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1.
Paul R. Libby 《Archives of biochemistry and biophysics》1980,203(1):384-389
Two similar histone acetyltransferases have been separated from rat liver nuclei and purified 500-fold. Both enzymes also acetylate spermidine and spermine but diamines are not acetylated. Both enzymes preferentially acetylate histone 3; among the remaining histones H2A and H2B are good substrates, whereas H1 and histone 4 are poor substrates. Apparent Michaelis constants for spermidine were about 2 × 10?4m; apparent Michaelis constants for acetyl coenzyme A were 1.5 × 10?5 and 10?5m for enzymes A and B, respectively. At low concentrations DNA inhibits histone acetylation by enzyme A (50% inhibition at 25 μg/ml DNA). Enzyme B is relatively insensitive to DNA. This suggests the possibility of separate intranuclear localization of the two enzymes. 相似文献
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The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74 % decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50 %, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions. 相似文献
4.
We have previously reported [López-Rodas et al. (1989) J. Biol. Chem. 264, 19028-19033] that the yeast Saccharomyces cerevisiae contains four histone acetyltransferases, which can be resolved by ion-exchange chromatography, and their specificity toward yeast free histones was studied. In the present contribution we show that three of the enzymes are nuclear, type A histone acetyltransferases and they are able to acetylate nucleosome-bound histones. They differ in their histone specificity. Enzyme A1 acetylates H2A in chicken nucleosomes, although it is specific for yeast free H2B; histone acetyltransferase A2 is highly specific for H3, and histone acetyltransferase A3 preparations acetylate both H3 and H4 in nucleosomes. The fourth enzyme, which is located in the cytoplasm, does not accept nucleosomes as substrate, and it represents a canonical type B, H4-specific histone acetyltransferase. Finally, histone deacetylase activity is preferentially found in the nucleus. 相似文献
5.
Porcine liver nuclear histone acetyltransferase. Partial purification and basic properties 总被引:1,自引:0,他引:1
The major histone acetyltransferase activity from porcine liver nuclei has been isolated and partially purified by a simple, rapid, and reproducible method. Extraction of nuclei in buffered 30% saturated ammonium sulfate and subsequent ammonium sulfate fractionation, chromatography on DEAE-Sephacel and hydroxylapatite, and ultracentrifugation on linear 15-30% glycerol gradients provides an 8650-fold purification (over nuclei) in 42% yield. The molecular weight of the enzyme is approximately 94,000 as determined by glycerol gradient ultracentrifugation and gel filtration on Sephacryl S-200. The optimum pH for the reaction is 7.5 and the activity is inhibited by monovalent and divalent salts and by sulfhydryl blocking reagents. The enzyme activity is substantially protected from thermal denaturation at 37 degrees C by the addition of glycerol to the incubation medium. In the presence of the core histones, the enzyme catalyzes the acetylation reaction in the order H3 greater than H4 greater than H2B greater than H2A; the order for histones bound in nucleosome core particles is H4 greater than H2B greater than H3 greater than H2A. The high mobility group proteins 14 and 17 serve as substrates for the enzyme in vitro, suggesting a possible role for enzymatic high mobility group acetylation in chromatin function. 相似文献
6.
Yeast contains multiple forms of histone acetyltransferase 总被引:1,自引:0,他引:1
G López-Rodas V Tordera M M Sánchez del Pino L Franco 《The Journal of biological chemistry》1989,264(32):19028-19033
We have assayed several methods to quantitatively recover yeast histone acetyltransferases in an attempt to study the multiplicity of enzymatic activities. Two methods, namely (NH4)2SO4 precipitation and salt dissociation of chromatin in 0.5 M NaCl, yielded convenient preparations of total histone acetyltransferases. DEAE-Sepharose chromatography of the crude extracts resulted in the separation of three peaks of activity when total yeast histones were used as substrate. However, the scanning of the enzymatic activity toward individual histones along the chromatography, achieved by determining the specific activity of the individual histones after incubating whole histones and [14C]acetyl-CoA with the chromatographic fractions, yielded four peaks. The first two peaks showed specificity toward H2B and H3, respectively. Although they partially overlapped, rechromatography on cation exchangers allowed us to resolve the two activities, and several criteria were used to prove that they correspond to different enzyme molecules. The last two peaks were H4-specific, but the present data suggest that one of the activities is chromatin-bound, whereas the other surely corresponds to the cytoplasmic B-form of the enzyme. The enzyme specific for yeast H2B acetylates chicken erythrocyte H2A, rather than H2B. The detected multiplicity of yeast histone acetyltransferases may correspond to the multiplicity of roles proposed for histone acetylation. 相似文献
7.
The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 M 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells. 相似文献
8.
Nuclear histone acetyltransferase isolated from calf thymus was found to be inhibited by numerous salts at millimolar concentrations. Salts made up of monovalent ions caused 50% decrease in enzymatic activity at an average concentration of 51 +/- 14 mM while the same degree of inhibition was achieved by divalent salts at 15 +/- 5 mM. At the same ionic strength in the range from 5 to 70 mM, the divalent salts were 14-31% more inhibitory than the salts of monovalent ions. Kinetic analysis showed that NaCl and (NH4)2SO4 inhibited the enzyme competitively against both acetyl-CoA and histones. The inhibition constants for NaCl against acetyl-CoA and histones are respectively 30 and 34 mM. That for (NH4)2SO4 are 8 and 12 mM respectively. 相似文献
9.
Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases 总被引:3,自引:0,他引:3
G López-Rodas E I Georgieva R Sendra P Loidl 《The Journal of biological chemistry》1991,266(28):18745-18750
DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms. 相似文献
10.
An histone acetyltransferase has been purified from nuclei of 40-h-old Artemia salina larvae. The enzyme is very unstable at 0 degrees C, requires free -SH groups for activity and is rapidly inactivated at 40 degrees C. The optimal pH for activity is 8.5 and the activity is half inhibited by millimolar concentrations of Mn2+, Ca2+ or Mg2+ or decimolar concentrations of Na+ and K+. The molecular weight of the enzyme, determined by gel filtration chromatography, changed with the ionic strength of the medium (280,000 in 10 mM Tris . HCl, 170,000 in 0.2 M KCl). The very-lysine-rich histone H1 is a better substrate acceptor than the arginine-rich histones H3 or H4. Under proper conditions, the enzyme can modify all the internal lysyl residues in histones H1 and H4. The acetylation of H1 is inhibited when all the other histone fractions are present in the assay mixture. 相似文献
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Monoclonal antibodies selective for Drosophila melanogaster choline acetyltransferase 总被引:5,自引:0,他引:5
The newly developed monoclonal antibody technology was applied to the production of antibodies selective for Drosophila melanogaster choline acetyltransferase (EC 2.3.1.6). Two stable cell lines, 1C8 and 1G4, were isolated from NS-1/spleen cell hybrids by employing a choline acetyltransferase enzyme activity-screening method. Both cell lines were cloned twice and were maintained in continuous culture and as ascites tumors. Purified antibody was isolated from ascites fluids by pH elution after adsorption to Protein A-Sepharose. Both antibodies eluted from the Protein A-Sepharose as a single subclass, IgG1, and directly inhibited choline acetyltransferase activity. Scatchard analysis of titration data for choline acetyltransferase antibody-enzyme interaction generated linear curves for both antibodies: KA for 1C8 was 2.77 X 10(7) M-1 and KA for 1G4 was 0.78 X 10(7) M-1. Inclusion of the choline acetyltransferase substrate acetyl-CoA at 10 times the KM in the antibody-enzyme reaction mixture substantially reduced the level of inhibition observed with both antibodies; choline, however, exhibited no protective effect. Neither antibody reacted with choline acetyltransferase-containing extracts of vertebrates or other insect neural tissues. We conclude that the two antibodies are nonidentical, monoclonal, and highly selective for D. melanogaster choline acetyltransferase, both reacting at or near the acetyl-CoA binding region of the enzyme-active site. 相似文献
13.
Lau OD Courtney AD Vassilev A Marzilli LA Cotter RJ Nakatani Y Cole PA 《The Journal of biological chemistry》2000,275(29):21953-21959
p300/CBP-associated factor (PCAF) is a histone acetyltransferase that plays an important role in the remodeling of chromatin and the regulation of gene expression. It has been shown to catalyze preferentially acetylation of the epsilon-amino group of lysine 14 in histone H3. In this study, the kinetic mechanism of PCAF was evaluated with a 20-amino acid peptide substrate derived from the amino terminus of histone H3 (H3-20) and recombinant bacterially expressed PCAF catalytic domain (PCAF(cat)). The enzymologic behavior of full-length PCAF and PCAF(cat) were shown to be similar. PCAF-catalyzed acetylation of the substrate H3-20 was shown to be specific for Lys-14, analogous to its behavior with the full-length histone H3 protein. Two-substrate kinetic analysis displayed an intersecting line pattern, consistent with a ternary complex mechanism for PCAF. The dead-end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus H3-20. The dead-end analog inhibitor H3-20 K14A was competitive versus H3-20 and uncompetitive versus acetyl-CoA. The potent bisubstrate analog inhibitor H3-CoA-20 was competitive versus acetyl-CoA and noncompetitive versus H3-20. Taken together, these inhibition patterns support an ordered BiBi kinetic mechanism for PCAF in which acetyl-CoA binding precedes H3-20 binding. Viscosity experiments suggest that diffusional release of product is not rate-determining for PCAF catalysis. These results provide a mechanistic framework for understanding the detailed catalytic behavior of an important subset of the histone acetyltransferases and have significant implications for molecular regulation of and inhibitor design for these enzymes. 相似文献
14.
Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities 总被引:28,自引:2,他引:26
F. Fonnum 《The Biochemical journal》1969,115(3):465-472
1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-(14)C]acetate or sodium [(3)H]acetate by incubation with CoA, ATP, Mg(2+) and extract from acetone-dried pigeon liver were used. 3. [1-(14)C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [(3)H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [(3)H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively. 相似文献
15.
DEAE-Sepharose chromatography of extracts from plasmodia of the myxomycete Physarum polycephalum revealed the presence of multiple histone acetyltransferases and histone deacetylases. A cytoplasmic histone acetyltransferase B, specific for histone H4, and two nuclear acetyltransferases A1 and A2 were identified; A1 acetylates all core histones with a preference for H3 and H2A, whereas A2 is specific for H3 and also slightly for H2B. Two histone deacetylases, HD1 and HD2, could be discriminated. They differ with respect to substrate specificity and pH dependence. For the first time the substrate specificity of histone deacetylases was determined using HPLC-purified individual core histone species. The order of acetylated substrate preference is H2A much greater than H3 greater than or equal to H4 greater than H2B for HD1 and H3 greater than H2A greater than H4 for HD2, respectively; HD2 is inactive with H2B as substrate. Moreover histone deacetylases are very sensitive to butyrate, since 2 mM butyrate leads to more than 50% inhibition of enzyme activity. 相似文献
16.
It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets. 相似文献
17.
E P Candido 《Canadian journal of biochemistry》1975,53(7):796-803
Histone acetyltransferase activity of trout testis was studied both in intact nuclei, and in high salt nuclear extracts. With intact nuclei, the distribution of incorporated [14C]acetate in the various histones was similar to that observed in vivo; the arginine-rich histones H3 and H4 showed the highest specific activities, and lower amounts of label were detected in histones H2a and H2b. Histone H1 incorporated little or no label. Acetyltransferase activity could be detected in purified, sheared chromatin after the addition of MgCl2 or KCl, suggesting that the enzyme is bound to chromatin. Treatment of nuclei with 0.4 M NaCl caused the dissociation of acetyltransferase activity. Most of this solubilized activity failed to bind to DEAE Sephadex and behaved as a high molecular weight heterogeneous complex on Sephadex G-100, suggesting that the enzyme is present as an aggregate with other proteins in the extract. The pH optimum of this preparation was approximately 8.5, and the enzyme showed a preference for histones H3 and H4 as substrates. 相似文献
18.
Acetylation of lysine 56 of histone H3 (H3-Lys-56) occurs in S phase and disappears during G(2)/M phase of the cell cycle. However, it is not clear how this modification is regulated during the progression of the cell cycle. We and others have shown that the histone acetyltransferase (HAT) Rtt109 is the primary HAT responsible for acetylating H3-Lys-56 in budding yeast. Here we show that Rtt109 forms a complex with Vps75 and that both recombinant Rtt109-Vps75 complexes and native complexes purified from yeast cells acetylate H3 present in H3/H4/H2A/H2B core histones but not other histones. In addition, both recombinant and native Rtt109-Vps75 HAT complexes exhibited no detectable activity toward nucleosomal H3, suggesting that H3-Lys-56 acetylation is at least in part regulated by the inability of Rtt109-Vps75 complexes to acetylate nucleosomal H3 during G(2)/M phase of the cell cycle. Further, Rtt109 bound mutant H3/H4 tetramers composed of histones lacking their N-terminal tail domains less efficiently than wild-type H3/H4 tetramers, and Rtt109-Vps75 complexes displayed reduced HAT activity toward these mutant H3/H4 tetramers. Thus, the N termini of H3/H4 tetramers are required for efficient acetylation of H3 by the Rtt109-Vps75 complex. Taken together, these studies provide insights into how H3-Lys-56 acetylation is regulated during the cell cycle. 相似文献
19.
Two forms of a histone H1-specific S-adenosylmethionine:protein-lysine N-methyltransferase (protein methylase III) have been purified from Euglena gracilis 48- and 214-fold, respectively, with yields of 3.4 and 4.6%. The enzymes were purified on DEAE-cellulose and histone-Sepharose affinity chromatography and found to be highly specific toward histone H1 as a substrate. However, one of the enzymes also methylates other histone subfractions to a limited extent. Of the proteins other than histones, only myosin showed measurable methyl-accepting capability. Both enzymes were found to be inhibited by S-adenosylhomocysteine (D and L forms), S-adenosyl-L-ethionine, and sinefungin. While the Ki values for S-adenosyl-L-ethionine were similar for both enzymes, the values for S-adenosyl-L-homocysteine and sinefungin were 10-fold lower for the second form. The Km values for histone H1 and S-adenosyl-L-methionine were found to be 3.1 X 10(-7) and 2.7 X 10(-5) M, respectively, for the first enzyme, and 4.4 X 10(-7) and 3.45 X 10(-5) M for the second. Peptide analysis of methyl-14C-labeled H1 revealed that the two enzymes methylate different sites within the histone H1 molecule. The two enzymes were found to have molecular weights of 55,000 and 34,000, respectively. Both enzymes have an optimum pH of 9.0, which is identical to that of other protein-lysine N-methyltransferases thus far identified. 相似文献
20.
《DNA Repair》2019
The nucleotide excision repair (NER) pathway is critical for removing damage induced by ultraviolet (UV) light and other helix-distorting lesions from cellular DNA. While efficient NER is critical to avoid cell death and mutagenesis, NER activity is inhibited in chromatin due to the association of lesion-containing DNA with histone proteins. Histone acetylation has emerged as an important mechanism for facilitating NER in chromatin, particularly acetylation catalyzed by the Spt-Ada-Gcn5 acetyltransferase (SAGA); however, it is not known if other histone acetyltransferases (HATs) promote NER activity in chromatin. Here, we report that the essential Nucleosome Acetyltransferase of histone H4 (NuA4) complex is required for efficient NER in Saccharomyces cerevisiae. Deletion of the non-essential Yng2 subunit of the NuA4 complex causes a general defect in repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast; in contrast, deletion of the Sas3 catalytic subunit of the NuA3 complex does not affect repair. Rapid depletion of the essential NuA4 catalytic subunit Esa1 using the anchor-away method also causes a defect in NER, particularly at the heterochromatic HML locus. We show that disrupting the Sds3 subunit of the Rpd3L histone deacetylase (HDAC) complex rescued the repair defect associated with loss of Esa1 activity, suggesting that NuA4-catalyzed acetylation is important for efficient NER in heterochromatin. 相似文献