A Critical Tryptophan and Ca2+ in Activation and Catalysis of TPPI, the Enzyme Deficient in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis
Figure 2
Specific activity of TPPI for various W mutants relative to mature TPPI.
Freshly plated CHO cells at 70% confluency were transiently transfected with mutant plasmids using FugeneHD reagent. After 48 h, cells were pelleted by centrifugation at 800× g for 5 min, followed by homogenization in lysis buffer comprising 50 mM ammonium formate, pH 3.5, containing 0.15M NaCl and 0.1% Triton X-100. TPPI activity was measured as described in Experimental Procedures and related to the band intensity of mature TPPI by Western blot analysis.