Recently we constructed a Ribonuclease (RNase) Ti secreting Escherichia coli strain1. In analogy to a DNase test agar system2'3 we now established a rapid screening assay for the detection of the mentioned overproducer on agar plates.

Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase TI, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted. The mutants are His40 --f Thr, Glu58 + Asp, Glu58 + Gln, His92 + Ala and His92 + Phe. His92 + Ala and His92 + Phe mutants are inactive. On the basis of their k,,,/K, values, the mutants Glu58 + Asp and Glu58 + Gln show 10% and 7% residual activity, relative to wild-type RNase T1, whereas the His40 + Thr mutant shows 2% activity. The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three-dimensional structure of the enzyme.

Oxidative stress is regarded as a main causal factor for natural aging. This study tested the hypothesis that healthy elderly people show higher oxidative DNA damage levels and lower antioxidative enzymatic defense capacities than younger ones. In a cross-sectional study, blood samples of 20 older (62-79 years) and 20 younger adults (24-28 years) were compared with respect to oxidative DNA damage in lymphocytes (alkaline elution), oxidative status (serum peroxides), activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and concentrations of total glutathione. In accordance with our hypothesis, elderly males showed a tendency towards higher levels of oxidative DNA damage (single strand breaks). SOD activity inversely correlated with the amount of DNA damage (single-strand breaks and Fpg-sensitive modifications). Oxidative status was increased in older men and negatively correlated with glutathione concentrations. GPx activity was elevated and the SOD/GPx ratio lowered in older males. Subjects with lowered SOD/GPx ratio showed increased oxidative DNA damage. The results indicate age-related changes in the balance between first step (SOD) and second step (GPx) of the enzymatic antioxidant defense system. They support the assumption that a biological optimum between antioxidative enzymes might be more important than their absolute activities.