Units Mg-1 Protein Research Articles

Purification and characterization of a novel acid-tolerant and heterodimeric β-glucosidase from pumpkin (Cucurbita moschata) seed

A novel β-glucosidase was purified from pumpkin (Cucurbitamoschata) seed by anion exchange chromatography and gel permeation chromatography, and its molecular mass was determined to be 42.8kDa by gel permeation chromatography. The heterodimeric structure consisting of two subunits, free from disulfide bonds, was determined by native-PAGE analysis followed by zymography. The enzyme was maximally active at pH 4.0 and 70°C, and Vmax, Km, and kcat values were 0.078 units mg-1 protein, 2.22mM, and 13.29min-1, respectively, employing p-nitrophenyl-β-d-glucopyranoside as the substrate. The high content of glycine determined by amino acid analysis implies that the enzyme possesses flexible conformations and interacts with cell membranes and walls in nature. Circular dichroism studies revealed that the high stability of the enzyme within the pH range of 2.0-10.0 is due to its reversible pH-responsive characteristics for α-helix-antiparallel β-sheet interconversion.

Partial purification and characterization of fatty acid esterase from pearl millet

Aim: The present study aimed to identify a negative modulator of lipolytic enzyme fatty acid esterase (FAE) for exploring possibilities of increasing shelf life of pearl millet flour through arresting in-situ hydrolysis of lipids. Methodology: FAE was partially purified from flour of pearl millet hybrid HHB 234 by (NH4)2SO4 fractionation (30-60% saturation) and gel filtration chromatography using Sephadex G-75. The enzyme was characterized for physico-chemical properties viz., molecular mass, optimum pH, optimum temperature and thermal stability and kinetic properties viz., Km value and effect of modulators. Results: Crude extract contained 1008 units of activity and 421 mg proteins resulting in to specific activity of 2.4 units mg-1 protein. The enzyme was purified 10.7 fold with a recovery of 21.52% and specific activity of 25.7 units mg-1 protein by ammonium sulphate fractionation followed by gel filtration. The molecular weight of purified enzyme preparation was 60 kDa, as determined by gel filtration through Sephadex G-75. The enzyme exhibited maximum activity at pH 8.2 and 45°C. The enzyme was stable up to 60°C for 20 min and showed Km value of 0.65 µM for p-NPB. At 10 mM concentration, Mg2+ and Zn2+ altered the activity positively by 54% and negatively by 42% whereas EDTA, DTT, PMSF, ascorbic acid inhibited the activity by 75, 68, 50 and 48%, respectively. Interpretation: Partially purified lipolytic enzyme FAE from pearl millet flour was strongly inhibited by ascorbic acid. This novel information might be useful in developing processing technologies for improving shelf life of flour. Key words: Characterization, Fatty acid esterase, Pearl millet, Purification, Shelf life

The Effects of Hot and Cold Water Treatment on Quality Parameters and Enzymatic Activity in Chestnut

This study was carried out in order to limit the biochemical and enzymatic changes occurred after harvested in a variety of (Castanea sativa Mill. cv. 'Sariaslama') chestnuts to prolong the stored period by reduce the loss of quality. Before samples were stored, hot (46±2 °C, 45 minutes) and cold water (15±2 °C, 8 days) treatments were performed. After the hot and cold water treatments the fruits were stored in normal (NA) and controlled atmosphere (CA). In CA storage the fruits were kept in three different atmosphere combinations: (10% CO2, 2% O2; 15% CO2, 2% O2; 20% CO2, 2% O2). In both storage methods, chestnuts were stored for 5 months in 0±1 °C temperature and 90±5% relative humidity conditions. During the storage, parameters such as weight loss (%), content of relative water (%), polyphenol oxidase (PPO) (units mg-1 protein), soluble solids (°brix), starch (mg mL-1), total sugar (mg mL-1), vitamin C (mg 100 mL-1), macro (potassium, phosphorus, calcium, sodium) and micro (iron, magnesium) elements (mg 100 g-1) were examined in the fruit samples taken. Maximum weight loss was examined in the fruit kept in NA whereas total sugar and starch showed less of a change in CA in this present study. PPO enzymatic activity can successfully control with hot water treatment in 15% CO2, 2% O2 gas combination. When soluble solids was examined, a smaller degree of change was observed in the fruit stored in CA. Promising results were achieved with the fruit that was stored using hot water treatment and 15% CO2, 2% O2 combination in this study, which was conducted on the ‘Sariaslama’chestnut.

Mitigating effects of paclobutrazol on flooding stress damage by shifting biochemical and antioxidant defense mechanisms in mungbean(Vigna radiata L.) at pre-flowering stage

Flooding stress is one of the major obstacles which hamper plant growth and development in various ways. Two screened mungbean genotypes, HUM-16 (flooding tolerant) and HUM-12 (flooding susceptible), were selected to evaluate the effect of paclobutrazol @10ppm and 25ppm along with its combinations through pre-seed soaking and foliar spray at pre-flowering stage. The flooding stress shock was imposed for seven days at pre-flowering stage. Paclobutrazol (PBZ) treatments significantly enhanced chlorophyll, soluble sugar, starch, MSI, APX, and SOD in both HUM-16 and HUM-12 genotypes under flooding stress. Among all the treatments, PBZ @10ppm seed treatment + 10ppm foliar spray revealed higher chlorophyll (2.47 and 1.97 mg g-1 FW), soluble sugar (24.57 and 20.53 mg g-1 FW), MSI (49.16% and 38.67%), APX (16.13 and 6.50 Units mg-1 protein min-1) and SOD (38.00 and 18.30 Units mg-1 protein min-1) in both tolerant and susceptible genotype, respectively, while it minimized the production of MDA (18.30% and 49.00%) and H2O2 (19.05% and 25.06%) in both tolerant and susceptible genotype, respectively, under flooding stress as compared to control. Paclobutrazol treatments, as evident, minimized the flooding stress damage in tolerant genotype as compared to susceptible genotype by enhancing chlorophyll, soluble sugar and antioxidant enzymes activity (APX and SOD) and by reducing the generation of H2O2 and MDA contents under flooding stress.

Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD+-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg-1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg-1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg-1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 micromol g-1 (cell dry weight) min-1. In the presence of formate, the intracellular [NADH]/[NAD+] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.

A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups

The chemoenzymatic route to 2-deoxy-2-propionamido- d-mannose ( 1b), 2-butyramido-2-deoxy- d-mannose ( 2b) and 2-deoxy-2-phenylacetamido- d-mannose ( 3b) involved N-acylation of 2-amino-2-deoxy- d-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido- d-glucose ( 1a), 2-butyramido-2-deoxy- d-glucose ( 2a) and 2-deoxy-2-phenylacetamido- d-glucose ( 3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6- O-phospho- d-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy- d-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 °C and 8.0–9.0, respectively. 2-Acetamido-2-deoxy-6- O-phospho- d-glucose and 2-acetamido-2-deoxy-6- O-sulfo- d-glucose but not 2-acetamido-2-deoxy-1- O-phospho- d-glucose or 2-acetamido-2-deoxy- d-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al 3+, Ca 2+, Co 2+, Cu 2+, Mn 2+ or Zn 2+ and activated by 1 mM Mg 2+. The concentrated enzyme is highly stable at 4 °C in the presence of 0.1 M ammonium sulfate.

Purification and characterization of a thermostable β-Galactosidase from kidney beans ( Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.

Sunflower Nitrate Reductase: Purification and Characterization

A single isoform, NADH: nitrate reductase (NR), was purified 500 folds from sunflower leaves by affinity chromatography on Blue Sepharose CL-6B. Purified NR had a pH optima of 7.25 and a molecular weight of 210 kD. In SDS-PAGE, two bands of 47 and 56 kD were obtained. NADH: ferric citrate reductase activity was copurified with NR with a specific activity of 2. The Vmax of NADH: ferric citrate reductase was 8.69 units mg-1 protein and the apparent Km for ferric citrate was 0.435 mM.

RODENT ADRENAL ADAPTATION TO NINE DAY HYPOKINETIC MODEL EXPOSURE1140

The 30° head down tilt (HDT) tail suspension model in rodents has been used as a hypokinetic model to study general physiologic deconditioning. To what extent HDT induces adrenal hypoactivation as part of the deconditioning syndrome has not been elucidated. To determine if exposure to HDT for 9 days results in an adrenal hypoactivation the following hypothesis was tested: adrenal corticosterone content and catecholamine synthesizing enzyme activity, tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT), will decline in rodents exposed to 9 day HDT. Male Sprague-Dawley rats (95 days of age) were randomly assigned into the following groups: 1) cage control(CAGE-CN, n=20), 2) 9-day head down tilt (HDT, n=20). Tyrosine hydroxylase activity was determined by radiometric assay, PNMT activity was measured spectrophotometrically, and corticosterone content was determined via radioimmunoassay. All assay results were normalized to adrenal protein content. The enzyme PNMT activity (unitsmg-1 protein), and corticosterone content (pgug-1 protein) in adrenal tissue presented respectively were significantly lower in the HDT group(*0.052 ± 0.005, *264.9± 16) compared to the CAGE-CN group (0.073 ± 0.007, 483.7± 34). Tyrosine hydroxylase activity (CPM mg-1 protein) demonstrated no significant difference between SUS (2129 ± 342) and CAGE-CN (2161 ± 356) group. These findings suggest that 9 day exposure to simulated weightlessness results in adrenal hypoactivation which is consistent with general physiologic deconditioning.

Blockade of nitrergic transmission by hydroquinone, hydroxocobalamin and carboxy‐PTIO in bovine retractor penis: role of superoxide anion

1. The effects of inhibiting endogenous Cu/Zn superoxide dismutase (SOD) with diethyldithiocarbamate (DETCA) were examined on the ability of hydroquinone, hydroxocobalamin and carboxy-PTIO to block nitrergic relaxation in the bovine retractor penis (BRP) muscle. 2. Incubation of strips of BRP with DETCA (3 mM) for 2 h reduced SOD activity from 73.1 +/- 15.7 to 8.2 +/- 1.9 units mg-1 protein. 3. Hydroquinone (10 microM--1 mM) produced weak inhibition of nitrergic (4 Hz, 10 s) relaxation in control strips of BRP, but powerful inhibition in strips treated with DETCA (3 mM, 2 h). Exogenous SOD (250 units ml--1) produced a partial blockade of the ability of hydroquinone to inhibit nitrergic relaxation in DETCA-treated strips. 4. In an assay of SOD-inhibitable reduction of cytochrome C, hypoxanthine (0.1 mM)/xanthine oxidase (16 munits ml-1) and pyrogallol (10 microM), led to the rapid generation of superoxide anion. Hydroquinone (10 microM) also led to the generation of the free radical, although the rate of generation was slower. 5. Two NO-scavenging agents, hydroxocobalamin (0.1 microM--1 mM) and carboxy-PTIO (0.1-1 mM), produced concentration-dependent blockade of nitrergic relaxation of the BRP. The magnitude of the blockade induced by these agents was unaffected following treatment with DETCA or SOD. 6. The findings with hydroquinone support our previous proposal that endogenous Cu/Zn SOD plays a vital role in protecting nitrergic neurotransmission from inactivation by superoxide anion. Results with hydroxocobalamin and carboxy-PTIO are consistent with the known ability of these agents to scavenge NO. The nitrergic neurotransmitter in the BRP thus appears to have the properties of NO.

Aspects of antioxidant status in the polychaete Arenicola marina:tissue and subcellular distribution, and reaction to environmental hydrogen peroxide and elevated temperatures

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 143:141-150 (1996) - doi:10.3354/meps143141 Aspects of antioxidant status in the polychaete Arenicola marina: tissue and subcellular distribution, and reaction to environmental hydrogen peroxide and elevated temperatures Buchner T, Abele-Oeschger D, Theede H Solar radiation leads to hydrogen peroxide (H2O2) accumulation in shallow intertidal surface waters during daytime tidal emersion periods. The lugworm Arenicola marina irrigates its sedimentary tubes with surface water containing variable H2O2 concentrations. We studied aspects of the antioxidant status of the intertidal polychaete A. marina in response to seasonal variations of oxidative stress in its environment. Antioxidant enzyme activities [superoxide dismutase (SOD), glutathione reductase (GR) and catalase] and vitamin E were concentrated chiefly in the chloragog and to a lesser extent in body wall tissue. Response to experimental H2O2 exposure (5 µmol l-1) was confined to the chloragog tissue and consisted of elevated catalase activity. On a subcellular level, the major part of enzymatic antioxidants examined was found in the cytosolic (77% of SOD activity, 60% of GR activity, 41% of catalase activity, 87% of total glutathione) and the peroxisomal (56% of catalase activity) fractions, whereas the same antioxidants were virtually absent in mitochondria. Gradual acclimation of winter A. marina (5°C) to higher ambient temperatures (20°C) over 20 d resulted in a doubling of chloragog SOD activities, while catalase activity was not affected. Elevated in situ concentrations of photoproduced H2O2 during summer (1.7 µmol l-1) coincided with a significant increase of chloragog catalase activities in young (winter: 317.97 ± 78.3 U mg-1 protein, summer: 783.41 ± 192.7 U mg-1 protein, means ± SD) and in adult (winter: 480.09 ± 160.1 U mg-1 protein, summer: 1165.5 ± 207.5 U mg-1 protein) lugworms. Seasonal differences in the SOD activities of adult worms (winter: 16.06 ± 4.69 SOD units mg-1 protein; summer: 23.29 ± 3.31 SOD units mg-1 protein) were attributed to the elevated sediment temperatures in intertidal areas during summer. Arenicola marina · Hydrogen peroxide · Enzymatic antioxidants Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 143. Publication date: November 14, 1996 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1996 Inter-Research.

Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201.

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected in Escherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone STP2201 as a strong promoter in E. coli. Subcloning of a STP2201-melC DNA fragment into the pMEU-series S. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype to E. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase pro-anti-tyrosinase antiserum in S. thermophilus. Substituting melC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to an E. coli transformant at a level of (1.06 +/- 0.15) x 10(-7) units mg-1 protein. Introduction of this plasmid into S. thermophilus by electrotransformation yielded ChoA+ transformant that produced the enzyme at about 25% of the level found in E. coli.

Isolation and characterization of a microbial Arg/Lys carboxypeptidase, carboxypeptidase F.

Carboxypeptidase F was isolated from a fungal strain F-33 and characterized. The enzyme has the ability to release arginine and lysine from the carboxy terminus of peptides, and showed high specific activity against arginine (140 units mg-1 protein). Optimal temperature and pH for the enzyme reaction were 55 degrees C and pH 8.5, respectively. The enzyme possessed a high thermal stability. Native molecular weight was estimated to be approximately 450,000. Enzymatic activity was inhibited by Co2+, Cd2+, chelating agents and thiol inhibitors.

Breast cancer prognosis is poor when total plasminogen activator activity is low

Plasminogen activator (PA) is a serine protease which exists in two forms: tissue-type (t-PA) and urokinase-type (u-PA). The total PA activity was measured in tumour extracts of 235 breast cancer patients who were followed for a median of 8.5 years after surgery. Patients were initially divided into three groups with low (< 60 units mg-1 protein), intermediate (60-300 unit mg-1 protein), or high (> 300 unit mg-1 protein) total PA activity in tumour extracts. The PA activity was not significantly associated with the recognised prognostic factors of age, menstrual status, tumour size, lymph node involvement, histologic type, grade of anaplasia, and/or vessel involvement. A significant association was found between total PA activity and the oestrogen receptor (ER) or progesterone receptor (PgR) status. Among receptor-positive tumours, a significantly greater proportion of patients had high PA activity in their tumour extracts. Breast cancer patients with low total PA activity had a significantly shorter disease-free and overall survival rate when compared to those with intermediate or high PA activity. In univariate and multivariate analyses, total PA activity (< 60 unit mg-1 vs > or = 60 unit mg-1 protein) was found to be a significant prognostic factor for disease-free and overall survival of about the same import as lymph node involvement. Furthermore, the combination of total PA activity and nodal status could be even more precise in predicting survival times and probabilities in individual patients. This retrospective study demonstrates the total PA activity is a valuable prognostic factor in determining prognosis in human breast cancer.

Antioxidant enzymes of larvae of the cabbage looper moth, Trichoplusia ni: subcellular distribution and activities of superoxide dismutase, catalase and glutathione reductase.

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg-1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%), 142.3 units (23.8%), and 22.9 units (3.8%) mg-1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%), 2.46 units (23.0%), and 0.70 units (6.5%) mg-1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing .OH radical from all intracellular compartments by the destruction of H2O2 which together with O2- is a precursor of .OH.