The cytosolic superoxide dismutase (SOD) of cDNA collection was screened with

The cytosolic superoxide dismutase (SOD) of cDNA collection was screened with the SOD gene fragment as a probe. that the enzymes play a special defensive role for the parasite at the interface of the host-parasite interaction. Like the above-mentioned parasites, has developed SODs to defend itself from oxidative killing mechanisms of the host (48). However, at present there is little information indicating that SOD is associated with the pathogenicity of the parasite. Therefore, in order to elucidate a possible role of the SOD of in the defense 72-48-0 manufacture against the oxygen-dependent killing mechanisms of the host, we carried out the purification and characterization of cytosolic SOD from adult Rabbit polyclonal to BMPR2 worms were obtained from the liver of an infected bovine. The collected worms were washed several times with phosphate-buffered saline (PBS [pH 7.4]) and then incubated in the same buffer at 37C for 3 h to eliminate any residual host matter. After the parasites were washed with PBS several times, they were stored at ?70C until used. Preparation of parasite extract. adult worms were homogenized with a Teflon homogenizer in PBS supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetic acid, and 10 mM leupeptin. The homogenates were centrifuged at 28,000 for 30 min at 4C, and the supernatants were collected and used for further studies. The protein concentration was determined by the method of Lowry et al. (35), with bovine serum albumin as a standard. Purification of SOD. extract was applied to a diethylaminoethyl (DEAE)-Sephacel column (1.6 by 12 cm; Pharmacia, Uppsala, Sweden) equilibrated with 50 mM sodium phosphate buffer (pH 7.5). The column was extensively washed with the same buffer, and the absorbed proteins were eluted with a linear gradient of 0.5 M NaCl. Fractions exhibiting SOD activity were pooled and purified further by successive carboxymethyl (CM) Sepharose Fast Flow chromatography (1.6- by 12-cm column; Pharmacia), equilibrated 72-48-0 manufacture with 0.1 M sodium acetate buffer (pH 5.5), and eluted with a linear gradient of 0.5 M NaCl. Fractions exhibiting SOD activity were collected, concentrated, and applied to Superose 12 molecular-sieve chromatography columns (1.6 by 30 cm; Pharmacia) equilibrated with 50 mM sodium phosphate buffer (pH 7.5) containing 0.15 M NaCl. Active fractions were collected, concentrated, and applied to Mono-Q ion-exchange chromatography columns (0.5 by 5 cm; Pharmacia) equilibrated with 10 mM Tris-HCl buffer (pH 7.0). After extensive washing with the same buffer, absorbed proteins were eluted with a linear gradient of 0.5 M NaCl. All the purification procedures were performed at 4C. Enzyme 72-48-0 manufacture assay. SOD activity was dependant on the neotetrazolium chloride (NTC) decrease assay predicated on the technique of Noridaka et al. (45). The assay mixtures (0.5 ml) contained 50 l of 0.5 M sodium phosphate (pH 7.5), 25 l of 16% Triton X-100, 2.5 l of 10 mM EDTA, 75 l of just one 1.2 mM NTC, 2.5 l of xanthine oxidase (1.0 U), 10 l of test, 25 l of 4 mM hypoxanthine, and distilled drinking water. The mature worms had been homogenized in guanidium thiocyanate and split on the CsCl stage gradient, and total RNA was extracted by a way referred to previously (12). mRNA was chosen by oligo(dT) chromatography (23). Building of cDNA collection. cDNA collection of was built utilizing the Librarian Communicate cDNA collection kit (Invitrogen, NORTH PARK, Calif.). The pcDNA3.1+ vector was linearized with TOP10F (Invitrogen) and amplified by over night growth upon ampicillin plates. These plates had been pooled and scraped right into a glycerol share that was aliquoted, and 72-48-0 manufacture kept at ?70C. The amount of major recombinants within the library was established to be 1.38 106 by serial dilution of the unamplified library. Restriction analysis of 10 clones showed that 10 out of 10 contained inserts. The inserts had the sizes listed above, and the average size of the 10 clones was 0.74 kb. PCR. Two degenerate oligonucleotide primers were designed based on conserved amino acids of copper/zinc-containing (Cu/Zn-SODs) from various eukaryotic organisms. The sequence of the forward primer (primer 1) was 5-GC(T/G)GG(A/T)(G/C)C(T/G)CATTT(T/C)AATCC-3, and the sequence of the reverse primer (primer 2) was 5-CC(A/G)CA(A/T)GC(A/T)A(A/C)ACGA(G/C)(G/C)ACCAGCATT-3. Using the two primers, PCR analysis was performed on an cDNA library to check the presence of SOD cDNA sequence. Two microliters of library was heated at 90C for 5.