Bc0545

Pyruvate kinase activity and 3-phosphoglycerate kinase activity were determined using the assay kits (BC0545 and BC2255, Solarbio, China) following the instructions provided by the manufacturer. The enzymes activity was presented as units per milligram of protein (U/mg protein).

The enzymatic activities of HK, PFK and PK were assayed according to the instructions of the commercial kits (BC0745, Solarbio; BC0535, Solarbio; and BC0545, Solarbio). Cells or tissues were collected into centrifuge tubes and extracts were added in appropriate proportions. Cells were crushed with ultrasound (Scientz, Scientz-IID) and tissues were homogenized in ice bath. The extracts were then centrifuged at 8000 g for 10 min at 4°C. The supernatant was taken for testing. The reagents were added sequentially to the 96-well plate according to the instructions. After mixing well, the absorbance difference values were measured at different times on a microplate reader (MuitisKan, ThermoFisher) according to the instructions. The wavelength of the microplate reader was set to 340 nm.

The activity of PDH was measured as reported (75 (link)–77 (link)), and the PK enzymatic activity was assessed using a commercial assay kit (BC0545, Beijing Solarbio Science and Technology Co., Ltd.). The bacteria were treated as stated above. After incubation with serum or/and mannitol, the bacteria were collected and adjusted to OD600 of 1.0. Moreover, 20 ml of OD600 = 1.0 bacterial solution was collected in a 1.5-ml centrifugation tube, resuspended with 1 ml sterile saline, and disrupted by sonic oscillation (200 W total power with 35% output, 2 s pulse, and 3 s pause over ice) for 3 min. After centrifugation, the supernatants of the enzyme activity assay were collected. Protein concentration was measured with enhanced BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China).