Purification and characterization of DNA ligase I from the trypanosomatid Crithidia fasciculata.

A DNA ligase has been purified approximately 5000-fold, to near homogeneity, from the trypanosomatid Crithidia fasciculata. The purified enzyme contains polypeptides with molecular masses of 84 and 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides formed enzyme-adenylate complexes in the absence of DNA, contained an epitope that is highly conserved between human and bovine DNA ligase I and yeast and vaccinia virus DNA ligases, and were identified in fresh lysates of C. fasciculata by antibodies raised against the purified protein. Hydrodynamic measurements indicate that the enzyme is an asymmetric protein of approximately 80 kDa. The purified DNA ligase can join oligo(dT) annealed to poly(dA), but not oligo(dT) annealed to poly(rA), and can ligate blunt-ended DNA fragments. The enzyme has a low Km for ATP of 0.3 microM. The DNA ligase absolutely requires ATP and Mg2+, and is inhibited by N-ethylmaleimide and by KCI. Substrate specificity, Km for ATP, and the conserved epitope all suggest that the purified enzyme is the trypanosome homologue of DNA ligase I.     

Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase--have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo.     

Purification of a mitochondrial DNA polymerase from Crithidia fasciculata.

The mitochondrial DNA polymerase from Crithidia fasciculata has been purified to near homogeneity. SDS-PAGE analysis of the purified enzyme reveals a single polypeptide with a molecular weight of approximately 43,000. The protein is basic, with an isoelectric point between 7.6-8.0. Its Stokes radius of 22 A and its sedimentation coefficient of 4.1 S suggest a native molecular weight of 38,000, indicating that the protein is a monomer under our experimental conditions. Western blots and immunoprecipitations of crude extracts reveal a cross-reacting protein of 48 kDa, suggesting that the purified enzyme may be an enzymatically active proteolytic product. The mitochondrial origin of the polymerase was confirmed by cell fractionation. Our results indicate that the C. fasciculata enzyme may be among the smallest known mitochondrial polymerases.     

Purification and characterization of iron superoxide dismutase and copper-zinc superoxide dismutase from Acanthamoeba castellanii

Two superoxide dismutases (SOD I and SOD II) were purified from Acanthamoeba castellanii and characterized for several biochemical properties. Analysis of the primary structure and inhibition studies revealed that SOD I is iron SOD (Fe-SOD), with a molecular mass of 50 kDa, and SOD II is copper-zinc SOD (Cu,Zn-SOD), with a molecular mass of 38 kDa. Both enzymes have a homodimeric structure consisting of 2 identical subunits, each with a molecular mass of 26 and 19 kDa for SOD I and SOD II, respectively. The isoelectric points of SOD I and SOD II were 6.4 and 3.5, respectively, and there were no isoenzyme forms detected. Both enzymes show a broad optimal pH of 7.0-11.0. Because no differences were observed in the apparent molecular weight of SOD I after addition of the reducing agent 2-mercaptoethanol, the subunits do not appear to be linked covalently by disulfide bonds. However, the subunits of SOD II were covalently linked by intra- and interdisulfide bonds. Western blot analyses showed that the 2 enzymes have different antigenicity. Both enzymes occur as cytoplasmic and detergent-extractable fractions. These enzymes may be potential virulence factors of A. castellanii by acting both as antioxidants and antiinflammatory agents. These enzymes may be attractive targets for chemotherapy and immunodiagnosis of acanthamoebiasis.     

Purification and characterization of superoxide dismutase from chicken liver

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme has been isolated, purified and partially characterized from chicken liver. The following steps were carried out in order to purify chicken liver SOD. Initially, the liver was homogenized and hemoglobin was removed. Subsequently protein precipitation was effected with (NH(4))(2)SO(4), methanol, (NH(4))(2)SO(4)-methanol and polyethylene glycol methods. The product from polyethylene glycol-3350 precipitation was found to have the highest SOD activity. Polyethylene glycol was removed by chromatography using a PD-10 column. After passing through an ultrafilter, the superoxide dismutase was fractionated by DEAE-ion chromatography and then Sephadex G-75 gel filtration chromatography. During this purification procedure, a specific activity of 4818.2 IU/mg was reached, corresponding to 285.8-fold purification. The purified enzyme, which was characterized as cyanide-sensitive SOD, contained two subunits having Cu and Zn elements with a molecular weight of 16000+/-500 for each. The optimum pH of purified CuZnSOD was determined to be 8.9. The enzyme was found to have good pH stability in the pH range 6.0-7.5 at 25 degrees C over a 2-h incubation period and displayed good thermal stability up to 45 degrees C at pH 7.4 over a 1-h incubation period. The SOD enzyme was not inhibited by DTT and beta-mercaptoethanol, but inhibited by CN(-) and H(2)O(2). In the presence of 2 mM iodoacetamide, the enzyme showed an approximately 40% activity loss. Finally, the inhibitory effect of ionic strength on SOD was also investigated.     

Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.     

Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.     

Purification,characterization and amino terminal sequence of the superoxide dismutase from Babesia hylomysci

Babesia hylomysci was found to contain two superoxide dismutase (SOD) isoenzymes with isoelectric points (pI) of 4.9 and 5.2. The two isoenzymes (45 and 47 kDa) were composed of two subunits of 22 kDa. An unique amino terminal sequence was determined up to 34 residues from the pooled isoenzymes and was identified as a sequence of SOD. The comparison of this N-terminal sequence of B. hylomysci SOD with 29 known Fe- or Mn-SODs showed more homologies with Fe-SODs.     

Purification, characterization, and molecular cloning of a thermostable superoxide dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS-PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.     

Purification and characterization of Fe-containing superoxide dismutase from Methanobrevibacter arboriphilus strain AZ

Superoxide dismutase (SOD) was purified from cells of the strict anaerobic methanogenic archaeon Methanobrevibacter arboriphilus strain AZ. The four-step purification procedure resulted in enzyme with specific activity of 3970 units/mg and yield of 22%. It was shown that the SOD is a Fe-containing homotetramer composed of subunits of 21.2 kD each. Sodium azide (13.5 mM), unlike KCN, inhibits the activity of the SOD. Hydrogen peroxide (0.5 mM) inactivates the enzyme, which is consistent with the properties of the known Fe-containing SODs from methanogenic Archaea.     

Purification and properties of a soluble nicotinamide adenine dinucleotide-linked isocitrate dehydrogenase from Crithidia fasciculata.

A soluble NAD+-linked isocitrate dehydrogenase has been isolated from Crithidia fasciculata. The enzyme was purified 128-fold, almost to homogeneity, and was highly specific for NAD+ as the coenzyme. There is also a cytoplasmic NADP+-linked and a mitochondrial isocitrate dehydrogenase in the organism. Studies of the physical and kinetic properties of the soluble NAD+-isocitrate dehydrogenase from this organism showed that it resembled microbial NADP+-isocitrate dehydrogenases in general, all of which are cytoplasmic enzymes. The enzyme appeared not to be related to other NAD+-isocitrate dehydrogenases, which are found in the mitochondria of eukaryotic cells. The molecular weight of the soluble NAD+-isocitrate dehydrogenase was 105,000 which is within the range of the values for microbial NADP+-isocitrate dehydrogenases. Similar to the NADP+-isocitrate dehydrogenase in this organism, the enzyme was inhibited in a concerted manner by glyoxalate plus oxalacetate. Kinetic analysis revealed that Mn2+ was involved in the binding of isocitrate to the enzyme. Inhibition of the NAD+-linked isocitrate dehydrogenase by p-chloromercuribenzoate could be prevented by prior incubation of the enzyme with both Mn2+ and isocitrate; however, neither ion alone conferred protection. Free isocitrate, free Mn2+, and the Mn2+-isocitrate complex could all bind to the enzyme. Four different mechanisms with respect to the binding of isocitrate to the enzyme were tested. Of these, the formation of the active enzyme-Mn2+-isocitrate complex from (a) the random binding of Mn2+, isocitrate, and the Mn2+-isocitrate complex, or (b) the binding of Mn2+-isocitrate with free Mn2+ and isocitrate acting as dead-end competitors were both in agreement with these data.     

A DNase from the trypanosomatid Crithidia fasciculata.

We have purified to homogeneity a DNase from a Crithidia fasciculata crude mitochondrial lysate. The enzyme is present in two forms, either as a 32 kDa polypeptide or as a multimer containing the 32 kDa polypeptide in association with a 56 kDa polypeptide. Native molecular weight measurements indicate that these forms are a monomer and possibly an alpha 2 beta 2 tetramer, respectively. The monomeric and multimeric forms of the enzyme are similar in their catalytic activities. Both digest double-stranded DNA about twice as efficiently as single-stranded DNA. They introduce single-strand breaks into a supercoiled plasmid but do not efficiently make double-strand breaks. They degrade a linearized plasmid more efficiently than a nickel plasmid. Both enzymes degrade a 5'-32P-labeled double-stranded oligonucleotide to completion, with the 5'-terminal nucleotide ultimately being released as a 5'-mononucleotide. One difference between the monomeric and multimeric forms of the enzyme, demonstrated by a band shift assay, is that the multimeric form binds tightly to double-stranded DNA, possibly aggregating it.     

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.     

Purification and characterization of a manganese containing superoxide dismutase from bovine heart mitochondria

• 1.1. A manganese containing superoxide dismutase from bovine heart mitochondria was isolated and characterized.
• 2.2. It has a molecular weight of about 86,000 and is composed of 4 noncovalently bound subunits of equal size.
• 3.It appears to contain 2 mole manganese per mole enzyme.
• 4.The carbohydrate content is very low.
• 5.The specific activity and amino acid composition are similar to those of other mitoehondrial superoxide dismutases.
• 6.The enzyme forms complexes with ampholytes and can therefore not be analysed by isoelectric focusing.

     

Purification and characterization of extracellular superoxide dismutase in mouse lung

Extracellular superoxide dismutase (EC-SOD) is the major isozyme of SOD in arteries, but is also abundant in lungs. In particular, mouse lungs contain large amounts of EC-SOD compared to lungs in other mammals. This suggests that EC-SOD may have an amplified function in the mouse lung. This study describes the purification and characterization of mouse EC-SOD as well as its localization in mouse lung. Mouse EC-SOD exists primarily as a homotetramer composed of a pair of dimers linked through disulfide bonds present in the heparin-binding domains of each subunit. In addition, mouse EC-SOD can exist in active multimeric forms. We developed and utilized a polyclonal antibody to mouse EC-SOD to immunolocalize EC-SOD in mouse lung. EC-SOD labeling is strongest in the matrix of vessels, airways, and alveolar septa. This localization suggests that EC-SOD may have important functions in pulmonary biology, perhaps in the modulation of nitric oxide-dependent responses.