inhibitory activity was related to the defense mechanism of mulberry plants against insect herbivores.     

Effects of α-Synuclein Monomers Administration in the Gigantocellular Reticular Nucleus on Neurotransmission in Mouse Model

The aim of the study was to examine the Braak’s hypothesis to explain the spreading and distribution of the neuropathological changes observed in the course of Parkinson’s disease among ascending neuroanatomical regions. We investigated the neurotransmitter levels (monoamines and amino acid concentration) as well as tyrosine hydroxylase (TH) and transglutaminase-2 (TG2) mRNA expression in the mouse striata (ST) after intracerebral α-synuclein (ASN) administration into gigantocellular reticular nucleus (Gi). Male C57BL/10 Tar mice were used in this study. ASN was administrated by stereotactic injection into Gi area (4 μl; 1 μg/μl) and mice were decapitated after 1, 4 or 12 weeks post injection. The neurotransmitters concentration in ST were evaluated using HPLC detection. TH and TG2 mRNA expression were examined by Real-Time PCR method. At 4 and 12 weeks after ASN administration we observed decrease of DA concentration in ST relative to control groups and we found a significantly higher concentration one of the DA metabolites—DOPAC. At these time points, we also noticed the increase in DA turnover determined as DOPAC/DA ratio. Additionally, at 4 and 12 weeks after ASN injection we noted decreasing of TH mRNA expression. Our findings corresponds with the Braak’s theory about the presence of the first neuropathological changes within brainstem and then with time affecting higher neuroanatomical regions. These results obtained after administration of ASN monomers to the Gi area may be useful to explain the pathogenesis of Parkinson’s disease.