Rate Of Deacetylation Research Articles

PHYSICOCHEMICAL CHARACTERISTICS AND ANTIOXIDANT EFFICACY OF CHITOSAN FROM THE INTERNAL SHELL OF SPINELESS CUTTLEFISH Sepiella inermis

Cuttlefish chitosan was extracted from the cuttlebone of Sepiella inermis by demineralization and deproteinization and produced by deacetylation, and its physical and chemical parameters were also compared with that of commercial chitosan. Ash, moisture, and mineral and metal content of the chitosan was estimated by adopting standard methodologies. The rate of deacetylation was calculated as 79.64% by potentiometric titration. Through viscometry and gel permeation chromatography, the molecular weight of chitosan was found to be significantly lower than that of the commercial chitosan. Optical activity was found to be levorotatory. The structure of the chitosan was elucidated with spectral techniques such as Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. Cuttlefish chitosan showed a melting endothermic peak at 117.32°C. The x-ray diffraction (XRD) pattern of chitosan and standard chitosan exhibited the same crystalline peaks. Through scanning electron microscopy (SEM) the fine structure of chitosan was studied. The binding capacity (water and fat) of cuttlefish chitosan was found to be significantly higher than that of the commercial chitosan. The antioxidant efficacy of chitosan was determined through the conjugated diene method, scavenging ability on DPPH radicals, reducing power, and chelating ability on ferrous ions. This study has brought out the importance of shell as a potential source for obtaining another natural antioxidant.

A new nonpeptide substrate of human sirtuin in a capillary electrophoresis‐based assay. Investigation of the binding mode by docking experiments

Sirtuins are nicotinamide dinucleotide-dependent class III histone deacetylases catalyzing various physiological processes involved in cell proliferation, differentiation, apoptosis, and ageing. This makes them attractive targets in drug research. In order to simplify sirtuin substrates for assay development, two N(ɛ)-acetyllysine derivatives, N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine amide, and N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine methyl ester were synthesized and evaluated as substrates for human SIRT1 in a capillary electrophoresis-based enzyme assay. Substrate, deacetylated product, and the coproduct nicotinamide were separated in a 200 mM phosphate/Tris buffer at pH 2.85. Field-amplified sample injection was employed to achieve sufficient assay sensitivity. While the ester derivative was not recognized by the enzyme, the amide substrate was effectively converted to the deacetylated product. The assay was subsequently validated with respect to range, linearity, limit of detection, and limit of quantification. Michaelis-Menten kinetic parameters, K(m) = 83 μM and V(max) = 6.8 μM/min were determined. The applicability of the assay for inhibitor screening was demonstrated using the known inhibitors sirtinol and the suramin derivate NF258. Resveratrol did not increase the deacetylation rate at concentrations of up to 200 μM. Docking experiments revealed the necessity of an amide function at the C-terminus of nonpeptide substrates while more structural freedom is tolerated at the N-terminus of N(ɛ) -acetyllysine.

Kinetics of de-N-acetylation of the chitin disaccharide in aqueous sodium hydroxide solution

Chitosan is prepared from chitin, a process which is carried out at highly alkaline conditions, and that can be performed either on chitin in solution (homogeneous deacetylation) or heterogeneously with the chitin as a solid throughout the reaction. We report here a study of the de-N-acetylation reaction of the chitin dimer (GlcNAc–GlcNAc) in solution. The reaction was followed by 1H NMR spectroscopy in deuterated aqueous sodium hydroxide solution as a function of time, sodium-hydroxide concentration and temperature. The 1H NMR spectrum of GlcNAc–GlcNAc in 2.77M deuterated aqueous sodium hydroxide solution was assigned. The interpretation of the 1H NMR spectra allowed us to determine the rates of de-N-acetylation of the reducing and non-reducing ends, showing that the reaction rate at the reducing end is twice the rate at the non-reducing end. The total deacetylation reaction rate was determined as a function of the hydroxide ion concentration, showing for the first time that this de-N-acetylation reaction is second order with respect to hydroxide ion concentration. No significant difference in the deacetylation rates in deuterated water compared to water was observed. The activation energy for the reaction (26–54°C) was determined to 114.4 and 98.6kJ/mol at 2.77 and 5.5M in deuterated aqueous sodium hydroxide solution, respectively.

Modeling the Basal Dynamics of P53 System

BackgroundThe tumor suppressor p53 has become one of most investigated genes. Once activated by stress, p53 leads to cellular responses such as cell cycle arrest and apoptosis.Methodology/Principal FindingsMost previous models have ignored the basal dynamics of p53 under nonstressed conditions. To explore the basal dynamics of p53, we constructed a stochastic delay model by incorporating two negative feedback loops. We found that protein distribution of p53 under nonstressed condition is highly skewed with a fraction of cells showing high p53 levels comparable to those observed under stressed conditions. Under nonstressed conditions, asynchronous and spontaneous p53 pulses are triggered by basal DNA double strand breaks produced during normal cell cycle progression. The first peaking times show a predominant G1 distribution while the second ones are more widely distributed. The spontaneous pulses are triggered by an excitable mechanism. Once initiated, the amplitude and duration of pulses remain unchanged. Furthermore, the spontaneous pulses are filtered by ataxia telangiectasia mutated protein mediated posttranslational modifications and do not result in substantial p21 transcription. If challenged by externally severe DNA damage, cells generate synchronous p53 pulses and induce significantly high levels of p21. The high expression of p21 can also be partially induced by lowering the deacetylation rate.ConclusionsOur results demonstrated that the dynamics of p53 under nonstressed conditions is initiated by an excitable mechanism and cells become fully responsive only when cells are confronted with severe damage. These findings advance our understanding of the mechanism of p53 pulses and unlock many opportunities to p53-based therapy.

The effect of temperature and pH on the deacetylation of diamorphine in aqueous solution and in human plasma.

The effect of temperature on the kinetics of the deacetylation of diamorphine and 6-monoacetylmorphine was studied in human plasma. Diamorphine was rapidly and quantitatively degraded to 6-monoacetylmorphine with initial half-lives of 354, 18 and 3 min at temperatures of 4, 25 and 37 degrees C, respectively. Further deacetylation to morphine was not detected. In aqueous solution, diamorphine was quantitatively degraded to give 6-monoacetylmorphine as the major product and morphine as a minor product, the rate of deacetylation being dependent on temperature and pH. At pH 4.0 and 5.6 diamorphine had a half-life of greater than 14 days at all temperatures but at alkaline pH diamorphine was rapidly deacetylated. The rate of deacetylation of 6-monoacetylmorphine was consistently slower than that of diamorphine under identical conditions of pH and temperature. A method is described for the rapid stabilization and subsequent assay of diamorphine in plasma which will prevent errors in estimation of the drug due to unwanted hydrolysis.

CK2 Is the Regulator of SIRT1 Substrate-Binding Affinity, Deacetylase Activity and Cellular Response to DNA-Damage

SIRT1, an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase, protects cells from stress-induced apoptosis, and its orthologues delay aging in lower eukaryotes. SIRT1 increases survival in response to stress such as DNA damage by deacetylating a number of substrates including pro-apoptotic protein p53. The molecular mechanism by which DNA-damage activates SIRT1 is not known. By screening a kinase inhibitor library, we identified CK2 as a SIRT1 kinase. CK2 is a pleiotropic kinase with more than 300 substrates and well-known anti-apoptotic and pro-growth activities. We find that CK2 is recruited to SIRT1 after ionizing radiation (IR) and phosphorylates conserved residues Ser 154, 649, 651 and 683 in the N- and C-terminal domains of mouse SIRT1. Phosphorylation of SIRT1 increases its deacetylation rate but not if the four Ser residues are mutated. In addition, phosphorylation of SIRT1 increases its substrate-binding affinity. CK2-mediated phosphorylation increases the ability of SIRT1 to deacetylate p53 and protect cells from apoptosis after DNA damage. Based on these findings, we propose that CK2 protects against IR-induced apoptosis partly by phosphorylating and activating SIRT1. Thus, this work suggests that SIRT1 is a component of the expansive anti-apoptotic network controlled by CK2. Since expression of both CK2 and SIRT1 is upregulated with tumorigenesis and downregulated with senescence, the CK2-SIRT1 link sheds new light on how CK2 may regulate cancer development and aging.

Biodegradable Protections for Nucleoside 5′-Monophosphates: Comparative Study on the Removal of O-Acetyl and O-Acetyloxymethyl Protected 3-Hydroxy-2,2-bis(ethoxycarbonyl)propyl Groups

The applicability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups as biodegradable phosphate protecting groups for nucleoside 5'-monophosphates has been studied in a HEPES buffer at pH 7.5. Enzymatic deacetylation with porcine carboxyesterase triggers the removal of the resulting 3-hydroxy-2,2-bis(ethoxycarbonyl)propyl and 3-hydroxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups by retro-aldol condensation and consecutive half acetal hydrolysis and retro-aldol condensation, respectively. The kinetics of these multistep deprotection reactions have been followed by HPLC, using appropriately protected thymidine 5'-monophosphates as model compounds. The enzymatic deacetylation of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl 5'-triester (2) is 25-fold faster than the deacetylation of its 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart 1, and the difference in the deacetylation rates of the resulting diesters, 12b and 12a, is even greater. With 2, conversion to thymidine 5'-monophosphate (5'-TMP) is quantitative, while conversion of 1 to 5'-TMP is accompanied by formation of thymidine. Consistent with the preceding observations, quantitative release of 5'-TMP from 2 has been shown to take place in a whole cell extract of human prostate cancer cells.

Mouse aminoacylase 3: A metalloenzyme activated by cobalt and nickel

Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn 2+-metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained ∼ 0.35 zinc atoms per monomer. Incubation with Co 2+ and Ni 2+ activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn 2+, Mn 2+ or Fe 2+ restored initial activity. Co 2+ and to a lesser extent Ni 2+ increased activity several times in comparison with intact wt-AA3. Co 2+ drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co 2+ and Ni 2+.

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.

Influences of mechanical properties and permeability on chitosan nano/microfiber mesh tubes as a scaffold for nerve regeneration

We have developed chitosan nonwoven micro/nanofiber mesh tubes and examined the effects of their mechanical strength and permeability on nerve regeneration. Chitosan nano/microfibrous tubes with a deacetylation rate (DAc) of 78% or 93% were prepared by electrospinning. A chitosan film tube with a DAc of 93% was also fabricated and combined with the nano/microfibrous tubes to form bilayered tubes with a nano/microfiber mesh inner structure and a film outer layer. Nano/microfiber mesh tubes with a DAc of 78% or 93%, bilayered tubes with a nano/microfiber mesh inner structure with a DAc of 78% or 93% and a film outer layer with a DAc of 93%, and film tubes with a DAc of 93% were each tested as bridge grafts into injured rat sciatic nerve. Isografting was performed as a control. Although the functional recovery of motor activity was delayed in each group, sensory function reemerged first in the isograft group followed by the group receiving nano/microfiber mesh tubes with a DAc of 93%. Histological analysis was consistent with these results. The chitosan nano/microfiber mesh tubes with a DAc of 93% have sufficient mechanical properties to preserve tube space, provide a better scaffold for cell migration and attachment, and facilitate humoral permeation to enhance nerve regeneration.

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40±0.070min−1 and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using 1H NMR, had a Km of 1.49±0.16mM and Vmax of 2.61±0.08μmolL−1min−1. Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of ∼10μM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were ∼5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.

Nucleocytosolic Acetyl-Coenzyme A Synthetase Is Required for Histone Acetylation and Global Transcription

Metabolic enzymes rarely regulate informational processes like gene expression. Yeast acetyl-CoA synthetases (Acs1p and 2p) are exceptional, as they are important not only for carbon metabolism but also are shown here to supply the acetyl-CoA for histone acetylation by histone acetyltransferases (HATs). acs2-Ts mutants exhibit global histone deacetylation, transcriptional defects, and synthetic growth defects with HAT mutants at high temperatures. In glycerol with ethanol, Acs1p is an alternate acetyl-CoA source for HATs. Rapid deacetylation after Acs2p inactivation suggests nuclear acetyl-CoA synthesis is rate limiting for histone acetylation. Different histone lysines exhibit distinct deacetylation rates, with N-terminal tail lysines deacetylated rapidly and H3 lysine 56 slowly. Yeast mitochondrial and nucleocytosolic acetyl-CoA pools are biochemically isolated. Thus, acetyl-CoA metabolism is directly linked to chromatin regulation and may affect diverse cellular processes in which acetylation and metabolism intersect, such as disease states and aging.

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.

Mutations of acetylcholinesterase which confer insecticide resistance in insect populations

Resistance-modified acetylcholinesterases have been described in many insect species and sequencing of their genes has allowed several point mutations to be described. Most mutations line the active site gorge. Each mutation provides a specific resistance pattern: it confers resistance to one insecticide but may increase sensitivity to another. Most mutations alter hydrolysis of the substrate by decreasing the rate of enzyme deacetylation and by diminishing the stability of the enzyme. Mutations are often found in combination in the same protein. This has several consequences: it increases the level of resistance, it enlarges the spectrum of resistance and it may restore the catalytic efficiency of the enzyme. Natural populations are heterogeneous, composed of a mixture of different alleles.

Effect of deacetylation rate on gelation kinetics of konjac glucomannan

Effect of deacetylation rate on the gelation behaviors on addition of sodium carbonate for native and acetylated konjac glucomannan (KGM) samples with a degree of acetylation (DA) range of 1.38–10.1 wt.% synthesized using acetic anhydride in the presence of pyridine as catalyst was studied by dynamic viscoelastic measurements. At a fixed alkaline concentration ( C Na), both the critical gelation times ( t cr) and the plateau values of storage moduli ( G ′ sat ) of the KGM gels increased with increasing DA. While at a fixed ratio of alkaline concentrations to values of DA ( C Na/DA), the similar t cr and G ′ sat values independent of DA were observed. On the whole, increasing KGM concentration or temperature shortened the gelation time and enhanced the elastic modulus for KGM gel. The effect of deacetylation rate related to the C Na/DA on the gelation kinetics of the KGM samples were discussed.

Deoxy and deoxyfluoro analogues of acetylated methyl β- d-xylopyranoside––substrates for acetylxylan esterases

Four modified substrates for acetylxylan esterases, 2-deoxy, 3-deoxy, 2-deoxy-2-fluoro, and 3-deoxy-3-fluoro derivatives of di- O-acetylated methyl β- d-xylopyranoside were synthesized via 2,3-anhydropentopyranoside precursors. Methyl 2,3-anhydro-4- O-benzyl-β- d-ribopyranoside was transformed into methyl 2,3-anhydro-4- O-benzyl-β- d-lyxopyranoside in three steps. The epoxide ring opening of 2,3-anhydropentopyranosides was accomplished either by hydride reduction or hydrofluorination. Methyl β- d-xylopyranoside 2,3,4-tri- O-, 2,4-di- O-, and 3,4-di- O-acetates, and the prepared diacetate analogues were tested as substrates of acetylxylan esterases from Schizophyllum commune and Trichoderma reesei. Measurement of their rate of deacetylation pointed to unique structural requirements of the enzymes for the substrates. The enzymes differed particularly in the requirement for the trans vicinal hydroxy group in the deacetylation at C-2 and C-3 and in the tolerance to the presence of trans vicinal acetyl groups esterifying the OH group at C-2 and C-3.

Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.

Effect of degree of acetylation on gelation of konjac glucomannan.

Effect of the degree of acetylation (DA) on the gelation behaviors on addition of sodium carbonate for native and acetylated konjac glucomannan (KGM) samples with a DA range from 1.38 to 10.1 wt % synthesized using acetic anhydride in the presence of pyridine as catalyst was studied by dynamic viscoelastic measurements. At a fixed alkaline concentration (CNa), both the critical gelation times (tcr) and the plateau values of storage moduli (G'sat) of the KGM gels increased with increasing DA, while at a fixed ratio of alkaline concentrations to values of DA (CNa/DA), similar tcr and values independent of DA were observed. On the whole, increasing KGM concentration or temperature shortened the gelation time and enhanced the elastic modulus for KGM gel. The effect of deacetylation rate related to the CNa/DA on the gelation kinetics of the KGM samples was discussed.

Mode of action of acetylxylan esterase from Streptomyces lividans: a study with deoxy and deoxy-fluoro analogues of acetylated methyl beta-D-xylopyranoside.

Streptomyces lividans acetylxylan esterase removes the 2- or 3-O-acetyl groups from methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl beta-D-xylopyranoside. When the free hydroxyl group was replaced with a hydrogen or fluorine, the rate of deacetylation was markedly reduced, but regioselectivity was not affected. The regioselectivity of deacetylation was found to be independent of the prevailing conformation of the substrates in solution as determined by 1H-NMR spectroscopy. These observations confirm the importance of the vicinal hydroxyl group and are consistent with our earlier hypothesis that the deacetylation of positions 2 and 3 may involve a common ortho-ester intermediate. Another possible role of the free vicinal hydroxyl group could be the activation of the acyl leaving group in the deacetylation mechanism. Involvement of the free hydroxyl group in the enzyme-substrate binding is not supported by the results of inhibition experiments in which methyl 2,4-di-O-acetyl beta-D-xylopyranoside was used as substrate and its analogues or methyl beta-D-xylopyranoside as inhibitors. The enzyme requires for its efficient action the trans arrangement of the free and acetylated hydroxyl groups at positions 2 and 3.

Global Regulation of Post-translational Modifications on Core Histones

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.