Lactate dehydrogenase

To measure extracellular lactate, media was mixed with 6% perchloric acid at 2:1 ratio (Sigma). Samples were centrifuged and the supernatant was neutralized using 1/6 volume of 2 M Na₂CO₃ (Sigma). Enzymatic reaction was carried out in 0.4 M hydrazine (Sigma), 0.5 M glycine (Bio-Rad), pH 9.0 buffer with 2 mM NAD and 2 U/ml lactate dehydrogenase (Sigma). Absorbance at 340 nm was measured with 2 min interval for 15 cycles. To measure intracellular lactate, cells were trypsinized and snapped frozen. The frozen cell pellet was then lysed instantaneously with H₂O with aid of three pulses of 5 s 20% amplitude sonication. Sample was then mixed with 6% perchloric acid and neutralized with 2 M Na₂CO₃. Enzymatic reaction was performed in 0.5 M glycine, pH 9.0 buffer with 2 mM NAD, 2 U/ml lactate dehydrogenase, 0.05 mg/ml MTT and 0.1 mM phenazine thiosulfate (PES, Sigma). Absorbance at 570 nm was monitored with 2 min interval for 15 cycles. Initial velocity gradient was determined from the linear portion of kinetic curve. Lactate concentration was calculated from a lactate standard curve constructed using sodium lactate solution (Sigma).

The ATPase assays were carried out in DAB (50 mM K‐Ac, 30 mM HEPES, pH 7.4, 2 mM Mg(Ac)₂, 1 mM EGTA) as follows. We mixed dynein (monomeric for all constructs) to a final concentration of 10–20 nM with 2 mM Mg‐ATP (Sigma), 0.2 mM NADH (Sigma), 1 mM phosphoenolpyruvate (Sigma), 0.01 U pyruvate kinase (Sigma), 0.03 U lactate dehydrogenase (Sigma), 10 μM Taxol, 1 mM DTT, and 0–5 μM microtubules in DAB. Absorbance at 340 nm was continuously measured in an Eppendorf Spectrophotometer (UV‐Vis BioSpectrometer), and the data were fit to the following equation (Bhabha et al, 2014) using an excel curve fitting routine: $k_{\mathrm{obs}}=\left(k_{\mathrm{cat}}-k_{\mathrm{basal}}\right)\frac{[\text{MT}]}{K_{\mathrm{M}}+[\text{MT}]}+k_{\mathrm{basal}}.$
The vanadate inhibition of dynein ATPase activity was performed as previously described (Höök et al, 2005). Briefly, we mixed dynein [monomeric of wild‐type and mutant 5 (from the same batch that was used to solve the structure of mutant 5 in the presence of ADP‐vi)] to a final concentration of 20 nM with 1 mM Mg‐ATP (Sigma), 0.2 mM NADH (Sigma), 1 mM phosphoenolpyruvate (Sigma), 0.01 U pyruvate kinase (Sigma), 0.03 U lactate dehydrogenase (Sigma), 10 μM Taxol, 1 mM DTT, 6 μM microtubules, and 0–100 μM vanadate (Sigma) in DAB. The vanadate was boiled for 10 min before usage. Absorbance at 340 nm was continuously measured in an Eppendorf Spectrophotometer (UV‐Vis BioSpectrometer), and the turnover rate was calculated as described above.