L ldh

SEC-purified nanomaterials, as well as the trimeric aldolases I53-40T₃ and 4e38 were diluted into “40 buffer” lacking CHAPS such that the total aldolase concentration was 10 μM; 100 μL aliquots of these samples were subjected to a range of incubation temperatures for 1 h. After incubation, samples were centrifuged at 5000 × g for 1 h and then diluted to 0.1 μM. Aldolase activity was measured via an LDH-coupled colorimetric assay. A master mix containing 0.1 mg/mL L-LDH (Sigma), 0.1 mM NADH (Sigma), 1 mM 2-keto-3-deoxy6-phosphogluconic acid (KDPG, Sigma) was prepared and 90 μL aliquots were added to a Corning UV transparent half-area 96-well plate. Ten microliters of diluted aldolase sample was added to each well to initiate the coupled reaction, and the consumption of NADH was monitored via A_339 nm on a SpectraMax M5e (Molecular Devices). The relative initial reaction velocity over 100–200 s was calculated via regression analysis (Softmax Pro).

Lactate content in cell lysates and lactate released into the culture medium were measured using the NADH optical test according the reference [27 (link)]. For determining the intracellular lactate levels, 100 µL of a lysate from 1 × 10⁶ trypsinized cells, prepared with hypotonic buffer indicated above, was precipitated by adding 33.3 µL 4 M perchloric acid on ice for 20 min. Following centrifugation at 13,000× g, 100 µL of supernatant was collected and adjusted to pH 6.5–8.5 by adding 64.6 µL of ice-cold 2 M KOH for 20 min. The resulting precipitate was centrifuged and the supernatant was collected for lactate determination. An aliquot of supernatant and lysate samples was added to a buffer containing 1.1 M hydrazine (pH 9.0), 5 mM NAD+, and L-LDH (rabbit muscle, approximately 1000 units/mL, Sigma). After incubating for 30 min at room temperature, the change in fluorescence was measured at the excitation and emission wavelengths of 355 and 460 nm, respectively (Victor 3, Perkin Elmer). Lactate concentrations were calculated from a reference curve. The intracellular lactate concentration was calculated based on the cell number of the lysate and a volume of 2 × 10⁻¹² L per cell, as determined by microscopy and the size of distribution given by the Casy counter.