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. 2021 Jan 21;296:100205. doi: 10.1074/jbc.RA120.015103

Figure 1.

Figure 1

PCK1 is chemically acetylated in the presence of acetyl-CoA.A, top, the ability of rat recombinant PCK1 to be acetylated by p300 in vitro was tested using different controls. However, PCK1 (α-PCK1) exhibited a high susceptibility to react and be acetylated (α-AcK) in the presence of only 1 mM acetyl-CoA, as determined by Western blotting. Numbers on the left represent molecular weight (kDa). Bottom, relative acetylation levels of PCK1 determined from Western blots above in the presence of acetyl-CoA or in the presence of acetyl-CoA and p300 after 18 h (n = 3). B, histone H2A was acetylated by p300 under the same experimental conditions described in (A). C, determination of PCK1 activity in both reaction directions for samples acetylated in the presence of acetyl-CoA versus acetyl-CoA+p300 obtained in (A) (n = 3); ∗∗p < 0.01. D–E, Western blot shows that SIRT1 (α-SIRT1) deacetylates (α-AcK) chemically (AcCoA) and enzymatically (AcCoA+p300) acetylated PCK1 (α-PCK1) (D), without recovery in enzyme activity, compared with recombinant PCK1 incubated in the absence of acetyl-CoA (E) (n = 3). F, chemical acetylation of human recombinant PCK1 after 18 h of incubation in the presence of 1 mM acetyl-CoA, as described in (A). G–H, similar experiment to that described for (F) but performed at 37 °C for 2 or 4 h (G). After 2 h, enzyme activity was considerably decreased, compared with the freshly thawed enzyme (control), but differences between no acetyl-CoA (control no AcCoA) and acetyl-CoA (AcCoA) treatment were observed (n = 3); ∗p < 0.05; ∗∗p < 0.01. I, determination of the ability of CoA to interfere with chemical acetylation of PCK1 by Western blot analysis. The acetylation reaction was carried out in the absence of CoA or in the presence of 0.5, 1, 2, or 5 mM CoA, with 1 mM acetyl-CoA in all cases. J, determination of PCK1 activity in both reaction directions in the presence of different concentrations of CoA. PCK1 was preincubated in the absence of CoA or in the presence of the indicated concentrations of CoA for 30 min at 30 °C and enzyme activity was assayed in buffer containing the same concentrations of CoA. PCK1, phosphoenolpyruvate carboxykinase.