Figure 7. Structural perturbation of Asp-NAT by salt (A) and CHAPS (B) and its impact on enzyme complex size and activity. Salt concentration seems to decrease the Asp-NAT activity by interference (A) whereas detergent concentration decreases the activity by destroying the hydrophobic interactions within the Asp-NAT complex. The columns were pre-equilibrated and eluted with the respective media. The HPLC column size in Fig. A was 14 ml and fraction size was 1 ml each, and, in Fig. B, the column size was 100 ml and fraction size was 2 ml each. Asp-NAT activity was determined after dialyzing the sample against 0.15 M NaCl medium. The recovery of activity in Fig. A was 95%, 80% and 64% for 0.15M, 0.5 M and 2.0 M NaCl media, respectively. Asp-NAT activity was determined after dialyzing the sample against 1 mM CHAPS containing medium. About 85% of the activity was recovered in 1 mM CHAPS treated samples and only 5% of the activity was recovered in 10 mM CHAPS treated samples. The y-axis represents the activity recovered, relative to the input activity normalized to 100.
Table 2. Steady state kinetic parameters and inhibitory concentrations of NAA and CoA for Asp-NAT.
Substrate/ Km (|iM) Vmx (nmol/h/mg IC-50% /max (%)"
Product protein) (l^M)
L-Asp 580 27
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