|dc.description.abstracten||Copper-Zinc superoxide dismutase (SOD-1) is an ubiquitously occurring eukaryotic enzyme with a variety of important effects on respiring organisms. This enzyme has been studied on several species of eukaryotic and non-eukaryotic organisms. However, the Cu,Zn SOD from molds and yeast have not been studied extensively. Today, the only fungí specie from which the SOD-1 has been characterized is Saccharomyces cerevisiae, although the nucleotide sequences of sod-1 gene from Schizosaccharomyces pombe, Neurospora crassa andAspergillus Japonicus, are included on data banks.
We have isolated a cytosolic Cu,Zn SOD from Debaryomyces hansenii showing a subunit mass of 15.6 kDa. The preparation was found to be heterogeneous by IMAC chromatography, native- and IF-electrophoresis (showing two pl ranges: 5.14 to 4.0 and 1.6 to 1.8), suggesting the existence of two Cu,Zn SOD enzymes in Deb. hansenii. Differences on specific activity and amino acid composition oftwo Cu,Zn SOD IMAC fractions (32 and 37) supports this conclusion. Major differences are observed on the number of histidine and tyrosine residues; one form (IMAC 32, -17 300 U/mg of protein) presents 6 histidines, while the other (IMAC 37, -8 000 U/mg of protein) only 3 residues; the number of tyrosine residues on form one are 2 while on form two is increased to 5. We suggest that the 3 tyrosine residues could be play the role of the histidines on the active site of form one. Under stress conditions promoted by CIO2 concentration, the presence of two genes could be suggested.
The enzyme preparation had a remarkably strong stability at pH 6.0 to 7.0, surviving boiling periods of 1 O minutes without losing more than 60 % of activity. On Western blots these enzymes were recognized by antibodies raised in rabbits against Deb. hansenii extracts, while only a weak cross-reaction was detected using antibodies generated against either Sacch. cerevisiae and/or bovine erythrocyte Cu,Zn SODs. When rnice were immunized with acrylarnide-gels slices containing the Deb. hansenii Cu,Zn SOD enzymes a severe effect on animals' health was observed (-25% lower weight compared to controls). In sequencing analysis, a peptide obtained by trypsin digestion was found to correlate 85 % in homology with the Sacch. cerevisiae Cu,Zn SOD, as well as, by amino acid composition analysis. The pure Deb. hansenii-SOD form one enzyme, is 300% more active than other Cu,Zn S0Ds used as a reference. Considering all these properties, we concluded that the cytosolic Cu,Zn SOD preparation from Deb. hansenii could be an interesting altemative source for such enzyme.
A gene (dh sod-1) encoding a Cu,Zn SOD of the marine yeast Deb. hansenii was cloned by use of mRNA by RT-PCR technique. Through this procedure two identical clones containing fragments of 470 bp were obtained. From nucleotide sequence we determined that both clones contained a 462 bp coding region, which encodes a 154 amino acids protein with a predicted molecular weight of 15.92 kDa. The encoding region in the dh sod-1 gene has no introns, and no longer than 470 bp PCR products were obtained when partially digested genornic DNA was used as template on PCR arnplification. The amino acid composition deduced from amino acid sequence of dh sod-1, corresponds to that of fraction IMAC 32, suggesting that the cloned gene corresponds to the form one of Cu,Zn SOD on Deb. hansenii.
Comparing the deduced amino acid sequence with that of cytosolic superoxide dismutases from
Sacch. cerevisiae and Neurospora crassa -70% homology was observed. Lower homologies (among 55 - 65%) were observed with the corresponding enzyme of other eukaryotic organisms. However, although the obtained encoding clones (dh sod-1) contains ali the necessary information to produce a functional protein, when an expression experiment was done in a bacterial system, recombinant products were not detected.||es_MX