Amino acid sequences of pyridoxal 5'-phosphate binding sites and fluorescence resonance energy transfer in chicken liver fatty acid synthase

The amino acid sequences associated with pyridoxal 5'-phosphate binding sites in chicken liver fatty acid synthase have been determined: a site whose modification causes selective inhibition of the enoyl reductase activity and a site (site I) that is not associated with enzymatic activity. The amino acid sequences of peptides obtained by trypsin hydrolysis of the pyridoxamine 5'-phosphate labeled enzyme were determined. For the site associated with enoyl reductase activity, the sequence similarities between chicken and goose are extensive and include the sequence Ser-X-X-Lys, a characteristic structural feature of pyridoxamine enzymes. In addition, the spatial relationships between the pyridoxal 5'-phosphate binding sites and reductase site(s) have been studied with fluorescence resonance energy-transfer techniques. The distances between site I and the enoyl reductase and beta-ketoacyl reductase sites are greater than 50 and 41-44 A, respectively. The distance between the two reductase sites is greater than 49 A.     

Distribution of reaction intermediates on chicken liver fatty acid synthase

The distributions of covalent intermediates in the reaction cycle catalyzed by chicken liver fatty acid synthase were studied. In isotope-trapping experiments, 30% of [1-14C] acetyl-enzyme and 6.7% of [2-14C]malonyl-enzyme are converted to long-chain fatty acids, indicating the initiation reaction is partially random. The 3-hydroxybutyryl intermediate is located on the tryptic peptide which contains the 4'-phosphopantetheine (greater than 90%), while the C4-C18 saturated intermediates are distributed both on this peptide and on the peptide that contains the active cysteine. The ratio of intermediates on the two peptides is about unity for chain lengths less than C14, but the amount on the active cysteine progressively decreases for chain lengths of C14, C16, and C18 with trypsinized enzyme. The distributions of carbon chain lengths for the saturated or 3-keto intermediates when acetoacetyl-labeled trypsinized enzyme is incubated with limiting malonyl coenzyme A or NADPH, respectively, show large fractions both of unreacted enzyme and of C16 or longer intermediates. A detailed analysis suggests that the initial condensation and reduction steps are slower than the analogous reactions with longer chain length intermediates. The 3-keto intermediate comprises over 70% of each chain length intermediate detected when NADPH is the limiting substrate, indicating the reduction of the 3-keto intermediates is at least 2 times slower than the reduction of the unsaturated intermediates.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. Acylation of specific binding sites

The mechanism of acyl enzyme formation from acyl-CoA derivatives was studied for chicken liver fatty acid synthase in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA at 23 degrees C. Three mechanistically important acyl-binding sites exist: a cysteine, 4'-phosphopantetheine, and a hydroxyl (serine). The cysteine was specifically labeled with iodoacetamide, and chemical modification of this labeled enzyme with chloroacetyl-CoA resulted in additional covalent labeling of 4'-phosphopantetheine. Reaction of the enzyme with acetyl-CoA results in 47% oxyester formation, whereas with malonyl-CoA and butyryl-CoA, 57 and 80% are oxyesters, respectively, as judged by treatment of the denatured enzyme with hydroxylamine. Limited proteolysis with trypsin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the reactive hydroxyl and cysteine are on the same peptide. Butyryl-CoA is a relatively poor primer for steady state fatty acid synthesis, probably because transfer from the hydroxyl-binding site to 4'-phosphopantetheine is inefficient. Quenched flow studies indicate that the rate constants for transfer of acetyl from enzyme-bound acetyl-CoA to native, iodoacetamide-labeled, and iodoacetamide-chloroacetyl-labeled enzyme are 43, 110, and 150 s-1. These results can be interpreted in terms of a random acylation of the hydroxyl, 4'-phosphopantetheine, and cysteine by enzyme-bound acetyl-CoA with rate constants of 150 s-1, less than 110 s-1, and less than 43 s-1, respectively. Alternatively the latter two rate constants could be characteristic of intramolecular transfer between enzyme acylation sites. Structural constraints apparently prevent all three acylation sites from being occupied simultaneously. The rate of deacetylation of the acetylated enzyme by enzyme-bound CoA also is most rapid for the iodoacetamide-chloroacetyl-labeled enzyme.     

Effects of osmolytes on unfolding of chicken liver fatty acid synthase

Urea-induced aggregation of chicken liver fatty acid synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating,oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85 ] was studied. The aggregation was facilitated at increased ionic strength. Methyl--cyclodextrin and some osmolytes, such as glycerol, sucrose, proline, glycine, and heparin, could effectively prevent the aggregation, implying an artificial chaperone role of those substances during fatty acid synthase unfolding. The osmolytes also protected the enzyme from inactivation.     

Stereochemistry of the reactions catalyzed by chicken liver fatty acid synthase

The stereochemistry of the four partial reactions catalyzed by chicken liver fatty acid synthase that lead to the synthesis of palmitic acid has been determined. The reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by NADPH proceeds with the transfer of the pro-4S hydrogen of NADPH to form D-3-hydroxybutyryl-CoA. During the subsequent dehydration of D-3-hydroxybutyryl-CoA the pro-2S hydrogen and the 3-hydroxyl group are removed in a syn elimination to form crotonyl-CoA. Crotonyl-CoA is reduced to butyryl-CoA by NADPH, with the transfer of the pro-4R hydrogen of NADPH to the pro-3R position in butyryl-CoA and the transfer of a solvent hydrogen to the pro-2S position. The occurrence of the syn dehydration, when combined with the results of a previous study [ Sedgwick , B., & Cornforth , J. W. (1977) Eur. J. Biochem. 75, 465-479], implies that the condensation of the enzyme-bound malonyl moiety with the enzyme-bound saturated fatty acid to form a 3-keto intermediate proceeds with inversion at C-2 of the malonyl. The stereochemistry of the hydration was derived from an analysis of the spin-spin coupling constant of 3-hydroxy[2-2H]butyric acid benzylamides obtained from 3-hydroxy[2-2H]butyryl-CoA synthesized by fatty acid synthase. The elucidation of the stereochemistry of the reduction of crotonyl-CoA relied on the previously established stereochemistry of pork liver acyl-CoA dehydrogenase. The source of all 28 prochiral hydrogens of the palmitic acid synthesized by chicken liver fatty acid synthase was inferred from the results of this work.     

Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

The relationships between the aggregation state and the enzymatic activities of chicken liver fatty acid synthase have been explored by monitoring the changes in light scattering, fluorescence, and the overall, beta-ketoacyl synthase, beta-ketoacyl reductase and enoyl reductase activities during dissociation and reassociation of the enzyme. The data obtained indicate that the enzyme dissociates at low temperature in both 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 5 mM Tris(hydroxymethyl)aminomethane, 35 mM glycine (pH 8.3) and 1 mM EDTA, but the extent of dissociation is less in the phosphate buffer. The assay conditions influence the assessment of the degree of dissociation and association: high temperatures, phosphate (high salt), NADPH and acetoacetyl-coenzyme A promote association of the monomeric enzyme, whereas dilution in the Tris-glycine buffer (low salt) and low temperature promote dissociation. Both the rate and extent of association and dissociation are altered by substrates. The monomeric enzyme does not possess beta-ketoacyl synthase and beta-ketoacyl reductase activities. Results obtained with the 1,3-dibromo-2-propanone cross-linked enzyme, which lacks beta-ketoacyl synthase activity, indicate that the NADPH-binding site of beta-ketoacyl reductase is disrupted at low ionic strength. In contrast, changes in ionic strength have little effect on the enoyl reductase activity. The dimer is stabilized by both electrostatic and hydrophobic interactions, with the former being of special importance for maintenance of the beta-ketoacyl reductase active site. site.     

Inhibitive effect of zinc ion on fatty acid synthase from chicken liver

Fatty acid synthase (FAS; acyl-CoA:malonyl-CoA C-acyltransferase [decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing], EC 2.3.1.85) is an important enzyme participating in energy metabolism in vivo which is related to adiposis and cancer [Cancer Lett. 167 (1) (2001) 99; Nat. Med. 8 (4) (2002) 335]. Tests of fast- and slow-binding inhibitions showed that fatty acid synthase of chicken liver is rapidly and irreversibly inactivated by low Zn(2+) concentrations. Electrophoresis and FPLC results showed that FAS cross-links occurred in the presence of high Zn(2+) concentrations (>4 microM) which may be another reason that FAS lost its activity. The modification velocity of FAS by DTNB decreased with increasing Zn(2+) concentration, which confirmed that Zn(2+) interacted with SH groups. Substrate protective experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all three substrates tested had some protective effects on FAS in the presence of Zn(2+), and malonyl-CoA was the most effective of the three substrates. In the presence of malonyl-CoA, the activity loss of FAS decreased sharply and almost no cross-link was observed in SDS-PAGE. This suggests that the phosphopantetheine SH group is the critical group in the cross-link and inhibition of FAS in the presence of Zn(2+).     

Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate

Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.     

Complete amino acid sequence of the thioesterase domain of chicken liver fatty acid synthase

The complete amino acid sequence of thioesterase domain of chicken liver fatty acid synthase has been determined by sequencing peptides produced by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage. The thioesterase domain consists of 300 amino acid residues. All of the tryptic peptides of the thioesterase domain were isolated and sequenced, except the segment covered from position 109 to position 124. Peptides resulting from digestion by Staphylococcus aureus V8 protease and cyanogen bromide cleavage filled the missing part and overlapped the complete sequence of the entire thioesterase domain. The NH2 terminus of the thioesterase domain was determined to be lysine by sequencing the whole domain up to 20 residues while the COOH terminus was identified as serine through carboxyl peptidase Y cleavage. The active site of the thioesterase domain of chicken fatty acid synthase was suggested to be the serine on position 101 according to its homology with other serine-type esterases and proteases which have a common structure of -Gly-X-Ser-Y-Gly- with the variable amino acids X and Y disrupting the homology.     

Small-angle neutron-scattering and electron microscope studies of the chicken liver fatty acid synthase

A structural model for the chicken liver fatty acid synthase is proposed based on electron microscope and small-angle neutron-scattering studies of the enzyme. The model has the overall appearance of two side by side cylinders with dimensions of 160 X 146 X 73 A, with each subunit 160 A in length and 73 A in diameter. The model was constructed by dividing each cylinder into three domains having lengths of 32, 82, and 46 A, with the domain structures in the two subunits being related to each other by a dyad axis. The model is consistent with chemical cross-linking studies which indicated that the subunits are arranged in a head to tail fashion. The cross-linking studies further showed that the beta-ketoacyl synthase active site contains a cysteine and a pantetheine residue from adjacent subunits. It is proposed that the domains which catalyze the addition of C2 units from malonate to the growing fatty acid chain lie in the crevice between the two subunits and that the two independent sets of fatty acid-synthesizing centers lie on the major axis of the model on opposite ends of the molecular dyad.     

Unfolding and inactivation of fatty acid synthase from chicken liver during urea denaturation

The inactivation and conformational changes of the multifunctional fatty acid synthase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85) from chicken liver have been studied in urea solution. The results show that complete inactivation of the fatty acid synthase occurs before obvious conformational changes with regard to the overall, beta-ketoacyl reduction and acetoacetyl-CoA reduction reactions. Significant conformational changes indicated by the changes of the intrinsic fluorescence emission and the circular dichroism spectra occurred at higher urea concentrations. The kinetic rate constants for the two phase inactivation and unfolding reactions were measured and semilogarithmic plots of the activity versus time gave curves which could be resolved into two straight lines, indicating that both the inactivation and unfolding processes consisted of fast and slow phases as a first-order reaction. The results from Lineweaver-Burk plots indicated that urea is a competitive inhibitor for acetyl-CoA and malonyl-CoA, with K(m) increasing with increasing urea concentrations. However, urea is a noncompetitive inhibitor for NADPH, the substrate of the overall reaction and beta-ketoacyl reduction reaction, and acetylacetate, the substrate of the beta-ketoacyl reduction reaction. Activation by low concentrations of urea was observed although this activation was only temporarily induced in an early stage of inactivation. The aggregation phenomenon of the fatty acid synthase in a certain concentration range of urea (3-4 M) was also observed during unfolding. This result shows that this multifunctional enzyme unfolds with competition with misfolding in the folding pathway. Comparison of inactivation and conformational changes of the enzyme as well as aggregation imply that unfolding intermediates may exist during urea denaturation. The possible unfolding pathway of fatty acid synthase is also discussed in this paper.     

Interaction of spin-labeled nicotinamide adenine dinucleotide phosphate with chicken liver fatty acid synthase

The spatial relationships between the four reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding sites on chicken liver fatty acid synthase were explored with electron paramagnetic resonance (EPR) and spin-labeled analogues of NADP+. The analogues were prepared by reaction of NADP+ with 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid, with 1,1'-carbonyldiimidazole as the coupling reagent. Several esterification products were characterized, and the interaction of the N3' ester of NADP+ with the enzyme was examined in detail. Both 1H13, 14N and 2H13, 15N spin-labels were used: the EPR spectrum was simpler, and the sensitivity greater, for the latter. The spin-labeled NADP+ is a competitive inhibitor of NADPH in fatty acid synthesis, and an EPR titration of the enzyme with the modified NADP+ indicates four identical binding sites per enzyme molecule with a dissociation constant of 124 microM in 0.1 M potassium phosphate and 1 mM ethylenediaminetetraacetic acid (pH 7.0) at 25 degrees C. The EPR spectra indicate the bound spin-label is immobilized relative to the unbound probe. No evidence for electron-electron interactions between bound spin-labels was found with the native enzyme, the enzyme dissociated into monomers, or the enzyme with the enoyl reductase sites blocked by labeling the enzyme with pyridoxal 5'-phosphate. Furthermore, the EPR spectrum of bound ligand was the same in all cases. This indicates that the bound spin-labels are at least 15 A apart, that the environment of the spin-label at all sites is similar, and that the environment is not altered by major structural changes in the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and mapping of the beta-hydroxyacyl dehydratase activity of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is cleaved by kallikrein into polypeptides ranging in molecular weight from 10,000 to 100,000. Fractionation of the digest by ammonium sulfate and chromatography on a Matrix Red A affinity column resulted in the isolation of a polypeptide (Mr = 26,000) containing the beta-hydroxyacyl dehydratase activity, but no other partial activities normally associated with the fatty acid synthase. The specific activity of the dehydratase increased 9 to 12 times in this fraction, an increase that is within the expected range based on relative molecular weight. Kinetic parameters of the purified dehydratase toward the model substrate, crotonyl-CoA, showed no change in apparent Km values and a 12-fold increase in Vmax values as compared to dehydratase activity of the intact synthase. However, the purified fragment did not catalyze the hydration of the crotonyl-N-acetylcysteamine derivative, a substrate that is readily hydrated by the intact synthase. Antibodies against the purified 26-kDa fragment cross-react with the intact synthase and the hydratase-containing fragments produced at all stages of digestion with kallikrein or trypsin as shown by Western blot analyses. The results show that the beta-hydroxyl dehydratase activity of the fatty acid synthase is located in the reduction Domain II (Tsukamoto, Y., Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15312-15322) of the synthase subunit.     

Fluorescence studies of chicken liver fatty acid synthase. Segmental flexibility and distance measurements

The 4'-phosphopantetheine of chicken liver fatty acid synthase was specifically labeled with the fluorescent substrate analog coenzyme A 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminohexanoate at low salt concentrations. A serine at the active site of the thioesterase was specifically labeled with the fluorescent compounds 6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]aminopentylmethylphosphono fluoridate and/or pyrenebutyl methylphosphonofluoridate. Dynamic anisotropy measurements indicate the thioesterase has considerable segmental flexibility, whereas the fluorescent labeled 4'-phosphopantetheine does not display detectable local or segmental flexibility. Fluorescence resonance energy transfer measurements indicate that the distance between the fluorescent label at the end of the 4'-phosphopantetheine and NADPH bound to the beta-ketoacyl reductase or enoyl reductase site on the same polypeptide chain is essentially the same, approximately 38 A. The two types of reductases were distinguished by specifically blocking enoyl reductase with pyridoxal 5'-phosphate. No significant energy transfer occurs between sites on different polypeptide chains so that the distances must be greater than 55 A. The distance between the serine on the thioesterase and the 4'-phosphopantetheine on the same polypeptide is 48 A; again no interpolypeptide chain energy transfer was observed. The distance between the serines of the two thioesterases within a fatty acid synthase molecule is greater than 56 A. The monomeric enzyme obtained at 1 degree C does not have beta-ketoacyl synthase and reductase activities. Also fluorescent titrations indicate NADPH is not bound to beta-ketoacyl reductase in monomeric enzyme. The addition of potassium phosphate to the monomers at 1 degree C rapidly dimerizes the enzyme and restores the beta-ketoacyl reductase activity. The beta-ketoacyl synthase activity is slowly restored when the dimer is raised to room temperature. The results obtained suggest that relatively large conformational changes may be part of the catalytic cycle.     

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Amino acid sequence of chicken liver cathepsin L

The complete amino acid sequences of the heavy and light chains of chicken liver cathepsin L have been determined by automated gas-phase Edman degradation. The heavy and light chains contained 176 and 42 amino acid residues respectively. A glucosamine-based oligosaccharide group was attached to Asn-109 of the heavy chain. Chicken liver cathepsin L had high sequence homology with rat cathepsin H, but exhibited less similarity with rat cathepsin B. Comparisons of cathepsin L with plant cysteine proteinases, such as papain, actinidin and aleurain, reveal high degree of homology.     

Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1984) J. Biol. Chem. 259, 2466-2473). Both isozymes share a number of properties including multiple phosphorylation of the enzyme subunit. In the present study, we determined the amino acid sequences surrounding phosphorylation sites in the rabbit liver isozyme recognized by cyclic AMP-dependent protein kinase. Two dominant phosphopeptides (P-1 and P-2) were generated from tryptic digestion. Amino acid sequences of the purified peptides were determined by automated Edman degradation using a gas-phase sequenator. The locations of phosphorylated residues were identified by measuring 32Pi release during Edman degradation cycles. The NH2-terminal sequence of peptide P-1 is S-L-S(P)-V-T-S-L-G-G-L-P-Q-W-E-V-E-E-L-P-V-D-D-L-L-L-P-E-V. This sequence exhibits a strong homology to the site 2 region in the NH2 terminus of the muscle isozyme. The NH2-terminal sequence of peptide P-2 is M-Y-P-R-P-S(P)-S(P)-V-P-P-S-P-L-G-S-Q-A. This sequence shows strong homology to the site 3 region in the COOH terminus of the muscle isozyme. However, some interesting sequence differences were revealed in this region. For example, substitution of serine for alanine at position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. Phosphorylation of the proline/serine-rich site 3 region correlated with inactivation of the liver isozyme and suggests an important role for this segment of the molecule in the regulation of glycogen synthase. No phosphorylation sites corresponding to sites 1a and 1b of the muscle isozyme were detected. In addition, the results provide definitive chemical proof that glycogen synthase from rabbit liver and muscle are isozymes encoded by distinct messages.