Glycerol suppresses glucose consumption in trypanosomes through metabolic contest
Fig 2
The glycerol preference is the consequence of the high excess of GK activity.
(A) Enzymatic assays used for the quantification of hexokinase (HK) and glycerol kinase (GK) activity. The bold and underlined substrates and enzymes are included in the assay for production of NADPH (HK assay) and consumption of NADH (GK assay) that are detected by spectrometry at 350 nm. 6PG, 6-phosphogluconate; G6PDH, glucose-6-phosphate dehydrogenase; Gly3P, glycerol 3-phosphate; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; PYK, pyruvate kinase. (B) HK (left panel) and GK (right panel) activity in total cell extracts (wild-type [WT], RNAiGK.i and RNAiGK.ni) determined in the presence of glucose (Glc), glycerol (Glyc) or equal amounts of glucose and glycerol (Glc/Glyc). (C) GK and HK activity in different combinations (indicated in the table below the graph) of total cell extracts from the parental (WT) and the RNAiGK.i cell lines. The amount of HK remained the same in all samples (see S2 Fig), while the amount of GK present in the parental samples was diluted with the GK-depleted RNAiGK.i samples. The HK and GK activity were determined in the presence of both glucose and glycerol, as in the Glc/Glyc condition (see [B]). (D) Expression of HK activity as a function of GK activity. The values in parentheses indicate the rate of glycerol consumption in the RNAiGK.ni and RNAiGK.i cells compared to the parental cells (100%) (see Fig 1F and 1G). (E) HK activity in the presence of 10 mM acetate and increasing amounts of acetate kinase. Data supporting the results described in this figure can be found at https://zenodo.org/record/5075637#.YORd2B069yA.