FIGURE 5.

Exogenous Hsc70 increases TH activity in MN9D cells and purified synaptic vesicles. A, TH activity was measured in purified SVs obtained from mouse brain. The SVs were incubated with 10 μg of recombinant Hsc70, the closely related recombinant Hsp70 or Hsp60, or denatured Hsc70 or denatured Hsp70. The TH activity within the purified SV2 without recombinant protein (Control) was considered 100%. B, purified SVs were incubated without (Control) or with 0.1, 1, or 10 μm recombinant Hsc70. TH activity increased in a dose-dependent manner in the presence of recombinant Hsc70. C, TH activity was also assessed in the purified SVs in the presence of GST-only or GST-Hsc70 constructs. The GST-Hsc70 fusion constructs that interact with TH and AADC (GST-373–650, GST-373–540, and GST-540–650) were able to enhance TH activity in comparison with the TH activity in purified SVs alone (control, considered 100%). In contrast, non-interacting GST-Hsc70 fusion constructs (GST-1–540) did not alter TH activity. Recombinant Hsc70 was used as a positive control, and GST-only was used as a negative control. D, TH activity was assessed post-nuclear preparation from MN9D WT cells. The PNS was incubated with recombinant Hsc70, the closely related recombinant Hsp70 or Hsp60, or denatured Hsc70 or denatured Hsp70. The TH activity within the PNS alone in the control was considered 100%. *, p < 0.05.