Effect of cerebrocrast on the lymphocyte blast transformation activity in normal and streptozotocin‐induced diabetic rats

Both IDDM and NIDDM are characterized by deviations in peripheral T and B lymphocyte count, T_helper:T_suppressor ratio, as well as by impaired T_suppressor function. These abnormalities may promote insulin antibody and other antibody production, contributing to overt diabetes mellitus development in early stage of the disease. In the present study we explored the effects of cerebrocrast (1,4‐dihydropyridine derivative) administration on Con A‐ and IL‐2‐stimulated tissue lymphocyte blast transformation activity and on the thymus and lymph node mass in normal and streptozotocin (STZ)‐induced diabetic rats. It was established that cerebrocrast, administered four times at the doses of 0·05 and 0·5 mg kg⁻¹, has long‐term (up to 14 days) effects on the immune system and protects against the toxic effect of STZ in STZ‐induced diabetic rats, preventing thymus and lymph node mass loss. We conclude that cerebrocrast administration leads to the increase in number and activity of T_helper and T_suppressor lymphocytes. Glycolysis and DNA synthesis in these cells is augmented under the influence of cerebrocrast administration. We propose that the increase in lymphocyte suppressive activity caused by cerebrocrast administration may prevent the development of IDDM and NIDDM in patients with pre‐diabetes, but in patients with early and overt diabetes mellitus the drug administration may prevent the overexpression of insulin antibodies and other antibodies. The effect of cerebrocrast on the de novo production of insulin and IL‐2 receptors may be beneficial for IDDM and NIDDM patients. Copyright © 1999 John Wiley & Sons, Ltd.     

Effect of new and known 1,4-dihydropyridine derivatives on blood glucose levels in normal and streptozotocin-induced diabetic rats

Analysis of the effect of several 1,4-DHP Ca(2+) channel antagonists on experimental and clinical diabetes shows that structurally similar Ca(2+) channel antagonists can exert opposite effects on Ca(2+) influx, glucose homeostasis and insulin secretion. The influence of the Ca(2+) channel antagonists on pancreatic beta cell functions is dependent on lipophilicity, interactions with the cell membrane lipid bilayer, with SNAREs protein complexes in cell and vesicle membranes, with intracellular receptors, bioavailability and time of elimination from several organs and the bloodstream. In the present work we studied the effect at several doses of new compounds synthesized in the Latvian Institute of Organic Synthesis on blood glucose levels in normal and STZ-induced diabetic rats. The compounds tested were: 1,4-DHP derivatives cerebrocrast (1), etaftoron (2), OSI-1190 (3), OSI-3802 (4), OSI-2954 (5) and known 1,4-DHP derivatives: niludipine (6), nimodipine (7) and nicardipine (8) which possess different lipophilicities. Analysis of the structure-function relationships of the effect of 1,4-DHP derivatives on glucose metabolism showed that cerebrocrast could evoke qualitative differences in activity. Insertion of an OCHF(2) group in position 2 of the 4-phenylsubstituent and propoxyethylgroup R in ester moieties in positions 3 and 5 of the DHP structure, as well as an increase in the number of carbon atoms in the ester moiety, significantly modified the properties of the compound. Thereby cerebrocrast acquired high lipophilicity and membranotropic properties. Cerebrocrast, in a single administration at low doses (0.05 and 0.5 mg x kg(-1), p.o.), significantly decreased the plasma level of glucose in normal rats and in STZ-induced diabetic rats returned plasma glucose to basal levels. This effect was characterized by a slow onset and a powerful long-lasting influence on glucose metabolism, especially in STZ-induced diabetic rats.     

Enantiomeric Recognition of Racemic 4‐Aryl‐1,4‐dihydropyridine Derivatives via Chiralpak AD‐H Stationary Phases

The chromatographic chiral resolution of two new series of racemic 4‐substituted‐1,4‐dihydropyridine derivatives was studied on a commercial Chiralpak AD‐H column. Analytes without 5,5‐dimethyl substituents ( 1–15 ) are more efficiently resolved than analytes with 5,5‐dimethyl groups ( 16–30 ). The AD‐H column discriminated between enantiomers through both hydrogen bonding attractions and π–π interactions. This interpretation is in accord with plots of the logarithm of separation factors, log(α), versus σ (Hammett–Swain substituent parameter) and σ⁺ (Brown substituent constant) plots. By elucidating the effects of the remote substituents on these chiral separations, it was shown that the influence of π–π interaction forces increase when steric bulk effects act to decrease the hydrogen bonding attractive forces on the AD‐H column. Chirality 24:854–859, 2012. © 2012 Wiley Periodicals, Inc.     

1,4‐Dihydropyridine derivatives without Ca2+‐antagonist activity up‐regulate Psma6 mRNA expression in kidneys of intact and diabetic rats

Impaired degradation of proteins by the ubiquitin–proteasome system (UPS) is observed in numerous pathologies including diabetes mellitus (DM) and its complications. Dysregulation of proteasomal degradation might be because of altered expression of genes and proteins involved in the UPS. The search for novel compounds able to normalize expression of the UPS appears to be a topical problem. A novel group of 1,4‐dihydropyridine (1,4‐DHP) derivatives lacking Ca2+‐antagonists activities, but capable to produce antidiabetic, antioxidant and DNA repair enhancing effects, were tested for ability to modify Psma6 mRNA expression levels in rat kidneys and blood in healthy animals and in rats with streptozotocin (STZ) induced DM. Psma6 gene was chosen for the study, as polymorphisms of its human analogue are associated with DM and cardiovascular diseases. 1,4‐DHP derivatives (metcarbatone, etcarbatone, glutapyrone, J‐9‐125 and AV‐153‐Na) were administered per os for three days (0.05 mg/kg and/or 0.5 mg/kg). Psma6 gene expression levels were evaluated by quantitative PCR. Psma6 expression was higher in kidneys compared to blood. Induction of diabetes caused increase of Psma6 expression in kidneys, although it was not changed in blood. Several 1,4‐DHP derivatives increased expression of the gene both in kidneys and blood of control and model animals, but greater impact was observed in kidneys. The observed effect might reflect coupling of antioxidant and proteolysis‐promoting activities of the compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Resolution of some 4‐benzofurazanyl and 4‐benzofuroxanyl 1,4‐dihydropyridine derivatives by chiral HPLC on Whelk‐o1 and some polysaccharide chiral stationary phases

The chromatographic chiral resolution of racemic methyl 1,4‐dihydro‐2,6‐dimethyl‐5‐nitro‐4‐benzofurazanyl‐3‐carboxylates 1 and 2 and their benzofuroxanyl analogues 3 and 4 were studied on Whelk‐O1, Chiralcel OD‐H, Chiralcel OJ, and Chiralpak AD and AS. These CSPs were selected on the basis of the results of structural searches in Chirbase. Examination of the data and cluster analysis pointed out the influence of benzofurazane–benzofuroxane change versus α–β connection change on retention and enantioselectivity, respectively. The major contribution to the retention change arose from the type of heterocycle, whereas the major contribution to the enantioselectivity change came from the mode of connection (α or β) almost irrespective of the nature of the heterocycle. It resulted in a similarity of behaviour between 1 and 2 on one hand and 3 and 4 on the other as far as capacity factors were concerned, and in a similarity of behaviour between 1 and 3 on the one hand and 2 and 4 on the other as far as enantioselectivities were concerned. Chiralpak AS was selected for semipreparative resolution of the enantiomers. The study of several CSPs allowed us to obtain correlations of structure with retention and enantioselectivity as well as the choice of a semipreparative support to provide the quantities for biological tests. Chirality 11:602–608, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of β‐1,4‐galactosyltransferase I in rat Schwann cells

Glycosylation is one of the most important post‐translational modifications. It is clear that the single step of β‐1,4‐galactosylation is performed by a family of β‐1,4‐galactosyltransferases (β‐1,4‐GalTs), and that each member of this family may play a distinct role in different tissues and cells. In the present study, real‐time PCR revealed that the β‐1,4‐GalT I mRNA reached peaks at 2 weeks after sciatic nerve crush and 3 days after sciatic nerve transection. Combined in situ hybridization for β‐1,4‐GalT I mRNA and immunohistochemistry for S100 showed that β‐1,4‐GalT I mRNAs were mainly located in Schwann cells after sciatic nerve injury. In conclusion, β‐1,4‐GalT I might play important roles in Schwann cells during the regeneration and degeneration of the injured sciatic nerve. In other pathology, such as inflammation, we found that LPS administration affected β‐1,4‐GalT I mRNA expression in sciatic nerve in a time‐ and dose‐dependent manner, and β‐1,4‐GalT I mRNA is expressed mainly in Schwann cells. These results indicated that β‐1,4‐GalT I plays an important role in the inflammation reaction induced by intraperitoneal injection of LPS. Similarly, we found that β‐1,4‐GalT I in Schwann cells in vitro was affected in a time‐ and concentration‐dependent manner in response to LPS stimulation. All these results suggest that β‐1,4‐GalT I play an important role in Schwann cells in vivo and vitro during pathology. In addition, β‐1,4‐GalT I production was drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that Schwann cells which regulated β‐1,4‐GalT I expression after LPS stimulation were via ERK, SAPK/JNK, and P38 MAP kinase signal pathways. J. Cell. Biochem. 108: 75–86, 2009. © 2009 Wiley‐Liss, Inc.     

In vitro studies on the degradation of poly(cis‐1,4‐isoprene)

Cleavage of the backbone of poly(cis‐1,4‐isoprene) (IR) in solid rubber material was accomplished by the addition of partially purified latex clearing protein (Lcp1_VH2) using a 200‐mL enzyme reactor. Two strategies for the addition of Lcp1_VH2 were studied revealing that the daily addition of 50 µg mL^?1 of Lcp1_VH2 for 5 days was clearly a more efficient regime in comparison to a one‐time addition of 250 µg of Lcp1_VH2 at the beginning. Soluble oligo(cis‐1,4‐isoprene) molecules occurred as degradation products and were identified by ESI‐MS and GPC. Oxygenase activity of Lcp1_VH2 with solid IR particles as substrate was shown for the first time by measuring the oxygen consumption in the reaction medium. A strong decrease of the dissolved oxygen concentration was detected at the end of the assay, which indicates an increase in the number of cleavage reactions. The oligo(cis‐1,4‐isoprene) molecules comprised 1 to 11 isoprene units and exhibited an average molecular weight (M_n) of 885 g mol^?1. Isolation of the oligo(cis‐1,4‐isoprene) molecules was achieved by using silica gel column chromatography. The relative quantification of the isolated products was performed by HPLC‐MS after derivatization with 2,4‐dinitrophenilhydrazyne yielding a concentration of total degradation products of 1.62 g L^?1. Analysis of the polymer surface in samples incubated for 3 days with Lcp1_VH2 via ATR‐FTIR indicated the presence of carbonyl groups, which occurred upon the cleavage reaction. This study presents a cell‐free bioprocess as an alternative rubber treatment that can be applied for the partial degradation of the polymer. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:890–899, 2018     

In vitro and in vivo effects of iron on the expression and activity of glucose 6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase,and glutathione reductase in rat spleen

Iron is an indispensable element for vital activities in almost all living organisms. It is also a cofactor for many proteins, enzymes, and other essential complex biochemical processes. Therefore, iron trafficking is firmly regulated by Hepcidin (Hamp), which is regarded as the marker for iron accumulation. The disruption of iron homeostasis leads to oxidative stress that causes various human diseases, but this mechanism is still unclear. The aim of this study is to provide a better in vivo and in vitro understanding of how long‐term iron overload affects the gene expression and activities of some antioxidant enzymes, such as glucose 6‐phosphate dehydrogenase (G6PD), 6‐phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) in the spleen. The findings of this study show that iron overload reduces the gene expression of G6pd, 6pgd, and Gr, but its actual effect was on the protein level.     

Combined effect of X‐ray radiation and static magnetic fields on reactive oxygen species in rat lymphocytes in vitro

The aim of this study was to investigate the effect of static magnetic fields (SMF) on reactive oxygen species induced by X‐ray radiation. The experiments were performed on lymphocytes from male albino Wistar rats. After exposure to 3 Gy X‐ray radiation (with a dose rate of 560 mGy/min) the measurement of intracellular reactive oxygen species in lymphocytes, using a fluorescent probe, was done before exposure to the SMF, and after 15 min, 1 and 2 h of exposure to the SMF or a corresponding incubation time. For SMF exposure, 0 mT (50 µT magnetic field induction opposite to the geomagnetic field) and 5 mT fields were chosen. The trend of SMF effects for 0 mT was always opposite that of 5 mT. The first one decreased the rate of fluorescence change, while the latter one increased it. Bioelectromagnetics 34:333–336, 2013. © 2012 Wiley Periodicals, Inc.     

A simple and sensitive spectrofluorimetric method for the determination of furosemide using zinc(II)–1,4‐bis(imidazol‐1‐ylmethyl)benzene complexes

A novel spectrofluorometric method for the determination of furosemide (FUR) is described. The method is based on enhancement of fluorescence emission of FUR in the presence of zinc (II) complexes of 1,4‐bis(imidazol‐1‐ylmethyl)benzene. Under optimum conditions, the enhanced fluorescence intensity is linearly related to the concentration of FUR. The proposed method has been successfully applied to the determination of FUR in pharmaceutical preparations. The possible mechanism of this reaction is discussed briefly based on data from fluorescence spectroscopy, UV–vis absorption and infrared spectroscopy. Copyright © 2012 John Wiley & Sons, Ltd.     

A new xanthene‐based two‐photon fluorescent probe for the imaging of 1,4‐dithiothreitol (DTT) in living cells

1,4‐Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two‐photon fluorescence probe (Rh‐DTT) to detect DTT in living systems for the first time. Rh‐DTT showed high selectivity and sensitivity to DTT. Rh‐DTT can be successfully used for the two‐photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.     

Golgi α1,4‐fucosyltransferase of Arabidopsis thaliana partially localizes at the nuclear envelope

We analyzed plant‐derived α1,4‐fucosyltransferase (FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc‐green fluorescent protein (GFP) or tomato LeFucTc‐GFP restored Lewis‐a formation in a fuctc mutant, confirming functionality in the trans‐Golgi. AtFucTc‐GFP partly accumulated at the nuclear envelope (NE) not observed for other homologs or truncated AtFucTc lacking the N‐terminus or catalytic domain. Analysis of At/LeFucTc‐GFP swap constructs with exchanged cytosolic, transmembrane and stalk (CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino‐acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N‐terminal GFP copies did, indicating localization at the inner nuclear membrane (INM). Tunicamycin treatment of AtFucTc‐GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N‐glycosylation. Yet neither expression in protoplasts of Arabidopsis N‐glycosylation mutants nor elimination of the N‐glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum (ER)‐to‐Golgi transport by co‐expression of Sar1(H74L) trapped tunicamycin‐released AtFucTc‐GFP in the ER, however, without NE localization. Since recovery after tunicamycin‐washout required de novo‐protein synthesis, our analyses suggest that AtFucTc localizes to the NE/INM due to interaction with an unknown (glyco)protein.      

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.     

Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme on which the pentose phosphate pathway was checked. In this study, purification of a G6PD enzyme was carried out by using rat erythrocytes with a specific activity of 13.7 EU/mg and a yield of 67.7 and 155.6‐fold by using 2′,5′‐ADP Sepharose‐4B affinity column chromatography. For the purpose of identifying the purity of enzyme and molecular mass of the subunit, a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out. The molecular mass of subunit was calculated 56.5 kDa approximately. Then, an investigation was carried out regarding the inhibitory effects caused by various metal ions (Fe²⁺, Pb²⁺, Cd²⁺, Ag⁺, and Zn²⁺) on G6PD enzyme activities, as per Beutler method at 340 nm under in vitro conditions. Lineweaver–Burk diagrams were used for estimation of the IC₅₀ and K_i values for the metals. K_i values for Pb⁺², Cd⁺², Ag⁺, and Zn⁺² were 113.3, 215.2, 19.4, and 474.7 μM, respectively.     

Chiral separations of some β‐adrenergic agonists and antagonists on AmyCoat column by HPLC

Sixteen β‐adrenergic antagonists namely acebutalol, alprenolol, atenolol, bisoprolol, bopindolol, bufurolol, carazolol, celiprolol, indenolol, metaprolol, nebivolol, oxprenolol, practolol, propranolol, tertalol, and timolol, and two β‐adrenergic agonists namely cimeterol and clenbuterol were resolved on AmyCoat (150 × 46 mm, 3 μm size of silica particle) by using (85:15:0.1, v/v/v), (90:10:0.1, v/v/v), and (95:05:0.1, v/v/v) combinations of n‐heptane, ethanol, and diethylamine solvents, respectively. The flow rates used were 0.5, 1.0, 2.0, and 3.0 ml/min with detection at 225 nm. The values of capacity, separation, and resolution factors ranged from 0.38 to 19.70, 1.08–2.33, and 1.0 and 4.50, respectively. The maximum and minimum resolutions were achieved for celiprolol and bufurolol, respectively. The chiral recognition mechanisms were also discussed. The values of validation parameters were calculated. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

QM/MM studies on the calcium‐assisted β‐elimination mechanism of pectate lyase from bacillus subtilis

Pectate lyase utilizes the anti‐β‐elimination chemistry to catalyze the cleavage of α‐1,4 glycosidic bond between _D‐galacturonate regions during the degradation of plant polysaccharide pectin. We report here detailed mechanistic studies of the Bacillus subtilis pectate lyase (BsPel) using QM/MM calculations. It was found that the residue Arg279 serves as the catalytic base to abstract the α‐proton from C₅₂ atom of substrate Ada2 subsite, forming an unstable carbanion intermediate. The glycosidic bond of this intermediate is scissile to generate the 4,5‐unsaturated digalacturonate product and a negatively charged β‐leaving group. Two active site residues (Lys247 and Arg279) and two Ca²⁺ ions (Ca2 and Ca3) form hydrogen‐bonding and coordination interactions with C₅₂? COO^? of Ada2, respectively, which facilitate the proton abstraction and stabilize the generated carbanion intermediates. Arg284 is not the potential proton donor to saturate the leaving group. Actually, the proton source of leaving group is the solvent water molecule rather than any active site acidic residues. In addition, the calculation results suggest that careful selections of QM‐ and Active‐regions are essential to accurately explore the enzymatic reactions. Proteins 2016; 84:1606–1615. © 2016 Wiley Periodicals, Inc.