This lack of change in M. smegmatis NADP+-GDH reaction activity P005091 nmr is in contrast to a recent study in which NADP+-GDH animating activity was found to increase significantly in response to nitrogen starvation in a related Actinomycete, Corynebacterium glutamicum [36]. In other bacterial species, NADP+-GDH forward reaction activity has been shown to be down-regulated in response to nitrogen excess [37, 38] or not regulated at all [39]. Figure 2 Specific activities

of the (A) NADP + -specific forward reaction in which NADPH was added as co-factor, (B) NADP + -specific reverse reaction in which NADP + was utilised as co-factor, (C) NAD + -specific forward reaction with NADH as co-factor and (D) NAD + -specific reverse CAL-101 concentration reaction in which NAD + was utilised as co-factor. Each enzyme reaction was assayed under conditions of nitrogen limitation (3 mM (NH4)2SO4) and in response to an ammonium pulse (60 mM (NH4)2SO4). One unit of enzyme activity was defined as the oxidation/reduction of 1 nmole co-factor per minute per milligram ofprotein. The mean specific

activity with standard deviations is included. Table 1 Specific activities of the both the aminating and deaminating reactions for NADP- and NAD-glutamate dehydrogenase enzymes in response to nitrogen starvation conditions (3 mM (NH4)2SO4).   Specific Activity (U) Time (hours)   p-value*   p-value*   p-value*   p-value*   NADPH   NADP +   NADH   NAD +   0 227 ± 24   49 ± 2   281 ± 67   0.02 ± 3   0.5 222 ± 9 0.76 94 ± 5 0.01 264 ± 51 0.01 2.57 ± 3 0.99 1 229 ± 2 0.71 101 L-NAME HCl ± 4 0.69 309 ± 21 0.00 4 ± 3 0.91 2 231 ± 10 0.91 103

± 9 0.80 309 ± 41 0.98 2 ± 2 0.91 4 209 ± 11 0.20 102 ± 25 0.85 356 ± 19 0.01 0.16 ± 3 1.00 *P-values less than 0.05 (in bold print) were regarded as statistically significant changes in specific activity from the previous time point. Upon analysis of the NADP+-GDH reverse or deaminating reaction activity, our results showed that there was a significant change in activity in response to nitrogen availability in M. smegmatis (Figure 1B) thereby suggesting NADP+-GDH is indeed regulated in response to varying nitrogen concentration conditions. When exposed to ammonium starvation conditions, there was a 2 fold increase (Figure 2B, ● and Table 1, p = 0.01) in NADP+-GDH deaminating reaction activity (i.e. glutamate catabolism), which remained constant throughout an extended period of nitrogen starvation (Table 1). The converse effect was observed under conditions of nitrogen excess, namely a rapid, approximately 2 fold decrease in reaction activity (Figure 2B, ■). Since NADP+-GDH performs a reversible reaction, it is interesting to note that a change only in the deaminating reaction activity in response to nitrogen availability was detected. The functional significance of the observed change in glutamate deamination is Selleckchem AMN-107 unclear.