ABSTRACT
Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo.
Subject(s)
DNA, Single-Stranded/metabolism , Fabaceae/enzymology , Plant Proteins/antagonists & inhibitors , Plant Proteins/analysis , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitors , Single-Strand Specific DNA and RNA Endonucleases/analysis , Spectrometry, Fluorescence/methods , Biosensing Techniques/methods , Enzyme Assays/methods , Enzyme Inhibitors/metabolism , Fabaceae/drug effects , Fabaceae/growth & development , Fluorescence , Graphite/chemistry , Limit of Detection , Oxides/chemistry , Plant Proteins/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolismABSTRACT
S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uµl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uµl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis.
Subject(s)
Fungal Proteins/analysis , Metal Nanoparticles , Single-Strand Specific DNA and RNA Endonucleases/analysis , DNA, Single-Stranded/metabolism , Diphosphates/pharmacology , Enzyme Inhibitors/pharmacology , Feasibility Studies , Fluorescent Dyes , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/metabolism , Metal Nanoparticles/chemistry , Oligonucleotides , Poly C , Silver , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitors , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Spectrometry, Fluorescence/methodsABSTRACT
Carbon nanotubes (CNTs) can efficiently quench the fluorescence of the adsorbed fluorophores and nonconvalently interact with soft single-stranded DNA (ssDNA). Upon disruption of CNTs-fluorescent oligonucleotides hybrid by nuclease S1, fluorescence turn-on was observed. Using this strategy, a platform based on fluorescence signal for monitoring the activity of nuclease with advantages of high sensitivity and commonality was established, and a linear relationship between initial cleavage reaction rate and nuclease S1 concentration is found in the range of 0.6-8.0 U mL(-1) with a detection limit of 0.08 U mL(-1). Furthermore, by taking pyrophosphate as an example, we use the assay to evaluate the prohibition effect on nuclease, and the extent of fluorescence recovery decreased linearly with increasing the concentration of pyrophosphate in the range of 0.2-1.4 mM, implying that the cleavage reaction by nuclease S1 was prohibited, and therefore this fluorescence assay can also be conveniently utilized for inhibitor screening of nuclease.
Subject(s)
DNA, Single-Stranded/metabolism , Enzyme Inhibitors/chemistry , Nanotubes, Carbon/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Spectrometry, Fluorescence , DNA Cleavage , DNA, Single-Stranded/chemistry , Diphosphates/chemistry , Fluorescent Dyes/chemistry , Kinetics , Oligonucleotides/chemical synthesis , Oligonucleotides/metabolism , Rhodamines/chemistry , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitorsABSTRACT
The active trypsin inhibiting component, SPC1, was obtained during the synthesis of a 22-residue peptide with three disulfide bridges according to the mimic mung bean Bowman-Birk type inhibitor. The K(i) value of SPC1 is 1.2x10(-7) M. In order to determine the topological structure of SPC1, X-ray diffraction studies were carried out on the complex of SPC1 with bovine beta-trypsin. Only the binding loop of SPC1 resolved at 2.2 A resolution due to conformational flexibility of the other residues [1]. The amino acid sequence was re-determined and electrospray mass spectroscopy was also performed to ensure that no cleaving occurred on SPC1 and the primary sequence of SPC1 is correct. Because the protein is more rigid in nonaqueous medium as has been proved by others [2], we treated the complex of SPC1 with neat cyclohexane and then subjected it to X-ray diffraction analysis, and the result showed that all the 22 residues of SPC1 were located in the electron density map. So the topological structure of SPC1 has been determined, suggesting that crystal treatment with cyclohexane may be used as a method to determine the conformation of the disordered regions in protein crystal structures.
Subject(s)
Peptides/chemistry , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitors , Trypsin Inhibitors/chemistry , Amino Acid Sequence , Animals , Cattle , Cyclohexanes , Models, Molecular , Molecular Sequence Data , Molecular Structure , Peptides/chemical synthesis , Peptides/pharmacology , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/pharmacology , X-Ray Diffraction/methodsABSTRACT
It has recently been reported that bilirubin forms a complex with Cu(II). In this paper we show that the formation of the complex results in the reduction of Cu(II) to Cu(I) and the redox cycling of the metal gives rise to the formation of reactive oxygen species, particularly hydroxyl radical. The bilirubin-Cu(II) complex causes strand breakage in calf thymus DNA and supercoiled plasmid DNA. Cu(I) was shown to be an essential intermediate in the DNA cleavage reaction by using the Cu(I) specific sequestering reagent neocuproine. Bilirubin-Cu(II) produced hydroxyl radical and the involvement of active oxygen species was established by the inhibition of DNA breakage by various oxygen radical quenchers.
Subject(s)
Bilirubin/chemistry , Copper/chemistry , DNA Damage , DNA/chemistry , Animals , Bilirubin/pharmacology , Cattle , Chelating Agents , Copper/pharmacology , DNA, Superhelical/chemistry , Free Radical Scavengers , Hydroxides/chemistry , Oxidation-Reduction , Phenanthrolines , Plasmids/chemistry , Reactive Oxygen Species , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitors , Spectrophotometry, UltravioletSubject(s)
Molecular Biology/methods , Plant Proteins/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolism , DNA/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Hydrogen-Ion Concentration/drug effects , Molecular Weight , Plant Proteins/antagonists & inhibitors , Salts/pharmacology , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitors , Substrate Specificity/drug effects , TemperatureABSTRACT
Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3'-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5'-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5'-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5'-AMP effectively, suggests the involvement of histidine in catalysis.
Subject(s)
Single-Strand Specific DNA and RNA Endonucleases/chemistry , Aspergillus oryzae/enzymology , Binding Sites , Catalysis , Circular Dichroism , Diethyl Pyrocarbonate/chemistry , Histidine/chemistry , Hydrogen-Ion Concentration , Oxidation-Reduction , Photochemistry , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitorsABSTRACT
A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3'-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.
Subject(s)
Single-Strand Specific DNA and RNA Endonucleases/metabolism , Animals , Binding Sites , Buffaloes , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Kinetics , Liver/metabolism , Methylation , Molecular Weight , Oxidation-Reduction , Single-Strand Specific DNA and RNA Endonucleases/antagonists & inhibitors , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/isolation & purification , Substrate SpecificityABSTRACT
Mouse vasopressin (VP) and oxytocin (OT) genes were isolated from a genomic library and the nucleotide sequences of the two genes were determined. The two genes have similar three exon structures and a high similarity in the part of exon 1 encoding vasopressin or oxytocin nonapeptide and in exon 2 encoding the central core of neurophysin. They are linked together in a tail to tail orientation separated by a short 3.5 kb intergenic sequence and are transcribed from opposite strands. Both genes have a single transcription initiation site downstream from a TATA-like sequence and a single polyadenylated transcript of about 760 bp for the vasopressin mRNA and about 700 bp for the oxytocin mRNA.
