BMC Biochemistry最新文献

Background: The details of a precise, accurate, and sensitive spectrophotometric method for measuring catalase activity are presented here. The assay was established for biological samples and depends on the rapid formation of a stable and colored carbonato-cobaltate (III) complex. Samples exhibiting catalase activity are incubated with hydrogen peroxide solution for 2 min prior to rapid mixing of the incubation enzymatic reaction mixture with cobalt-bicarbonate reagent, which assesses non-reacting hydrogen peroxide. Catalase activity is always directly proportional to the rate of dissociation of hydrogen peroxide. Hydrogen peroxide acts to oxidize cobalt (II) to cobalt (III) in the presence of bicarbonate ions; this process ends with the production of a carbonato-cobaltate (III) complex ([Co (CO₃)₃]Co). The formed end product has two maximum absorbance peaks: 440 nm and 640 nm. The 440-nm peak has been utilized for assessing catalase activity.

Results: The catalase activity results of the current method for erythrocyte lysate homogenates were computationally identical to those of the dichromate method (r = 0.9950). The coefficient of variation was calculated to determine the imprecision of the current assay. The within-run and between-run results were 2.96 and 3.83%, respectively.

Conclusion: This method is appropriate for analyzing bacteria, red blood cells and liver and kidney tissue homogenates.

Background: Endonucleases play critical roles in maintaining genomic stability and regulating cell growth. The purpose of this study was to evaluate detection of endonuclease activity as an indicator in the early diagnosis and prognosis of lymphoma.

Results: The method of endonuclease activity determination was successfully established and applied to compare cancer patient and control cohorts. Endonuclease activities of cancer tissues were significantly higher than those of adjacent control tissues (P < 0.001). We next investigated endonuclease activity in peripheral blood of enrolled patients and the controls, which were also significantly higher in patients than in controls (P = 0.015). Additionally, endonuclease activities were elevated in the metastasis subgroup compared with the non-metastasis subgroup(P = 0.038), whereas no significant difference was found between age(≤ 56y, > 56y) and gender (P = 0.736 > 0.05 and P = 0.635 > 0.05, respectively). Although there was no significant difference between control group with the non-metastatic cancer patients (P = 0.800 > 0.05), endonuclease activities were lower in the control group compared with the non-metastatic cancer patients with lymphoma (P = 0.033). The progression-free survival probability of patients with elevated R ratios(R ratio ≥ 1.4) was significantly lower than that of patients with lower R ratios (R ratio < 1.4).

Conclusions: An assay was established to detect the endonuclease activity,which might be useful for the prognosis of cancers, especially lymphoma.

Background: In this study Aesculus indica fruit was subjected to isolation of phytochemicals. Two antioxidants quercetin and Mandelic acid were isolated in pure state. The free radical scavenging and acetyl choline esterase inhibitory potential of the crude extract and sub fractions were also determined.

Results: The antioxidant capacity of crude extract, fractions and isolated compounds were determined by DPPH and ABTS methods. Folin-Ciocalteu reagent method was used to estimate the total phenolic contents and were found to be 78.34 ± 0.96, 44.16 ± 1.05, 65.45 ± 1.29, 37.85 ± 1.44 and 50.23 ± 2.431 (mg/g of gallic acid) in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fractions respectively. The flavonoid concentration in crude extract, ethyl acetate, chloroform, n-hexane and aqueous fraction were; 85.30 ± 1.20, 53.80 ± 1.07, 77.50 ± 1.12, 26.30 ± 1.35 and 37.78 ± 1.25 (mg/g of quercetin) respectively. The chloroform fraction was more potent against enzymes, acetyl choline esterase and butyryl choline esterase (IC₅₀ = 85 and 160 μg/ml respectively). The phenolic compounds in the crude extract and fractions were determined using HPLC standard method. Chlorogenic acid, quercetin, phloroglucinol, rutin, mandelic acid and hydroxy benzoic acid were detected at retention times 6.005, 10.062, 22.623, 30.597, 35.490 and 36.211 in crude extract and different fractions. The ethyl acetate fraction was rich in the targeted compounds and was therefore subjected to column isolation. The HPLC chromatogram of isolated compounds showed single peak at specified retention times which confirms their isolation in pure state. The isolated compounds were then characterized by FTIR and NMR spectrophotometric techniques.

Conclusion: The Aesculus indica fruit extracts showed antioxidant and anticholine esterase inhibitory potentials. Two bioactive compounds were isolated in the pure form ethyl acetate fraction. From results it was concluded that the fruit of this plant could be used to minimize oxidative stress caused by reactive oxygen species.

Background: Previous studies have demonstrated that members of Trichoderma are able to generate appreciable amount of extracellular amylase and glucoamylase on soluble potato starch. In this study the α-amylase was purified and characterized from Trichoderma pseudokoningii grown on orange peel under solid state fermentation (SSF).

Results: Five α-amylases A1-A5 from Trichodrma pseudokoningii were separated on DEAE-Sepharose column. The homogeneity of α-amylase A4 was detected after chromatography on Sephacryl S-200. α-Amylase A4 had molecular weight of 30 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had a broad pH optimum ranged from 4.5 to 8.5. The optimum temperature of A4 was 50 °C with high retention of its activity from 30 to 80 °C. The thermal stability of A4 was detected up to 50 °C and the enzyme was highly stable till 80 °C after 1 h incubation. All substrate analogues tested had amylase activity toward A4 ranged from 12 to 100% of its initial activity. The Km and Vmax values of A4 were 4 mg starch/ml and 0.74 μmol reducing sugar, respectively. The most of metals tested caused moderate inhibitory effect, except of Ca²⁺ and Mg²⁺ enhanced the activity. Hg²⁺ and Cd^+ 2 strongly inhibited the activity of A4. EDTA as metal chelator caused strong inhibitory effect.

Conclusions: The properties of the purified α-amylase A4 from T. pseudokoningii meet the prerequisites needed for several applications.

Background: Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes.

Results: In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Conclusions: Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.

Background: An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA₃^Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein.

Results: In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA₃^Lys.

Conclusions: These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA₃^Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA₃^Lys into HIV-1 particles.

Background: Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size.

Results: The performance of a commercially available feruloyl esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that K_m was not affected by the immobilization but k_cat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction.

Conclusions: These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.

Background: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear.

Results: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant.

Conclusions: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.

Background: Peptides with cytoprotective functions, including antioxidants and anti-infectives, could be useful therapeutics. Carnosine, β-alanine-histidine, is a dipeptide with anti-oxidant properties. Tripeptides of Ala-His-Lys, Pro-His-His, or Tyr-His-Tyr are also of interest in this respect.

Results: We synthesized several histidine-containing peptides including glycine or alanine, and tested their cytoprotective effects on hydrogen peroxide toxicity for PC12 cells. Of all these peptides (Gly-His-His, Ala-His-His, Ala-His-Ala, Ala-Ala-His, Ala-Gly-His, Gly-Ala-His (GAH), Ala-His-Gly, His-Ala-Gly, His-His-His, Gly-His-Ala, and Gly-Gly-His), GAH was found to have the strongest cytoprotective activity. GAH decreased lactate dehydrogenase (LDH) leakage, apoptosis, morphological changes, and nuclear membrane permeability changes against hydrogen peroxide toxicity in PC12 cells. The cytoprotective activity of GAH was superior to that of carnosine against hydrogen peroxide toxicity in PC12 cells. GAH also protected PC12 cells against damage caused by actinomycin D and staurosporine. Additionally, it was found that GAH also protected SH-SY5Y and Jurkat cells from damage caused by hydrogen peroxide, as assessed by LDH leakage.

Conclusion: Thus, a novel tripeptide, GAH, has been identified as having broad cytoprotective effects against hydrogen peroxide-induced cell damage.

Background: Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis-a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial cells and cancer cells in vitro, presumably by binding to its cellular receptor, cytoskeleton associated-protein 4 (CKAP4); however, the biophysical interaction of APF with CKAP4 has not been characterized previously. In this study, we used surface plasmon resonance (SPR) to explore the binding kinetics of the interaction of APF and as-APF (a desialylated APF analogue with full activity) to CKAP4.

Results: We immobilized non-glycosylated APF (TVPAAVVVA) to the Fc1 channel as the control and as-APF to Fc2 channel as the ligand in order to measure the binding of CKAP4 recombinant proteins encompassing only the extracellular domain (Aa 127-602) or the extracellular domain plus the transmembrane domain (Aa 106-602). Positive binding was detected to both CKAP4_126-602 and CKAP4_106-602, suggesting that as-APF can bind specifically to CKAP4 and that the potential binding site(s) are located within the extracellular domain. To identify the primary APF binding site(s) within the CKAP4 extracellular domain, deletion mutants were designed according to structural predictions, and the purified recombinant proteins were immobilized on a CM5 chip through amine-coupling to measure as-APF binding activity. Importantly, both CKAP4_127-360 and CKAP4_361-524 exhibited a fast association rate (k _on ) and a slow dissociation rate (k _off ), thus generating high binding affinity and suggesting that both regions contribute relatively equally to overall as-APF binding. Therefore, two or more as-APF binding sites may exist within the Aa 127-524 region of the CKAP4 extracellular domain.

Conclusions: We determined that the CKAP4_127-360 and CKAP4_361-524 mutants exhibit improved binding activity to as-APF as compared to the full-length extracellular domain, making it possible to detect low concentrations of as-APF in urine, thereby establishing a foundation for a non-invasive diagnostic assay for IC. Further, these data have revealed novel APF binding site(s) suggesting that targeting this region of CKAP4 to inhibit APF binding may be a useful strategy for treating IC-related bladder pathology.