No abstract

Protein phosphatases-2Ao, 2A1 and 2A2 have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase-2Ao and 2A1, and 0.5 mg of phosphatase-2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases-2Ao and 2A1 each comprised three subunits, termed A, B' and C (2A0) or A, B and C (2A1), while phosphatase-2A2 contained only two subunits, A and C. The A and C components of phosphatases-2Ao, 2A1 and 2A2 had indistinguishable mobilities on sodium dodecyl sulphate/ polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase-2A purified from ethanol-treated muscle extracts. The electrophoretic mobilities of the B and B' subunits were slightly different, and their peptide maps were distinct.The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 6 kDa (2A0), 202 f 6 kDa (2A1) and 107 f 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B') and 36 kDa (C).These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2 (2A0), ABC2 (2A1) and AC (2A2). Protein phosphatase-2A2 appears to be derived from 2Ao and/or 2A1 during purification through degradation or dissociation of the B' and/or B subunits.Protein phosphatases-2Ao, 2A1 and 2A2 were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5 = 0.25 pM), histone H1 (A0.5 = 0.3 pM) and polylysine (A0.5 = 0.04 pM). Activation by protamine varied over 5 -20-fold for phosphatase-2Ao and 5 -7-fold for phosphatases-2Al and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5 -10-fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2-fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B' subunits, or substrates phosphorylase and glycogen synthase.We have recently investigated the nature of the protein phosphatases involved in the control of metabolism [l -101. Based on experiments with about 20 phosphoprotein substrates, we were only able to identify four protein phosphatase catalytic subunits capable of dephosphorylating the major proteins involved in the control of glycogen metabolism, glycolysis and gluconeogenesis, aromatic amino acid breakdown, and fatty acid, cholesterol and protein synthesis. The four phosphatases have been classified into two types depending on whether they dephosphorylate the P-subunit of phosphorylase kinase and are inhibited by the thermostable proteins termed inhibitor-1 and inhibitor-2 (protein phosphatase-1), or whether ...

Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-M, S-adenosyl-L-methionine synthetase and low-M, S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-M, S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-M, S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-M, S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-M, and low-M , S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-M, S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-M, S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.ATP : methionine-S-adenosyl-L-transferase (AdoMet synthetase) catalyzes the formation of AdoMet using methionine and ATP as substrates. Pyrophosphate and phosphate are generated in this reaction [l]. AdoMet is the methyl donor in most transmethylation reactions and also the propylamino donor in the biosynthesis of polyamines [l -31. Of the plasma methionine 48% is metabolized in liver [4] and

We have measured the activity of S-adenosyl-L-methionine synthetase, the ratio between the high- and low-molecular-weight forms of this enzyme and the concentration of S-adenosyl-L-methionine in liver biopsies from a group of controls (n = 6) and in six cirrhotics (five posthepatitic and one alcoholic). The total activity of S-adenosyl-L-methionine synthetase was markedly reduced in cirrhosis (37.5% of that found in the control group). This was due to a specific reduction in the high-molecular-weight S-adenosyl-L-methionine synthetase in the group of cirrhotics (73.9 pmoles per min per mg protein) when compared with that observed in controls (460.3 pmoles per min per mg protein). Despite this reduction in the rate of synthesis of S-adenosyl-L-methionine (the high-molecular-weight form of the enzyme is 15 times more active than the low-molecular-weight form at physiological concentration of substrates), the concentration of this metabolite was the same in the control group (17.3 +/- 2.6 microM) and in the group of cirrhotics (17.8 +/- 3.1 microM). To explain these findings, it is postulated that in human liver, where the concentration of S-adenosyl-L-methionine is lower than the Km values of a variety of enzymes that use this metabolite (around 50 to 100 microM), a reduction in the synthesis of S-adenosyl-L-methionine is compensated by a reduction in the rate of utilization of this molecule without affecting the intrahepatic concentration of S-adenosyl-L-methionine.(ABSTRACT TRUNCATED AT 250 WORDS)

SIRT1 protein levels are downregulated by IL1β/NFκB signaling in APAP hepatotoxicity, resulting in inflammation and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16:293-296, 2012) with the following serving as open reviewers: Rafael de Cabo, Joaquim Ros, Kalervo Hiltunen, and Neil Kaplowitz. Antioxid. Redox Signal. 28, 1187-1208.