Nonenzymatic acetylation of histones with acetyl phosphate and acetyl adenylate.

Nonenzymatic acetylation of calf-thymus lysine- and arginine-rich histones was demonstrated to occur when these proteins were incubated with [14C]acetyl phosphate and [14C]acetyl adenylate. The levels of acetylation depend on both pH and on reagent concentration. When acetyl [33P]phosphate and acetyl [3H]adenylate were used as reagents, we found neither histone phosphorylation nor adenylylation. Most of the radioactivity of 14C-labeled acetylated histones was recovered as Ne-acetyllysine. Furthermore, only a small amount of O-bound radioactivity was released by the 14C-labeled acetylated arginine-rich histone during treatment with hydroxylamine. Experiments on the acetylation of histones, in the presence of increasing salt concentration, gave different results for the two acetylating agents.     

Effect of insulin on association of acetyl CoA carboxylase phosphatase and acetyl CoA carboxylase

Insulin promotes an association between acetyl CoA carboxylase and acetyl CoA carboxylase phosphatase. The association between rat epididymal fat tissue carboxylase and the phosphatase occurs in both a tissue culture system and in vivo and is accompanied by an increase in acetyl CoA carboxylase activity.     

An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans

Summary An acetyl esterase was purified from Trichoderma reesei by cation and anion exchange chromatography. The enzyme had a molecular weight of 45 000 as determined by SDS-electrophoresis, or 67 000 as determined by gel filtration. In chromatofocusing the enzyme was shown to consist of two isoenzymes with isoelectric points of 6.8 and 6.0. The enzyme showed activity towards naphthyl acetate, triacetin and glucose-and xylose acetates. However, it liberated acetic acid from acetylated xylo-oligomers only to a small extent. The liberation of acetic acid from the oligomeric substrate was enhanced by addition of endoxylanase and -xylosidase.     

Hyperthermostable acetyl xylan esterase

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90°C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl⁻¹ in the absence of substrate was about 13 h at 98°C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100–104°C and 107.5°C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.     

Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation

Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.     

Distribution in brain and retina of four enzymes of acetyl CoA synthesis in relation to choline acetyl transferase and acetylcholine esterase

Eleven regions of mouse brain and twelve layers of monkey retina were assayed for choline acetyl transferase (ChAT), acetylcholine esterase (AChE), and 4 enzymes that synthesize acetyl CoA. The purpose was to seek evidence concerning the source of acetyl CoA for acetylcholine generation. In brain ATP citrate lyase was strongly correlated with ChAT as well as AChE (r=0.914 in both cases). Weak, but statistically significant correlation, was observed between ChAT and both cytoplasmic and mitochondrial thiolase, whereas there was a significant negative correlation between ChAT and acetyl thiokinase. In retina ChAT was essentially limited to the inner plexiform and ganglion cell layers, whereas substantial AChE activity extended as well into inner nuclear, outer plexiform and fiber layers, but no further. ATP citrate lyase activity was also highest in the inner four retinal layers, but was not strongly correlated with either ChAT or AChE (r=0.724 and 0.761, respectively). Correlation between ChAT and acetyl thiokinase was at least as strong (r=0.757), and in the six inner layers of retina, the correlation between ChAT and acetylthiokinase was very strong (r=0.932).Special issue dedicated to Dr. Lawrence Austin     

Detergents inhibit chloramphenicol acetyl transferase.

Potent inhibition of chloramphenicol acetyl transferase (CAT) by Triton X-100 and Nonidet P-40 was observed. The CAT activity was also moderately inhibited by sodium deoxycholate and sodium dodecyl sulfate, and least by Tween 20. Detergents should, therefore, not be used for cell lysate preparation when CAT activity is used as the reporter in a transient expression experiment.     

ATPase of bovine heart mitochondria. Modulation of ITPase activity by ATP, ADP, acetyl ATP and acetyl AMP

(1) Mitochondrial ATPase (F1) is influenced by specific nucleotides in its kinetic behavior towards its substrates. In this work, initial hydrolysis rates, as well as continuous reaction progress, were measured by recording proton production (equivalent to triphosphate hydrolysis). (2) After preincubation with ATP, F1 hydrolyzes MgITP partly as if it were MgATP, with respect to temperature dependence and 2,4-dinitrophenol inhibition/stimulation. (3) Acetyl ATP is a competitive inhibitor versus ATP on the F1-ATPase. With F1 which has been freed of ambient ATP by repeated precipitations with ammonium sulfate the Ki of acetyl ATP is 400 nM. (4) F1-ATPase which was depleted of bound nucleotides in the presence of glycerol (Garret, N.E. and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647) was preincubated with ADP and acetyl ATP. These preparations were assayed for hydrolytic activity with MgITP as substrate. Compared to a nonpreincubated control enzyme, the hydrolysis with these preparations was first stimulated, then inhibited. This stimulation/inhibition effect is most pronounced at 10 degrees C, but is also observed at 20 degrees C. (5) When nucleotide-depleted enzyme is preincubated with acetyl AMP, its ability to hydrolyze MgITP slowly decreases to approx. 50% after 60 min. This effect is reversed by further preincubation with acetyl ATP. It is speculated that under appropriate conditions AMP may exist or arise in a buried position on F1-ATPase, and act there as an inhibitor of MgITP hydrolysis.     

Reaction of acetyl hypofluorite with pyranoid and furanoid glycals

The regiospecific syn-addition of acetyl hypofluorite to glycals derived from pentopyranoses led to mixtures of stereoisomers. Stereospecific reactions occurred with furanoid glycals, the direction of addition being governed by the nature of the substituent at C-3. Whereas a benzyloxy group caused attack from the opposite, less-hindered face of the double bond, a hydroxyl group induced addition from the same side. From these reactions, 2-deoxy-2-fluoro derivatives of β-d-arabino-, α-d-ribo-, β-d-lyxo-, and α-d-xylo-pyranose as well as β-d-manno-, α-d-gluco-, α-d-ribo-, and β-d-arabino-furanose were obtainedl their ¹H-, ¹³C-, and ¹⁹F-n.m.r. data are given.     

Purification and characterization of acetyl esterase from Candida guilliermondii

An extracellular acetyl esterase (EC 3.1.1.6) from Candida guilliermondii NRRL Y-17257 was purified to homogeneity by acetone precipitation and QAE sepharose anion-exchange chromatography. The enzyme was a monomer with an apparent molecular weight of 67 kDa and a pI of 7.6. It had maximum activity at pH 7.5 and at 50-60 degrees C. It was relatively stable over a pH range of 5.8-8.0 and exhibited thermal stability up to 60 degrees C. The Km and Vmax values on alpha-naphthylacetate were 2.63 mM and 213.3 micromol alpha- naphthol min-1 mg-1 protein, respectively.