**3.5 General properties of NDD activities**

DD activities were assayed only in crude cell-free extracts of aerobically-naphtahalene grown mycelia cells because at these moments, we were strongly interested in the NDD activity.

In all cases, the DD activities were tested in cell-free extracts of *M. circinelloides* YR-1.grown in 0.5% of naphthalene as sole carbon source for 22 h at 28 ºC. Cytosolic fraction was separated in a native electrophoresis and the amount of protein loaded per track equalized and was equivalent to 300 g, NADP+ as electron acceptor and 100 mM *cis-*naphthalene-diol as enzyme substrate were used to reveal the zymograms.


Table 5. Activities of *cis-*naphthalene-diol and alcohol dehydrogenase of cytosolic fraction of *M. circinelloides* YR-1 grown in the best inducer for each one. Densitometric analysis was carried out as described in Materials and Methods. The enzymatic determination was on the gel with *cis-*naphthalene-diol or ethanol as the substrate and NADP+ or NAD+ as the electron acceptor.

a Relative units were obtained by densitometry, using the value from iNDD as 100% when naphthalene was the carbon source.

#### **3.5.1 pH dependence**

The Fig. 3 shows that the optima pH value for all five activities expressed with nnaphthalene as carbon source and NADP+ and naphthalene-diol in the enzymatic reaction was 8.5. It is noticeable that only the iNDD show activity at pH 3 and little DD activities were showed at pH values below 8.5. It is important to say that the background in the lane for activity revealed at pH 9, was darken because of pH.

Fig. 3. Effect of pH on dihydrodiol dehydrogenase activities present in cytosolic fraction of *M. circinelloides* YR-1grown in naphthalene. Each track was cut and stained at the indicated pH value.
