Time course of Arginine Deprivation Response (ADR).

<p>A) Time-course of LdAAP3 protein abundance increase as function of arginine deprivation time in wild type (left four lanes, lower bands) and <i>Δldaap24</i> (right four lanes, lower bands) <i>L</i>. <i>donovani</i> promastigotes. Mid-log phase parasites were suspended at a density of 0.5–1×10⁷ cells/ml in medium 199 without arginine and further incubated at 26°C. Aliquots were collected at 0, 10, 30 and 60 minutes, cellular proteins separated on 9% SDS-PAGE, transferred to nitrocellulose paper and subsequently probed with anti LdAAP3 antiserum. B) Rate of arginine pool depletion following arginine deprivation of <i>L</i>. <i>donovani</i> promastigotes wild type and <i>Δldaap24</i>. Aliquots of wild type (●) and <i>Δldaap24</i> (■) collected in (A) were subjected to amino acid analyses as described in Materials and Methods. Values are mean ± S.D. (n = 3). Inset is a table that indicates intracellular arginine concentration values at zero and 60 minutes. C) Northern analysis of <i>LdAAP3</i> mRNA abundance change during first 30 minutes of arginine deprivation. As a probe we used LdAAP3.2 (LinJ.31.0910). D) Arginine (0.45 mM) added to promastigotes after 2 hours of arginine deprivation induced rapid degradation of LdAAP3. Degradation was inhibited by the addition of 1 mM MG132 (Last two right lanes).