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. 2009 May 6;10(5):2041–2053. doi: 10.3390/ijms10052041

Figure 5.

Figure 5.

The effect of human Tim/Pam proteins on the ATPase activity of yeast mtHsp70.

The ATPase assay was carried using the pyruvate kinase/lactate dehydrogenase-coupled assay as described previously [33]. The reaction mixture (total volume of 300 μl) contained the following components: 50 mM Tris-HCl pH=7.4, 50 mM K-Acetate pH=7.4, 10 mM Mg-Acetate pH=7.4, 0.3 mM NADH, 0.2 mM phosphoenolpyruvate, 20 units of pyruvate kinase, 10 units of lactate dehydrogenase and the indicated combinations of the following proteins mtHsp70 (2.5 μM), Mge1 (5 μM), full length Tim14/Pam18 and the soluble domain of Tim16/Pam16 from either Saccharomyces cerevisiae or human (2.5 μM). The reaction was initiated by the addition of 2 mM ATP. Rates were extracted from the linear phase of the reaction. Each column represents at least four independent repeats of the experiment. 1) mtHsp70. 2) mtHsp70+Mge1. 3) mtHsp70+Mge1+yTim14/Pam18 (full length). 4) mtHsp70+Mge1+yTim14/Pam18 (full length)+yTim16/Pam16s. 5) mtHsp70+Mge1+yTim14/Pam18 (full length)+hTim16/Pam16s.