Pk ldh

Adenylate kinase activity assay was performed as described in^{37 (link)}. In brief, an enzyme coupled ATPase assay was performed in a 96-well plate at 30 °C and measured in a Tecan Infinite 200 PRO reader (Tecan). The reaction volume of 200 µl was composed of 300 mM Tris-glycine buffer pH 8, 0.004% (w/v) trans-PCC-α-M, 5 mM MgCl₂, 4 mM PEP, 0.6 mM NADH, 3.5 µl pyruvate kinase/lactic dehydrogenase (PK/LDH) (Sigma-Aldrich). The reaction was started by the addition of 0–4 mM ATP. For detection of adenylate kinase activity, 0–2 mM AMP were added at saturating concentrations of ATP (4 mM). The absorbance of NADH was detected at 340 nm for 20 minutes. The decrease in NADH absorbance is proportional to the increase in ADP.

The catalytic domains of Src and Hck were expressed in bacteria and purified as previously described by Seeliger, Kuriyan, and colleagues [21 (link)]. The catalytic domains of Ack1 and IGF1R kinases were expressed in Sf9 cells using recombinant baculoviruses, as previously described [29 (link), 30 (link)]. ATP, adenosine 5′-(3-thiotriphosphate) (ATPγS), and PK/LDH were purchased from Sigma. The anti-Src (pY419; equivalent to chicken c-Src pY416) antibody was from Biosource, and anti-pTyr antibody (4G10) was from Millipore. [³⁵S]-labeled ATPγS was from Perkin-Elmer. Dithiothreitol (DTT), acetonitrile (ACN), ammonium bicarbonate, trifluoroacetic acid (TFA), and iodoacetamide (IAA) were from Thermo Fisher Scientific (Waltham, MA). Trypsin Gold, mass spectrometry grade, was from Promega (Madison, WI). Tris-HCl (10 %) non-denaturing gels were purchased from Bio-Rad.

The plasmid
that codes biotin-labeled kinesin 401 (K401) was a gift from Jeff
Gelles (pWC2 – Addgene plasmid #15960; http://n2t.net/addgene: 15960;
RRID_Addgene_15960)^{21 (link)} and was purified
according to previously published protocols.^{22 (link),23 (link)} To create active kinesin clusters, multimotor complexes were prepared
by mixing 0.2 mg/mL kinesin-1 and 0.1 mg/mL streptavidin (Sigma, S4762)
in M2B buffer (M2B: 80 mM PIPES, adjusted to pH = 6.8 using KOH, 1
mM EGTA, 2 mM MgCl₂) containing 0.9 mM DTT and incubated
on ice for 15 min. Four microliters of this mixture were combined
with 1% PEG and 2 mM ATP. The final concentration of kinesin in the
solution was 17 nM. To maintain a steady ATP concentration for the
entire experimental duration, an ATP regeneration system containing
32 mM phosphoenol pyruvate (PEP, Alfa Aesar B20358) and 1.7 μL
of pyruvate kinase/lactic dehydrogenase enzymes (PK/LDH, Sigma, P-0294)
was incorporated. To reduce photobleaching effects, an oxygen scavenging
mix containing 0.2 mg/mL glucose oxidase (Sigma, G2133), 0.05 mg/mL
catalase (Sigma, C40), 2.4 mM Trolox (Sigma, 238813), 0.5 mg/mL glucose,
and 0.65 mM DTT was included. For experiments with labeled motor clusters,
0.1 mg/mL Cy-3-labeled streptavidin (Sigma, S6402) was used to create
the kinesin complexes.

Quantification of ATP and AMP was based on the enzyme coupling method [26 (link)]. Twenty micrograms of total proteins was used. Briefly, ATP was assayed spectrophotometrically at 340 nm, following NADP⁺ reduction, at 25 °C. The reaction mixture contained the following: 1 mM NADP⁺, 10 mM MgCl₂, 5 mM glucose, and 100 mM Tris-HCl, pH 7.4, in 1 ml final volume. Samples were analyzed before and after the addition of purified hexokinase and glucose-6-phosphate dehydrogenase (4 μg; HK+G6PD, HKG6PDH-RO, Sigma-Aldrich, Italy). AMP was assayed spectrophotometrically at 340 nm, following NADH oxidation. The reaction mixture contained the following: 75 mM KCl, 5 mM MgCl₂, 0.2 mM ATP, 0.5 mM phosphoenolpyruvate, 0.2 mM NADH, 10 IU adenylate kinase (AK, M3003, Sigma-Aldrich, Italy), 25 IU pyruvate kinase plus 15 IU lactate dehydrogenase (PK+LDH, Sigma-Aldrich, Italy), and 100 mM Tris-HCl pH 8.0.

Kinase activity of the recombinant protein was measured using a continuous enzyme-coupled reaction system performed at 30 °C^{91 (link)}, with minor modifications. Briefly, the reaction buffer contained 20 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 mM PEP (Sigma Aldrich), 56 U/ml PK/LDH (Sigma Aldrich), 0.3 mg/ml NADH (Sigma Aldrich) and 1 mM ATP. The reaction was initiated by the addition of recombinant kinase to the final concentration, as indicated in the figure legends. Kinase phosphorylation was followed by recording the enzyme-coupled oxidation of NADH to NAD⁺, measured at 340 nm, on a VersaMax microplate reader (Molecular Devices), the data were plotted in Prism 7 (GraphPad).

ATPase activity was measured indirectly by monitoring NADH oxidation. The reaction buffer consisted of 50 mM Tris-Cl (pH 8.0), 2 mM MgCl2, 1 mM DTT and 10mM KCl. Each 100 μL reaction contained 0.4 mM NADH, 0.8 mM phosphoenolpyruvic acid, 1 μM RimK/RpsF protein, 0.7 μl PK/LDH (Sigma) and was initiated by the addition of 10 μL ATP. Enzyme kinetics were determined by measuring A₃₄₀ at 1 minute intervals. Kinetic parameters were calculated by plotting the specific activity of the enzyme (nmol ATP hydrolysed/ min/ mg of protein) versus ATP concentration and by fitting the non-linear enzyme kinetics model (Hill equation) in GraphPad Prism. 25 μM cdG or 1 μM RimB/RimA proteins were included as appropriate.