Biomedical subjects

M A Pajares

Publications and source records attributed to M A Pajares.

32 records · Page 2Linked to original sources

PubMed1992

S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.

Administration of carbon tetrachloride to rats resulted in induction of hepatic fibrosis and a 60% reduction of hepatic S-adenosylmethionine synthetase activity without producing any significant modification of hepatic levels of S-adenosylmethionine synthetase messenger RNA. The reduction of S-adenosylmethionine synthetase activity was corrected by treatment with S-adenosylmethionine (3 mg/kg/day, intramuscularly). Administration of carbon tetrachloride also produced a 45% depletion of liver glutathione (reduced form) that was corrected by S-adenosylmethionine treatment. After the rats received carbon tetrachloride, a 2.3-fold increase in liver collagen was observed; prolyl hydroxylase activity was 2.5 times greater than that seen in controls. These increases were attenuated in animals treated with carbon tetrachloride and S-adenosylmethionine. The attenuation by S-adenosylmethionine treatment of the fibrogenic effect of carbon tetrachloride was associated with a decrease in the number of rats in which cirrhosis developed.

Animals↗

PubMed1991

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.

Animals↗

PubMed1991

The role of cysteine-150 in the structure and activity of rat liver S-adenosyl-L-methionine synthetase.

The present paper reports the tryptic digestion of N-ethylmaleimide-treated S-adenosyl-L-methionine synthetase (high- and low-Mr forms) and the isolation of the modified peptides by h.p.l.c. There is only one site modified after 5 min incubation, and the modification at this site correlates with the main activity decrease. The amino acid composition of this peptide was determined, and its localization in the sequence shows the modified residue as cysteine-150, which is located close to the putative ATP-binding site. Modification of the enzyme for 20 min led to the appearance of a second labelled peptide, which seems to be responsible for about a further 10% of the activity loss. The modification by N-ethylmaleimide of the enzyme was partially prevented in the presence of adenosine 5'-[beta gamma-imido]triphosphate and methionine, further supporting the hypothesis that the modified residues lie within the active site. Urea treatment of the enzyme, followed by modification with N-ethylmaleimide, produces the modification of 7 of the 10 cysteine residues present in the sequence. The results obtained were the same for either of the isoforms.

Amino Acids↗

PubMed1991

Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species.

Binding Sites↗

PubMed1991

Inhibition of glutathione synthesis in the liver leads to S-adenosyl-L-methionine synthetase reduction.

The hepatic levels of glutathione in rats treated with buthionine sulfoximine (4 mmol/kg), an inhibitor of glutathione synthesis, were 72.5% +/- 4.9% of those determined in control animals. This decrease in glutathione concentration was prevented by the administration of glutathione monoethyl ester (7.5 mmol/kg). S-Adenosyl-L-methionine-synthetase activity in the liver of rats treated with buthionine sulfoximine was 39.4% +/- 6.5% of that determined in control animals. Again, glutathione monoethyl ester prevented the effect of buthionine sulfoximine on S-adenosyl-L-methionine-synthetase activity. There was a close correlation (r = 0.936) between the hepatic levels of glutathione and S-adenosyl-L-methionine-synthetase activity. The hepatic concentration of S-adenosyl-L-methionine in buthionine sulfoximine-treated animals was 59.7% +/- 3.7% of that measured in control rats. Contrasting with the protective effects mentioned above, glutathione monoester had no preventive action on buthionine sulfoximine-induced S-adenosyl-L-methionine depletion. Electron microscopic examination of liver samples of rats after buthionine sulfoximine administration showed evidence of liver degeneration, which was attenuated by glutathione monoethyl ester treatment. Glutathione (7.5 mmol/kg) treatment was less effective than glutathione monoethyl ester in attenuating buthionine sulfoximine effects on hepatic S-adenosyl-L-methionine metabolism and morphology. The reduction of S-adenosyl-L-methionine-synthetase activity observed after treatment with buthionine sulfoximine and its prevention by glutathione monoethyl ester, as well as the correlation between the activity of this enzyme and glutathione levels, indicate that glutathione plays an important role in maintaining S-adenosyl-L-methionine-synthetase activity in the liver.

Acetaminophen↗

PubMed1990

Inactivation and dissociation of S-adenosylmethionine synthetase by modification of sulfhydryl groups and its possible occurrence in cirrhosis.

Catalytically active human and rat liver S-adenosylmethionine synthetase exists mainly in tetramer and dimer form. In liver biopsy samples from cirrhotic patients a marked reduction in total S-adenosylmethionine synthetase activity and a specific loss of the tetrameric form of the enzyme exist. We have investigated the possible role of sulfhydryl groups in maintaining the structure and activity of S-adenosylmethionine synthetase. Both forms of S-adenosylmethionine synthetase are rapidly inactivated by N-ethylmaleimide, and the loss of enzyme activity correlates with the incorporation of approximately 2 moles N-ethylmaleimide per mole of subunit. In addition, reaction with N-ethylmaleimide resulted in displacement of the tetramer-dimer equilibrium of the enzyme toward the dimer, but no monomer was detected under these conditions. A catalytically active monomeric S-adenosylmethionine synthetase was detected in the cytosolic extract from a liver biopsy sample from a cirrhotic patient, supporting our model for the structure of S-adenosylmethionine synthetase. Because treatment of S-adenosylmethionine synthetase with N-ethylmaleimide resembles the situation of this enzyme in cirrhotic patients, it is proposed that impaired protection of the enzyme from oxidizing agents caused by a decreased synthesis of glutathione can explain the diminished synthesis of S-adenosylmethionine in liver cirrhosis.

Animals↗

PubMed1990

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.

Humans↗

PubMed1989

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.

Aldehydes↗

PubMed1989

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.

Alkaline Phosphatase↗

PubMed1989

Structural basis of protein kinase C activation by tumor promoters.

Protein kinase C (PKC) is an important enzyme that helps govern cell metabolism and growth. The enzyme is physiologically activated when an (S)-diglyceride binds to its own regulatory domain. The saturable binding site of the regulatory domain can also be bound by any of a group of structurally diverse tumor promoters, including debromoaplysiatoxins (DATs), phorbol esters, ingenols, teleocidins, and bryostatins. The question of how the same binding site can be the target of these structurally diverse molecules is of considerable importance and is addressed in this article. The relatively rigid structure of DAT and the fact that it possesses a diglyceride moiety renders it an ideal starting template. Structure-activity studies with PKC reveal that the C29 but not the C30 stereocenter of DAT is critical for activity. Furthermore, 3-deoxy-DAT and DAT are equipotent as PKC activators, hence the C3 hydroxyl group of DAT is not critical for activity. Straightforward structural considerations show that the C30 hydroxyl group of DAT matches the C3 hydroxyl group of diglyceride, the C29 stereocenter of DAT matches the C2 stereocenter of (S)-diglyceride, and the C1 ester moiety of DAT matches the C2 ester moiety of diglyceride. Based on these studies and on published structure-activity observations on other tumor promoters, a structural hypothesis is developed to account for the chemical mechanism of tumor promoter action. Experimentally testable predictions are made concerning the interactions with PKC of several classes of tumor PKC activators.

Animals↗

PubMed1987

Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.

Animals↗

PubMed1986

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.

Animals↗

PubMed1985

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.

Animals↗

PubMed1984

Purification and photoaffinity labelling of lipid methyltransferase from rat liver.

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.

Affinity Labels↗