Extended Data Fig. 1: PCr phosphatase activities of thermogenic fat and TNAP.
From: Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine
a, PCr phosphatase activities of total mitochondrial protein extracts from different tissues of cold-acclimated mice. Mitochondrial protein extract was prepared from tissues excised from 10 mice for BAT or 20 mice for iWAT. Each reaction contains 10 mM of PCr and 0.4 mg ml−1 of mitochondrial protein extract, except the buffer control. Data are presented as the estimated parameters ± uncertainties. Uncertainties are represented by the standard errors of nonlinear regression that fits a straight-line model to the initial linear phase of PCr hydrolysis kinetics measured by 31P NMR over 11 time points for BAT and iWAT and 6 time points for the buffer control (shown in Source Data). b, Ion-exchange chromatography of the active fraction of SEC. The PCr phosphatase activity of each fraction was measured by enzyme-coupled assay. The activity of the most active fraction was also verified by 31P NMR. Red and blue bars denote the fractions used for isobaric labelling (TMT) and quantitative mass spectrometric analysis. c, Western-blot analysis of the active SEC fraction prepared from cold-acclimated mice (cold), compared with the equivalent fraction prepared from room-temperature housed mice (RT). d, PCr phosphatase activities of total mitochondrial protein extracts from BAT of cold-acclimated mice treated with vehicle or SBI-425 (10 μM), measured by 31P NMR. n = 2 technical replicates per group. Data are presented as mean ± s.e.m. e, Stacked traces of 31P NMR spectra recorded at indicated time points, demonstrating the kinetics of PCr hydrolysis catalysed by recombinant TNAP. The minor peak marked with an asterisk on top is from glycerol-3-phosphate, a side-product of the phospho-transferase activity of TNAP50, that transfers the phosphoryl-group from PCr to glycerol present in the reaction buffer. f, Michaelis constant (Km) curves of hydrolysis of PCr (left) and PPi (right) catalysed by recombinant TNAP. Activities were measured by the enzyme-coupled assay; n = 2 technical replicates. Data are presented as mean ± s.e.m. g, Comparison of the Michaelis–Menten parameters extrapolated from f. Data are presented as the estimated parameters ± uncertainties. Uncertainties are represented by standard errors derived from the nonlinear regression fit of Michaelis–Menten model to the data in f.
