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PubMedDate not supplied

Comparative study of the second and third heterogeneous deacetylations of alpha- and beta-chitins in a multistep process.

Second and third heterogeneous deacetylations in a multistep process under argon atmosphere of alpha- and beta-chitins in the presence of 50% (w/v) NaOH, for temperatures ranging from 80 to 110 degrees C, were comparatively studied in order to optimize the multistep process of deacetylation. Along with the successive reactions, we observed important changes of chemical behavior with the crystalline state related to alpha- and beta-chitins, amorphous and partially deacetylated chitin, and chitosan. Thanks to the full reacetylation of all the deacetylated samples, we succeeded in estimating the oxidoreductive alkaline degradation occurring during deacetylation, whatever the degree of acetylation (DA) of the copolymer. It clearly appeared that the crystalline state of the samples was the key parameter on which depended the rate constants of both alkaline hydrolysis and deacetylation and, consequently, the activation energy Ea and the preexponential factor A. We may now propose optimal conditions allowing the production of well-defined chitosans with low DAs and higher molecular weights than those usually reported in the literature.

Acetylation↗

PubMed2004

Deacetylation of p53 after nerve growth factor treatment in PC12 cells as a post-translational modification mechanism of neurotrophin-induced tumor suppressor activation.

The tumor suppressor protein p53 is a transcription factor that regulates the response to cellular insults such as DNA damage and growth factor withdrawal. Transcriptional activity of p53 requires post-translational modification by phosphorylation and acetylation. This study used site-specific antibodies to demonstrate that nerve growth factor (NGF) treatment of PC12 cells results in p53 deacetylation at lysine (Lys) 382. Histone deacetylase (HDAC) activity, measured by a direct fluorescent assay, was increased after NGF treatment and peaked before p53 deacetylation. Inhibition of HDAC by trichostatin blocked the deacetylation of p53 and its transcriptional activity toward a reporter gene construct. Comparison of PC12 with PC12 cells containing a temperature-sensitive, dominant-negative construct showed that p53 deacetylation required functional p53. Inhibitors of MAP kinase that block p53 transactivation and inhibitors of TrkA receptor also abolished HDAC activation, indicating that deacetylation of p53 is an NGF-dependent post-translational mechanism of p53 activation. Finally, NGF or serum withdrawal did not lead to p53 deacetylation. A model is proposed in which the acetylation status of Lys 382 of p53 discriminates between cell cycle arrest and apoptosis.

Acetylation↗

PubMedDate not supplied

Deacetylation of cinobufagin by rat liver.

The enzyme system mediating the deacetylation of cinobufagin (CB) at the 16-position to give deacetylCB was characterized in the rat. Tissue distribution studies showed that the highest activity of CB deacetylation was mainly localized in the liver microsomal fraction. Some activity was also detected in the serum and intestine. Kinetic studies of the enzymatic reaction carried out by microsomes demonstrated that the formation of deacetyl-CB increased linearly with time up to 60 min and with protein content up to 10 mg. Apparent Km and Vmax values calculated from Lineweaver-Burk plots were 2.7 x 10(-4) M and 4.17 nmol/mg of protein/min, respectively. Low concentrations of several metal salts (AgNO3, MgCl2, CoSO4, and CuCl2) did not affect CB deacetylase activity. Microsomal CB deacetylation was inhibited by the organophosphorus compounds cyanox and fenitrothion at 1.0 x 10(-6) M. Eserine sulfate, disulfiram, rifampicin, and phenacetin at 1.0 x 10(-4) M also decreased CB deacetylase activity. Aspirin, sodium fluoride, and EDTA at 1.0 x 10(-3) M did not inhibit the deacetylation. In vivo treatment of rats with phenobarbital resulted in a 2-fold increase in microsomal CB deacetylase activity. All of these results suggest that the enzyme responsible for CB deacetylation is somewhat different from the other characterized esterases.

Acetylation↗

PubMedDate not supplied

Microsomal deacetylation of isomeric acetamidobiphenyls.

In vitro rates of deacetylation were measured with carcinogenic and noncarcinogenic isomeric arylamides using microsomal preparations from guinea pig, hamster, rat, mouse, rabbit and dog. Following incubation at 37 degrees, the substrate and hydrolysis products were extracted into ether and the corresponding aromatic amine quantified by gas-liquid chromatography. The data show that rates of deacetylation were both species and isomer dependent. In general, the highest rate of deacetylation in microsomes from all species was recorded with 2-acetamidobiphenyl (2-AABP); however, the mouse uniquely deacetylated 4-acetamidobiphenyl (4-AABP) most rapidly. The meta isomer 3-acetamidobiphenyl (3-AABP) was deacetylated at an intermediate rate in all species investigated. The variability in the rates of deacetylation within species may reflect the heterogeneity of the deacetylase enzymes and may be an important factor in amine/amide carcinogenesis.

Aminobiphenyl Compounds↗

PubMed1988

The metabolism of N-acetyl-3,5-dimethyl-p-benzoquinone imine in isolated hepatocytes involves N-deacetylation.

3,5-Dimethyl-N-acetyl-p-benzoquinone imine (3,5-dimethyl-NAPQI) was cytotoxic to isolated hepatocytes from Sprague Dawley rats at levels between 200 and 300 microM. It rapidly oxidized intracellular glutathione within 10 sec, with the formation of oxidized glutathione. The cytotoxicity of 3,5-dimethyl-NAPQI could be prevented over a 3.5-hr period with the carboxylesterase inhibitor bis(p-nitrophenyl) phosphate, indicating that cytotoxicity involved N-deacetylation. The N-deacetylated product could be trapped with glutathione as 3-(glutathion-S-yl)-4-amino-2,6-dimethylphenol in 3,5-dimethyl-NAPQI-treated hepatocytes but not in hepatocytes pretreated with bis(p-nitrophenyl) phosphate, indicating that N-deacetylation activity had been inhibited. 3,5-Dimethyl-NAPQI was readily N-deacetylated by rat liver microsomes, in contrast to 3,5-dimethylacetaminophen. The latter was also not cytotoxic to hepatocytes at up to 2 mM. The N-deacetylated product 4-amino-2,6-dimethylphenol rapidly underwent autoxidation to form 2,6-dimethylbenzoquinone imine and was highly cytotoxic to hepatocytes at 200-300 microM. The latter reacted with glutathione to give the above conjugate and no glutathione oxidation occurred. Dithioerythritol (2 mM) added at 10, 20, and 30 min after 3,5-dimethyl-NAPQI delayed but did not prevent cytotoxicity. Dithioerythritol also resulted in the partial restoration of GSH, presumably as a result of reduction of protein mixed disulphides. The mechanism of cytotoxicity of 3,5-dimethyl-NAPQI therefore appears to be a result of a combination of oxidative stress and deacetylation resulting in arylation.

Acetylation↗

PubMed1994

Comparative studies of adriamycin and 28-deacetyl sendanin on in vitro growth inhibition of human cancer cell lines.

The limonoid compound (28-deacetyl sendanin) isolated from the fruit of Melia toosendan SIEB. et ZUCC. was evaluated on anticancer activity. According to a standard in vitro cytotoxicity assay, eight human cancer cell lines and SRB assay were introduced for present evaluation. As a positive standard, adriamycin was tested in parallel. The cell lines were originated from six different organs. In view of dose-response profiles to 28-deacetyl sendanin, the most sensitive cells were SF-539 and PC-3 which were derived from CNS and prostate, respectively. In contrast, all the cell lines responded similarly to adriamycin to give rise to nearly identical dose-response profiles. By comparison of Gl50 between 28-deacetyl sendanin and adriamycin, six cell lines were more sensitive to 28-deacetyl sendanin and two were more resistant. As a result, 28-deacetyl sendanin had more sensitive and selective inhibitory effects on in vitro growth of human cancer cell lines in a comparison with adriamycin.

Antineoplastic Agents↗

PubMed2001

In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation.

A commercially available almond emulsin beta-glucosidase preparation has been reported to have chitobiose activity, and can hydrolyze chitin substrates due to a chitinase present in the enzyme preparation. This beta-glucosidase preparation was used to investigate hydrolytic activity on five chitosan samples with different molecular weight and degree of deacetylation. The degree of deacetylation and molecular weight of the chitosan samples were determined using a circular dichroism and a viscometric method, respectively. The hydrolytic activity of this beta-glucosidase preparation on chitosan was monitored viscometrically as the most convenient means of screening. Solutions of chitosan in pH 5.0 acetate buffer were prepared using the different viscosity grades of chitosan. The specific viscosity, measured after addition of beta-glucosidase to the above solutions, decreased dramatically over time in comparison to that of the respective control mixture without enzyme. Eadie-Hofstee plots established that hydrolysis of chitosan by this enzyme preparation obeyed Michaelis-Menten kinetics. Apparent Michaelis-Menten parameters and initial degradation rates were calculated and compared to determine the influences of the degree of deacetylation and molecular weight on the hydrolysis. The results show that higher molecular weight and higher degree of deacetylation chitosans possessed a lower affinity for the enzyme and a slower degradation rate. Faster degradation rates, then, are expected with lower molecular weight and low degree of deacetylation chitosans. Hydrolysis of these chitosan samples confirms the existence of a chitinase in the almond emulsin beta-glucosidase preparation, and further studies are warranted.

Chitin↗

PubMed2003

Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry.

Life span regulation and inhibition of gene silencing in yeast have been linked to nicotinamide effects on Sir2 enzymes. The Sir2 enzymes are NAD(+)-dependent protein deacetylases that influence gene expression by forming deacetylated proteins, nicotinamide and 2'-O-acetyl-ADPR. Nicotinamide is a base-exchange substrate as well as a biologically effective inhibitor. Characterization of the base-exchange reaction reveals that nicotinamide regulates sirtuins by switching between deacetylation and base exchange. Nicotinamide switching is quantitated for the Sir2s from Archeaglobus fulgidus (Sir2Af2), Saccharomyces cerevisiae (Sir2p), and mouse (Sir2alpha). Inhibition of deacetylation was most effective for mouse Sir2 alpha, suggesting species-dependent development of this regulatory mechanism. The Sir2s are proposed to form a relatively stable covalent intermediate between ADPR and the acetyl oxygen of the acetyllysine-protein substrate. During the lifetime of this intermediate, nicotinamide occupation of the catalytic site determines the fate of the covalent complex. Saturation of the nicotinamide site for mouse, yeast, and bacterial Sir2s causes 95, 65, and 21% of the intermediate, respectively, to return to acetylated protein. The fraction of the intermediate committed to deacetylation results from competition between the nicotinamide and the neighboring 2'-hydroxyl group at the opposite stereochemical face. Nicotinamide switching supports the previously proposed Sir2 catalytic mechanism and the existence of a 1'-O-peptidyl-ADPR.Sir2 intermediate. These findings suggest a strategy for increasing Sir2 enzyme catalytic activity in vivo by inhibition of chemical exchange but not deacetylation.

Acetylation↗

PubMedDate not supplied

Comparative study of the first heterogeneous deacetylation of alpha- and beta-chitins in a multistep process.

Heterogeneous deacetylation of alpha- and beta-chitins from shrimp shells and squid pens were comparatively studied. Each deacetylated sample, recovered after neutralization, was fractionated according to a water-soluble and insoluble fraction (pH 8.5). The systematic study of DAs, crystallinity changes, and distribution of N-acetylglucosamine residues were performed on the two kinds of fractions. For the two fractions resulting from a deacetylation in the presence of 50% (w/v) NaOH, for temperatures ranging from 80 to 110 degrees C, the activation energies of the reactions were found close to 39.9 +/- 1.0 and 42.8 +/- 1.8 kJ mol(-1) and the frequency factors of collision were of 7.2 +/- 2.4 and 54.4 +/- 18.5 10(3) min(-1), for alpha- and beta-chitins, respectively. Deacetylations of water-soluble and insoluble fractions were compared, and the major role played by the crystallinity level during deacetylation was evidenced and, thereafter, the role of the nature of the starting chitins on the chemical behavior. Thus, in some conditions, we observed critical values of DDA where the structures were becoming fully amorphous.

Acetylation↗

PubMed1980

Kinetics of N-acetylprocainamide deacetylation.

The kinetics of N-acetylprocainamide (NAPA) deacetylation to procainamide (PA) were determined in a normal subject using NAPA-13C, labeled in the acetyl group. The deacetylation clearance of NAPA (ClD) was found to be 6.5 ml/min whereas total NAPA elimination clearance was 231 ml/min, so that 2.8% of the administered NAPA-13C was metabolized by deacetylation. This estimated of ClD was shown to be representative of the rate of NAPA deacetylation in four patients on long-term NAPA therapy. Steady-state [PA]/[NAPA] ratios averaged 0.024, but would be expected to rise to 0.057 if functionally anephric patients were treated with NAPA. Despite reports that patients with the PA-induced systemic lupus erythematosus-like reaction have had symptomatic and immunologic remission when switched to NAPA, the demonstration that NAPA is deacetylated to PA indicates that the apparently greater immunologic safety of NAPA may be relative rather than absolute.

Acecainide↗

PubMed2006

Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice.

Errors in meiotic chromosome segregation are the leading cause of spontaneous abortions and birth defects. Almost all such aneuploidy derives from meiotic errors in females, with increasing maternal age representing a major risk factor. It was recently reported that histones are globally deacetylated in mammalian oocytes during meiosis but not mitosis. In the present study, inhibition of meiotic histone deacetylation was found to induce aneuploidy in fertilized mouse oocytes, which resulted in embryonic death in utero at an early stage of development. In addition, a histone remained acetylated in the oocytes of older (10-month-old) female mice, suggesting that the function for histone deacetylation is decreased in the oocytes of such mice. Thus, histone deacetylation may be involved in the fair distribution of chromosomes during meiotic division. The high incidence of aneuploidy in the embryos of older females may be due to inadequate meiotic histone deacetylation.

Acetylation↗

PubMed2000

Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product.

The Saccharomyces cerevisiae silencing protein Sir2 is the founding member of a universally conserved family of proteins that have been shown to possess NAD-dependent histone deacetylation and ADP-ribosylation activities. Here we show that histone deacetylation by Sir2 is coupled to cleavage of the high-energy bond that links the ADP-ribose moiety of NAD to nicotinamide. Analysis of the NAD cleavage products revealed the presence of nicotinamide, ADP-ribose, and a third product that appeared to be related to ADP-ribose. With the use of label transfer experiments, we show that the acetyl group in the histone substrate is transferred to this NAD breakdown product during deacetylation, forming a product that we conclude to be O-acetyl-ADP-ribose. Detection of this species strongly argues for obligate coupling of histone deacetylation to NAD breakdown by Sir2. We propose reaction mechanisms that could account for this coupling via acetyl-ADP-ribose formation. The unprecedented coupling of amide bond cleavage to cleavage of a high-energy bond raises the possibility that NAD breakdown by Sir2 plays an important role in silencing that is independent of its requirement for deacetylation.

Acetylation↗

PubMed2000

N-deacetylation of Sinorhizobium meliloti Nod factors increases their stability in the Medicago sativa rhizosphere and decreases their biological activity.

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16,2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.

Amidohydrolases↗

PubMed2005

Histone deacetylation is required for progression through mitosis in tobacco cells.

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.

Acetylation↗

PubMed1992

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.

Chromatography, High Pressure Liquid↗

PubMed2001

Regulation of transcription factor YY1 by acetylation and deacetylation.

YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. It activates or represses many genes during cell growth and differentiation and is also required for the normal development of mammalian embryos. Previous studies have established that YY1 interacts with histone acetyltransferases p300 and CREB-binding protein (CBP) and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3. Here, we present evidence that the activity of YY1 is regulated through acetylation by p300 and PCAF and through deacetylation by HDACs. YY1 was acetylated in two regions: both p300 and PCAF acetylated the central glycine-lysine-rich domain of residues 170 to 200, and PCAF also acetylated YY1 at the C-terminal DNA-binding zinc finger domain. Acetylation of the central region was required for the full transcriptional repressor activity of YY1 and targeted YY1 for active deacetylation by HDACs. However, the C-terminal region of YY1 could not be deacetylated. Rather, the acetylated C-terminal region interacted with HDACs, which resulted in stable HDAC activity associated with the YY1 protein. Finally, acetylation of the C-terminal zinc finger domain decreased the DNA-binding activity of YY1. Our findings suggest that in the natural context, YY1 activity is regulated through intricate mechanisms involving negative feedback loops, histone deacetylation, and recognition of the cognate DNA sequence affected by acetylation and deacetylation of the YY1 protein.

Acetylation↗

PubMedDate not supplied

Spironolactone metabolism in target tissues. Characteristics of deacetylation in kidney, liver, adrenal cortex, and testes.

Prior investigations demonstrated that many of the actions of spironolactone (SL) required deacetylation of the parent compound as the first step in the formation of biologically active metabolites. Studies were done to characterize the process of deacetylation in several target tissues. The reaction was catalyzed by microsomal and cytosolic fractions of livers, kidneys, adrenal glands, and testes. Microsomal activity was greatest in liver and kidney and far exceeded cytosolic metabolism in those tissues. In adrenal glands and testes, by contrast, deacetylation was greater in cytosolic than microsomal fractions. The metabolism-mediated destruction of adrenal microsomal cytochromes P-450 by SL was enhanced by coincubation of microsomes with cytosol, illustrating the potential importance of combined microsomal and cytosolic metabolism in the actions of SL. The deacetylation of SL was decreased by various esterase inhibitors; the organophosphate compounds were the most potent inhibitors. The effectiveness of the esterase inhibitors varied from tissue to tissue, as well as from microsomes to cytosol within each tissue. The results indicate that SL deacetylation is catalyzed by microsomal and cytosolic esterases in various target tissues; several isozymes appear to be involved. These and prior observations suggest that tissue metabolism of SL is of major importance in the actions of the drug.

Adrenal Cortex↗

PubMed1999

Histopathological and immunohistochemical studies of membranes of deacetylated chitin derivatives implanted over rat calvaria.

Membranes made of 65, 70, 80, 94, and 100% deacetylated chitin (chitosan) were implanted subperiosteally over the calvaria of 100 rats. Reactions were studied at 1, 2, 4, and 8 weeks after implantation. Membranes prepared with 65, 70, and 80% deacetylated chitin initially elicited marked inflammatory reactions that subsided in time with granulation tissue formation and osteogenesis. Osteocalcin-positive cells were detected immunohistochemically in the granulation tissue. On the other hand, membranes made of 94% deacetylated chitin and chitosan showed mild inflammation and minimal osteogenesis. The results indicate that membranes made of 65, 70, and 80% deacetylated chitin enhance osteogenesis at the site of their implantation. However, the initially severe inflammatory reaction associated with these materials needs to be controlled before the materials would be suitable for clinical application.

Acetylation↗