Guidelines for enhanced recovery after cardiac surgery. Consensus document of Spanish Societies of Anesthesia (SEDAR), Cardiovascular Surgery (SECCE) and Perfusionists (AEP). / Vía clínica de recuperación intensificada en cirugía cardiaca. Documento de consenso de la Sociedad Española de Anestesiología, Reanimación y Terapéutica del Dolor (SEDAR), la Sociedad Española de Cirugía Cardiovascular y Endovascular (SECCE) y la Asociación Española de Perfusionistas (AEP).

The ERAS guidelines are intended to identify, disseminate and promote the implementation of the best, scientific evidence-based actions to decrease variability in clinical practice. The implementation of these practices in the global clinical process will promote better outcomes and the shortening of hospital and critical care unit stays, thereby resulting in a reduction in costs and in greater efficiency. After completing a systematic review at each of the points of the perioperative process in cardiac surgery, recommendations have been developed based on the best scientific evidence currently available with the consensus of the scientific societies involved.

Structure-function relationships in methionine adenosyltransferases.

Methionine adenosyltransferases (MATs) are the family of enzymes that synthesize the main biological methyl donor, S-adenosylmethionine. The high sequence conservation among catalytic subunits from bacteria and eukarya preserves key residues that control activity and oligomerization, which is reflected in the protein structure. However, structural differences among complexes with substrates and products have led to proposals of several reaction mechanisms. In parallel, folding studies begin to explain how the three intertwined domains of the catalytic subunit are produced, and to highlight the importance of certain intermediates in attaining the active final conformation. This review analyzes the available structural data and proposes a consensus interpretation that facilitates an understanding of the pathological problems derived from impairment of MAT function. In addition, new research opportunities directed toward clarification of aspects that remain obscure are also identified.

Modulation by the ratio S-adenosylmethionine/S-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase.

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S-adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein.

S-adenosylmethionine synthesis: molecular mechanisms and clinical implications.

Methionine adenosyltransferase (MAT) is an ubiquitous enzyme that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. In mammals, there are two genes coding for MAT, one expressed exclusively in the liver and a second enzyme present in all tissues. Molecular studies indicate that liver MAT exists in two forms: as a homodimer and as a homotetramer of the same oligomeric subunit. The liver-specific isoenzymes are inhibited in human liver cirrhosis, and this is the cause of the abnormal metabolism of methionine in these subjects.

The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site.

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.

Recombinant rat liver S-adenosyl-L-methionine synthetase tetramers and dimers are in equilibrium.

Rat liver S-adenosyl-L-methionine synthetase is present in two oligomeric forms, tetramers and dimers, with different substrate kinetics and regulation. In vivo the relative amounts of both forms may change in some instances. The basis of this regulatory mechanism is not known. When rat liver cDNA was used to express the protein in Escherichia coli the two oligomeric forms were found. Gel filtration chromatography of the purified recombinant enzyme suggested that these two isoforms might be in equilibrium. This was confirmed by kinetic experiments which showed that the specific activity of the enzyme was dependent on the protein concentration. From these experiments, apparent equilibrium constants of (5.6 +/- 0.4) x 10(5) M-1 and (3.5 +/- 0.9) x 10(5) M-1 were obtained at 2mM and 60 microM methionine concentrations, respectively. Using hydrophobic chromatography on phenyl-Sepharose to separate the tetrameric and dimeric forms, an equilibrium constant of (4.9 +/- 0.7) x 10(5) M-1 was calculated. A rate constant for the dissociation of the tetramer of k-1 = (8.1 +/- 0.4) x 10(-4) s-1 at 4 degrees C was also calculated using the same approach. In summary, we have shown that the rat liver S-adenosyl-L-methionine synthetase produced in bacterial cells is present in two oligomeric forms, tetramers and dimers, which are in equilibrium. This system might be useful for studying the dynamics and the regulation of the distribution of oligomeric forms in the mammalian liver.

Glucocorticoid regulation of hepatic S-adenosylmethionine synthetase gene expression.

The effects of glucocorticoids on the regulation of rat liver S-adenosylmethionine synthetase were studied in vivo and in two culture systems. Livers from adrenalectomized animals were examined for enzyme activity, immunoreactive protein, and messenger RNA (mRNA) content. All three parameters showed a similar trend, i.e. they decreased 3-fold after adrenalectomy and increased over the control values upon triamcinolone replacement. These results suggested that glucocorticoid regulation of hepatic S-adenosylmethionine synthetase was mediated at the mRNA level. Triamcinolone and dexamethasone increased S-adenosylmethionine synthetase mRNA content in a time- and dose-dependent manner in both rat hepatoma H35 cells and primary cultures of adult rat hepatocytes. The kinetics of mRNA induction were identical in both culture systems, indicating that the hormone-mediated response is independent of the differentiated state of the cell. Insulin blocked the inducing effect of glucocorticoids on S-adenosylmethionine synthetase mRNA in a dose-dependent manner. On the other hand, the triamcinolone-dependent increase in mRNA levels was completely abolished by treatment with actinomycin D, whereas cycloheximide did not affect this response. The transcription rate of the gene, as measured by run-on assay, increased 3-fold after hormone addition. Transient transfections of H35 cells with 1.4 kilobases of the 5'-flanking region of the hepatic S-adenosylmethionine synthetase gene fused to a luciferase reporter gene showed that promoter activity is also increased 3-fold after triamcinolone treatment, suggesting that this promoter region contains the sequence elements necessary to confer glucocorticoid responsiveness. In addition to the transcriptional control of the hepatic S-adenosylmethionine synthetase gene, our results suggest that glucocorticoids may be acting at a posttranscriptional level.

Calcium-dependent binding between calmodulin and lysozyme.

Calmodulin, an acidic protein that binds calcium with high affinity, has multiple roles in the activation of many enzymes involved in cellular regulation of eukaryotes. In this study we show that calmodulin binding to hen egg-white lysozyme, in a Ca2+-dependent way, was observed using electroblots incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase or for affinity chromatography on a gel calmodulin column. Antimicrobial activity of lysozyme was not modified in the presence of Ca2+-calmodulin.

Role of thioltransferases on the modulation of rat liver S-adenosylmethionine synthetase activity by glutathione.

Rat liver S-adenosylmethionine synthetase, high- and low-Mr forms, are regulated in vitro by the GSH/GSSG ratio at pH 8. The inhibition and oxidation constants for both forms have been calculated in the presence of thioltransferases. The mechanism of the reaction appeared to involve the formation of intramolecular disulfides. Increases of 3- to 4-fold in the oxidation constants for both S-adenosylmethionine synthetase isoenzymes in the presence of protein disulfide isomerase suggested the possibility of a thiol-disulfide exchange regulatory mechanism for this enzyme in vivo. The significance of these results is discussed on the light of the data available relating glutathione changes and modulation of enzyme activities, either in vivo and in vitro.

Analysis of the 5' non-coding region of rat liver S-adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme.

A 3 kb cDNA coding for rat liver S-adenosylmethionine (AdoMet) synthetase has been isolated. The Mr of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl-Sepharose column. The length of the mRNA 5' non-coding region has been defined by primer-extension analysis. The rat liver cloned cDNA has been also used to detect S-adenosylmethionine synthetase mRNA in human liver.

How is rat liver S-adenosylmethionine synthetase regulated?

The in vivo regulation of S-adenosylmethionine synthetase, a key enzyme in methionine metabolism, is so far unknown. The enzyme activity has been shown to be modulated by glutathione and the oxidation state of its sulfhydryl groups. Analysis of the protein sequence has revealed the presence of putative phosphorylation sites. A mixed regulatory mechanism combining phosphorylation and the oxido/reduction of sulfhydryl groups is proposed. The role of glutathione in this mechanism is also discussed.

Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: Part I. Biochemical implications.

The energy-dependent conversion of methionine to S-adenosyl-L-methionine (SAMe) is catalysed by S-adenosyl-L-methionine synthetase (SAMe-synthetase) in the liver. In the hepatocyte, an equilibrium exists between the high and low molecular weight forms of SAMe-synthetase, which consist of a tetramer and a dimer, respectively, of a 48.5 kilodalton subunit. The 2 enzymic forms differ in their affinity for methionine and sensitivity to inhibition by pyrophosphate; 2 of the sulfhydryl groups of SAMe-synthetase have been identified as essential for the normal functioning of the enzyme. In patients with liver cirrhosis, a marked reduction in the utilisation of the high molecular weight SAMe-synthetase and displacement of the equilibrium occur, the molecular mechanism of which has yet to be established. This loss of activity is associated with a delay in methionine clearance and impairment of the trans-sulphuration pathway, which normally eliminates excess methionine by oxidising homocysteine to sulphate anion. It is hypothesised that in normal liver function the essential sulfhydryl groups of SAMe-synthetase are protected from oxidation by glutathione, a by-product of the trans-sulphuration pathway. However, glutathione levels are reduced in liver cirrhosis, and this may result in increased oxidation of the essential sulfhydryl groups, and consequent inactivation of the enzyme. Thus, the trans-sulphuration pathway may play an important role in the maintenance of normal SAMe-synthetase activity.

S-adenosyl-L-methionine synthetase and methionine metabolism deficiencies in cirrhosis.

Methionine metabolism impairment in human liver disease has been related with an alteration in SAM-synthetase. This deficiency is produced by a post-translational event since human liver cirrhosis presents normal levels of SAM-synthetase mRNA in spite of a more than 50% diminution in its activity. A series of different experiments on the structure and activity of this enzyme have provided strong evidence that SAM-synthetase is regulated by reduced/oxidized glutathione ratio. Restoration of glutathione levels by the addition of S-adenosyl-methionine or glutathione esters in various experimental conditions (buthionine sulfoximine and carbon tetrachloride intoxication) resulted in a normalization of the SAM-synthetase diminution caused by the toxics and an attenuation of the morfological alteration produced in the liver, including fiber production. This findings might have pharmacological implications in the treatment of liver diseases, since the possible beneficial effect of long term administration of SAM could include a reduction of fiber production.

Early effects of copper accumulation on methionine metabolism.

Wilson's disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.

Betaine homocysteine S-methyltransferase: just a regulator of homocysteine metabolism?

Betaine homocysteine methyltransferase (BHMT), a Zn(2+)-dependent thiolmethyltransferase, contributes to the regulation of homocysteine levels, increases in which are considered a risk factor for cardiovascular diseases. Most plasma homocysteine is generated through the liver methionine cycle, in which BHMT metabolizes approximately 25% of this non-protein amino acid. This process allows recovery of one of the three methylation equivalents used in phosphatidylcholine synthesis through transmethylation, a major homocysteine-producing pathway. Although BHMT has been known for over 40 years, the difficulties encountered in its isolation precluded detailed studies until very recently. Thus, the last 10 years, since the sequence became available, have yielded extensive structural and functional data. Moreover, recent findings offer clues for potential new functions for BHMT. The purpose of this review is to provide an integrated view of the knowledge available on BHMT, and to analyze its putative roles in other processes through interactions uncover to date.

The active-site environment of rhodopsin.

The 11-cis-retinal binding site of rhodopsin is of great interest because it is buried in the membrane but yet must provide an environment for charged amino acids. In addition, the active-site lysine residue must be able to engage in rapid Schiff base formation with 11-cis-retinal at neutral and lower pH values. This requires that this lysine be unprotonated. We have begun to study the environment of the active-site lysine using a reporter group adducted to it. Non-active-site permethylated opsin was reacted with 5-nitrosalicylaldehyde, and the resulting Schiff base was permanently fixed by borohydride reduction. The stoichiometry of incorporation was one. This chromophoric and pH-sensitive reporter group affords information on the active-site environment of rhodopsin by determining the ionization constants of its ionizable groups at different pH values. The pH titration of the modified protein showed a single pKa = 7.8 +/- 0.19 ascribable to the ionization of the phenol. The ionization of the modified lysine residue was not observed at all pH values studied. These studies are interpreted to mean that a negatively charged amino acid is propinquous to the active-site lysine residue and that this latter residue does not have an unusually low pKa.

Study of the rat liver S-adenosylmethionine synthetase active site with 8-azido ATP.

The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.

Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions. Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous. Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies. These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding. Maximal activity was obtained when chaotropic solubilization included the structural cation Mg(2+), the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers. Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties.

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase.

Rat liver methionine adenosyltransferase incorporated 8 mol of N-ethylmaleimide per mol of subunit upon denaturation in the presence of 8 M urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme.