Acetylation of Lys-373 in porcine pancreatic lipase after reaction of the enzyme or its C-terminal fragment [corrected] with p-nitrophenyl acetate

The reactions of lipase (449 amino acid residues) and lipase fragment (336-449) with p-nitrophenyl acetate have been studied from 2 different angles. In previous papers it has been shown that lipase and lipase fragment enzymatically hydrolyze p-nitrophenyl acetate. The amino acid residue of the catalytic site that is temporarily acetylated has not yet been characterized in lipase or lipase fragment. Besides this very fast enzymatic hydrolysis, acetylation reactions may take place on nucleophilic amino acid side-chain groups. In the present report, acetylated amino acid residues whose acetyl linkages were not cleaved after pH 7.5-8.5 incubations have been investigated. Several residues were acetylated in very low proportion, whereas lysine 373 was stoichiometrically acetylated in lipase and in lipase fragment. This specific acetylation may have been favored by the presence of a hydrophobic reversible binding site for p-nitrophenyl acetate near Lys-373. This acetylation did not greatly change the specific activity of lipase towards an emulsion of tributyrylglycerol in the presence of colipase, but under certain conditions it had an effect on the enzymatic hydrolysis of p-nitrophenyl acetate by the lipase fragment.     

Acetylated pectic acid as a substrate of Vi phages deacetylase

The conditions of obtaining [¹⁴C] acetylated pectic acid with a high specific activity are presented. On the basis of radioisotope measurements of the liberated, labelled acetic acid, the activity of deacetylase associated with the ViI, ViII and ViIII phage particle was determined. It has been shown that acetylated pectic acid is a substrate for the enzyme of those phages. The conditions of identifying the deacetylase are presented; it has been shown that the activity of Vi phage II enzyme is repressed by the phosphate buffer. In a system analogous to the one used for the Vipolysaccharide, the receptor activity of the acetylated pectic acid can not be shown.     

Effects of acetylation in vapor phase and mercerization on the properties of sugarcane fibers

Chemical modification of sugarcane bagasse fiber was achieved by mercerization reaction and esterification reaction with anhydride acetic vapor. This is a new acetylation procedure. The results show that the fiber length and diameter are reduced after the reactions. Fourier transform infrared spectroscopy (FT-IR) studies produced clear evidence of the partial acetylation reaction. Optical microscopy revealed fibrillation in the acetylated fiber attributed to hemicellulose dissolution. The thermal stability measured by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) increased after acetylation and decreased after mercerization. The higher thermal stability of the acetylated fiber as compared with modified fibers in liquid medium was attributed to the small quantity of water and acetic acid present for the reaction in vapor phase. The lesser tensile strength of the acetylated fiber was due to fibrillation. The porous structure obtained favors migration of the polymer chains into the fiber acetylated, and thus it should enhance the polymer–fiber adhesion in polymer composites.     

On the deacetylase activity of Vi bacteriophage III particles.

1. Using the complete phage particles as an enzyme, O-acetyl (1 leads to 4)-alpha-D-galacturonan (acetylated pectic acid) as a substrate, and gas-liquid-chromatography for the determination of the acid liberated, the virus-catalysed deacetylation of the polymer was studied. The activity was found to be stable up to about 50 degrees C, and from pH 4.5 to 9, with an optimum at pH 7.8; it was not affected by EDTA, or by 1,10-phenanthroline. The initial reaction velocity (at 37 degrees C) exhibited a simple hyperbolical dependence on the substrate concentration, with Km = 10.5 mM for O-acetyl (independent of virus concentration), and Vmax = 15 nmoles/min and 10(10) plaque forming units. The reaction was, however, rapidly inhibited by a partially deacetylated product (but neither by acetate, nor by pectic acid itself). 2. Using the natural substrate, acetylated (1 leads to 4)-2 amino-2-deoxy-alpha-D-galacturonan (Vi polysaccharide, Vi antigen), and a variety of structural analogues, the following conclusions about the substrate specificity of the Vi phage III deacetylase (acetyl-alpha-1,4-galacturonan acylhydrolase) were reached: (a) acetylated galacturonan is as good a substrate as acetylated aminogalacturonan; (b) of the two substrate diastereomers, acetylated alpha-L-guluronan (also 1 ax leads to 4 ax-linked units, but with axial acetyl residues at C-3), and beta-D-mannuronan (1 eq leads to 4 eq-linkages, and axial acetyl groups at C-2), only the former was acted upon, possibly indicating a specificity for the conformation of the polymer rather than for the configuration of the single residues; (c) all acyl analogues tested, O-monofluoroacetyl, O-propionyl, and O-butyryl galacturonan, were inert, showing a high degree of specificity for O-acetyl; (d) the oligomers, acetylated tri- and digalacturonic acid, as well as methyl-alpha-D-galacturonide, were still deacetylated, although more slowly, demonstrating tolerance of the enzyme of substrate size.     

A new core tetrasaccharide component from the lipopolysaccharide of Rhizobium trifolii ANU 843

A second core oligosaccharide fragment has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843. The oligosaccharide is a tetrasaccharide composed of galactose, galacturonic acid, mannose, and 3-deoxy-D-manno-2-octulosonic acid. The mannose residue is alpha-linked to the 4-position of 3-deoxy-D-manno-2-octulosonic acid and the galacturonic acid residue is alpha-linked to the 6-position of mannose. The galactose residue, which is acetylated at the 4-position, is attached to the 4-position of mannose by an alpha-linkage. All of the aldoses are in the pyranose form. The composition of the tetrasaccharide was determined by gas-liquid chromatography of the alditol acetate derivatives of the component monosaccharides. The configuration of anomeric linkages was determined by 1H NMR spectroscopy. Fast atom bombardment-mass spectrometry (FAB-MS) was performed on acetylated, per(trideutero)acetylated and underivatized tetrasaccharide giving sequence information in addition to information on the residue which was acetylated. Similar studies were performed on the oligosaccharide after reduction with sodium cyanoborohydride and peracetylation with labeled and unlabeled acetic anhydride as before. Further linkage and sequence analysis was obtained from methylation analysis, and from electron impact mass spectrometry of the per(trideutero)acetylated oligosaccharide and from collision-induced dissociation fast atom bombardment tandem mass spectrometry using linked scans at constant B/E on the cyanoborohydride-reduced, per (trideutero)acetylated oligosaccharide. The exact location of the acetyl group was deduced from 1H NMR double resonance experiments in conjunction with mass spectrometric data.     

4,8-anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination

A new, sialic-acid-derived compound was isolated from the acid hydrolysate of edible bird's nest by ion-exchange chromatography. Combined use of mass spectroscopy and 1H- and 13C-NMR spectroscopy established that it is the 4,8-anhydro derivative of N-acetylneuraminic acid and that in solutions it exists in two tautomeric forms. The formation of the new compound supports and earlier findings that in the glycoprotein of edible bird's nest at least a portion of N-acetylneuraminic acid is acetylated at HO-4.     

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.     

Comparison of the acetylation of proteins and nucleic acids

The possibility of acetylation of nucleic acids was examined. Although protein is actively acetylated with [1-(14)C]acetic acid in rat liver systems in vivo and in vitro and in a frog liver system in vivo, nucleic acids are not acetylated under these conditions; nucleic acids purified from these sources are without radioactivity. Requirements for acetylation in vitro of protein in rat liver are different from those in frog liver; GSH has no effect in the rat liver system and is inhibitory in the frog liver system. Among various acetylated proteins, proteins insoluble in 0.1m-sulphuric acid have the highest radioactivity.     

Molecular orientation of plastocyanin on spinach thylakoid membranes as determined by acetylation of lysine residues

To reveal the molecular orientation of plastocyanin (PC) on spinach thylakoid membranes, the position of Lys residues modified by acetic anhydride was compared between thylakoid-bound PC and the isolated one. Digestion of the isolated PC by a trypsin yielded a peptide map with seven spots prior to the acetylation of the protein; none of the spots appeared after the isolated PC was acetylated. On the other hand, there were two spots on the peptide map of the PC acetylated when it was bound to the thylakoids. Those spots were revealed by their amino acid compositions to correspond to the peptide fragments Phe 82-Lys 95 and Val 96-Asn 99. Thus, the Lys residues 81 and 95 of the thylakoid-bound PC were not acetylated. These results suggest that the PC molecule binds to the thylakoids with a specific region including the Lys's 81 and 95 in contact with the membranes. The Lys's 81 and 95 are located near Tyr 83, which has been thought to be the delivery site of electrons from the Cu2+ center.     

Histone H4 from cuttlefish testis is sequentially acetylated. Comparison with acetylation of calf thymus histone H4

The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.     

Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts

We have examined the distribution of acetylated alpha-tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meninges. Meningeal fibroblasts showed heterogeneous staining patterns with a monoclonal antibody against acetylated alpha-tubulin ranging from staining of primary cilia or microtubule-organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti-alpha-tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double-labeling experiments using an antibody against acetylated alpha-tubulin (6-11B-1) and antibodies against either tyrosinated or detyrosinated alpha-tubulin, it was found that acetylated alpha-tubulin and tyrosinated alpha-tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated alpha-tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated alpha-tubulin and was cold stable, and the other contained tyrosinated alpha-tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of alpha-tubulin are involved in the specification of stable microtubules.     

Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate

A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site.     

Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids

"Sialic acid engineering" refers to the strategy where cell surface carbohydrates are modified by the biosynthetic incorporation of metabolic intermediates, such as non-natural N-acetylmannosamine (ManNAc) analogues, into cellular glycoconjugates. While this technology has promising research, biomedical, and biotechnological applications due to its ability to endow the cell surface with novel physical and chemical properties, its adoption on a large scale is hindered by the inefficient metabolic utilization of ManNAc analogues. We address this limitation by proposing the use of acetylated ManNAc analogues for sialic acid engineering applications. In this paper, the metabolic flux of these "second-generation" compounds into a cell, and, subsequently, into the target sialic acid biosynthetic pathway is characterized in detail. We show that acetylated ManNAc analogues are metabolized up to 900-fold more efficiently than their natural counterparts. The acetylated compounds, however, decrease cell viability under certain culture conditions. To determine if these toxic side effects can be avoided, we developed an assay to measure the cellular uptake of acetylated ManNAc from the culture medium and its subsequent flux into sialic acid biosynthetic pathway. This assay shows that the majority ( > 80%) of acetylated ManNAc is stored in a cellular "reservoir" capable of safely sequestering this analogue. These results provide conditions that, from a practical perspective, enable the acetylated analogues to be used safely and efficaciously and therefore offer a general strategy to facilitate metabolic substrate-based carbohydrate engineering efforts. In addition, these results provide fundamental new insights into the metabolic processing of non-natural monosaccharides.     

Influence of dietary acetylated peptides on fermentation and peptidase activities in the sheep rumen

The predominant mechanism of peptide breakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminus of peptides inhibits their degradation by rumen micro-organisms in short-term incubations with rumen fluid in vitro . An experiment was undertaken to determine if adaptation of the rumen microbial population would take place when acetylated peptides were fed for a prolonged period, which would enable the microbial population to break down the protected peptides and thus decrease their nutritive value. Three adult sheep, fitted with permanent rumen cannulae, received a maintenance hay/concentrate diet to which was added, at each meal, 20 g of casein enzymic hydrolysate ('peptides') or 20 g of peptides previously treated with acetic anhydride. The diets were fed for 28 d in a 3 × 3 latin square and samples were taken during the last 7 d. Fermentation products and NH₃ concentrations indicated that acetylated peptides remained less degradable than untreated peptides. There was a trend towards increased proteolytic activity with acetylated peptides, and dipeptidase activity increased by 18% and 28%, respectively, compared with untreated peptides and control treatments. Activity against N-acetyl-Ala₂ also increased when acetylated peptides were fed, but it remained only 13% of the rate of Ala₂ hydrolysis. No increase was found in the rate of ammonia production from acetylated peptides in animals receiving acetylated peptides–this rate was 26% of that found with untreated peptides–and acetylated peptides continued to persist for longer in the rumen than untreated peptides after feeding. Thus it was concluded that the rumen microbial population did not adapt to utilize acetylated peptides.     

Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2. Why acetylated COX-1 does not synthesize 15-(R)-hete

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.     

Improved solid-phase synthesis of alpha,alpha-dialkylated amino acid-rich peptides with antimicrobial activity.

A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid-phase synthetic techniques. Each nonapeptide was rich in alpha,alpha-dialkylated amino acids [one 4-aminopiperidine-4-carboxylic acid (Api) and six alpha-aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9-fluorenylmethyloxycarbonyl (Fmoc)-Aib-Aib-OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3(10)-helical, amphipathic design of these peptides was born out most prominently in the N-terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations < or =100 microM) and the acetylated peptides (concentrations < or = 200 microM) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Acetylation of Nucleosides by Acetylsalicylic Acid (Aspirin)1

Abstract

Acetylsalicylic acid (aspirin) reacted with adenosine, cytidine, guanosine and their 2′-deoxynucleosides to give acetylated nucleosides. Cytidine and 2′-deoxycytidine gave N⁴-acetylated nucleosides in nitromethane while in pyridine fully acetylated products were obtained. Adenosine and 2′-deoxyadenosine also gave fully acetylated products. However, guanosine and 2′-deoxyguanosine gave 2′,3′,5′-tri-O-acetylribosyl and 3′,5′-di-O-acetyl-2′-deoxyribosyl nucleosides, respectively. The corresponding aglycons also gave acetylated heterocycles under various conditions.     

Insect growth inhibition by tocotrienols and hydroquinones from Roldana barba-johannis

The methanol extract from the aerial parts of Roldana barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and their corresponding acetylated and methylated derivatives showed insecticidal and insect growth regulatory activities against the Fall Armyworm [Spodoptera frugiperda J.E. Smith, (Lepidoptera: Noctuidae)], an important insect pest of corn. The most active compounds were sargachromenol and its acetylated derivative; sargahydroquinoic acid and its acetylated derivative; and a mixture of sargachromenol, sargahydroquinoic acid, and sargaquinoic acid (6:3:1) and the acetylated form of this mixture. All these compounds and mixtures had significant inhibitory effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. These substances may be useful as natural insecticidal agents.     

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.     

Radiolabeling of mammalian cells in tissue culture by use of acetic anhydride. Potential value for studying the dynamics of protein turnover in living cells.

When Chinese hamster ovary cells, growing in monolayer culture, were treated with trace quantities of radioactive acetic anhydride, label was very rapidly introduced into both the plasma membrane and intracellular components, all of which reached similar specific radioactivities. The cells themselves showed no measurable loss in viability. The incorporated label was recovered predominantly in the form of acetyl groups on proteins. Since tyrosyl hydroxyls were not extensively acetylated, it seems likely that substitution occurred mainly on amino groups. There was no indication that the acetyl groups once introduced were labile or that the modified proteins were discriminated against by the cells, since, in dividing cells, the acetylated proteins were as long-lived as those labeled with radioactive l-leucine. Nor was there any evidence for reutilization of labeled acetate following protein turnover in cells maintained in a nonproliferating state on a medium lacking the essential amino acid l-isoleucine. We suggest that acetylation could provide an ideal means for introducing a very short “pulse” of radioactivity into proteins of living cells. In addition, the method should be extremely useful for studying processes such as enzyme induction and protein turnover where the problem of amino acid reutilization has long been recognized as a possible basis for artifacts.