Sodium butyrate inhibits histone deacetylation in cultured cells

Sodium butyrate in millimolar concentrations causes an accumulation of acetylated histone species in a variety of vertebrate cell lines. In all lines tested, butyrate caused hyperacetylation of H3 and H4, and in rat IRC8 cells, H2A and H2B were also affected. In Friend erythroleukemic cells, butyrate also induces the synthesis of a nonhistone chromosomal protein, IP₂₅. Butyrate does not affect the rate of histone acetylation in cell-free extracts or nuclei of Friend cells. Rather, this fatty acid inhibits histone deacetylation. Cell-free extracts of either control cells or butyrate-grown cells contain comparable levels of histone-deacetylating activity. This in vitro activity is inhibited by the addition of butyrate to the extracts. Thus butyrate appears to be an inhibitor of histone deacetylases both in vivo and in vitro.     

Differential in vitro methylation and synthesis of the 480-kilodalton corrinoid protein in Methanosarcina barkeri grown on different substrates.

The 480-kDa corrinoid protein was significantly methylated in extracts of acetate- but not methanol-grown cells incubated with 14CH3OH, in part because of its decreased synthesis in cells grown on substrates other than acetate. In addition, a 200-kDa corrinoid protein was methylated in extracts of methanol- but not acetate-grown cells.     

Enzymes involved in crotonate metabolism in Syntrophomonas wolfei

Cell-free extracts of Syntrophomonas wolfei subsp. wolfei grown with crotonate in pure culture or in coculture with Methanospirillum hungatei contained crotonyl-coenzyme A (CoA): acetate CoA-transferase activity. This activity was not detected in cell-free extracts from the butyrate-grown coculture which suggests that the long lag times observed before S. wolfei grew with crotonate were initially due to the inability to activate crotonate. Cell-free extracts of S. wolfei grown in pure culture contained high specific activities of hydrogenase and very low levels of formate dehydrogenase. The low levels suggest a biosynthethic rather than a catabolic role for the latter enzyme when S. wolfei is grown in pure culture. CO dehydrogenase activity was not detected. S. wolfei can form butyrate using a CoA transferase activity, but not by a phosphotransbutyrylase or enoate reductase activity. A c-type cytochrome was detected in S. wolfei grown in pure culture or in coculture indicating the presence of an electron transport system. This is a characteristic which separates S. wolfei from other known crotonate-using bacteria.     

Alterations in the pyruvate dehydrogenase complex during adaptation to glucose by Neurospora

A 20-fold induction of the pyruvate dehydrogenase complex, pyruvate dehydrogenase (EC 1.2.4.1) plus dihydrolipoate S-acetyltransferase, (lipoyltransacetylase) (EC 2.3.1.12) plus dihydrolipoyl dehydrogenase, NADH : lipoamide oxidoreductase, (EC 1.6.4.3), from a specific activity of 3.5–65.0 was observed in mitochondrial extracts during adaptation of Neurospora to glucose from acetate media. The extent of ATP-dependent, time-dependent inactivation of the pyruvate dehydrogenase complex was approximately the same in both acetate- and glucose-grown cells, thereby indicating that the low pyruvate dehydrogenerase complex activities in acetate-grown cells did not represent phosphorylated pyruvate dehydrogenase complex molecules. High levels of dihydrolipoyl transacetylase (EC 2.3.1.12) were observed in mitochondrial extracts from acetate-grown cells; this lipoyltransacetylase was analyzed on sucrose density gradients and found to be associated with the pyruvate dehydrogenase complex. Digitonin fractionation of mitochondria revealed that both the pyruvate dehydrogenase complex and lipoyltransacetylase were primarily associated with the mitochondrial outer membrane.     

Protein content and enzyme activities in methanol- and acetate-grown Methanosarcina thermophila.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila TM-1 was examined by two-dimensional polyacrylamide gel electrophoresis. More than 100 mutually exclusive spots were present in acetate- and methanol-grown cells. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the carbon monoxide dehydrogenase complex were identified in acetate-grown cells. Activities of formylmethanofuran dehydrogenase, formylmethanofuran:tetrahydromethanopterin formyltransferase, 5,10-methenyltetrahydromethanopterin cyclohydrolase, methylene tetrahydromethanopterin:coenzyme F420 oxidoreductase, formate dehydrogenase, and carbonic anhydrase were examined in acetate- and methanol-grown Methanosarcina thermophila. Levels of formyltransferase in either acetate- or methanol-grown Methanosarcina thermophila were approximately half the levels detected in H2-CO2-grown Methanobacterium thermoautotrophicum. All other enzyme activities were significantly lower in acetate- and methanol-grown Methanosarcina thermophila.     

Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.     

Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts.

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.     

Linalyl Acetate Is Metabolized by Pseudomonas incognita with the Acetoxy Group Intact

Metabolism of linalyl acetate by Pseudomonas incognita isolated by enrichment culture on the acyclic monoterpene alcohol linalool was studied. Biodegradation of linalyl acetate by this strain resulted in the formation of linalool, linalool-8-carboxylic acid, oleuropeic acid, and Δ⁵-4-acetoxy-4-methyl hexenoic acid. Cells adapted to linalyl acetate metabolized linalyl acetate-8-aldehyde to linalool-8-carboxylic acid, linalyl acetate-8-carboxylic acid, Δ⁵-4-acetoxy-4-methyl hexenoic acid, and geraniol-8-carboxylic acid. Resting cell suspensions previously grown with linalyl acetate oxidized linalyl acetate-8-aldehyde to linalyl acetate-8-carboxylic acid, Δ⁵-4-acetoxy-4-methyl hexenoic acid, and pyruvic acid. The crude cell-free extract (10,000 g of supernatant), obtained from the sonicate of linalyl acetate-grown cells, was shown to contain enzyme systems responsible for the formation of linalyl acetate-8-carboxylic acid and linalool-8-carboxylic acid from linalyl acetate. The same supernatant contained NAD-linked alcohol and aldehyde dehydrogenases involved in the formation of linalyl acetate-8-aldehyde and linalyl acetate-8-carboxylic acid, respectively. On the basis of various metabolites isolated from the culture medium, resting cell experiments, growth and manometric studies carried out with the isolated metabolites as well as related synthetic analogs, and the preliminary enzymatic studies performed with the cell-free extract, a probable pathway for the microbial degradation of linalyl acetate with the acetoxy group intact is suggested.     

Peptide utilization in yeast studies on methionine and lysine auxotrophs ofSaccharomyces cerevisiae

A study was made of the growth response and cellular peptidase activity of several amino acid auxotrophs ofSaccharomyces cerevisiae to peptides containing the required amino acid. A methionine-requiring auxotroph grew on, and contained intra-cellular peptidase activity toward Met-Met, Met-Met-Met, and Met-Gly-Met-Met. In contrast, Gly-Met-Gly did not support the growth of this mutant nor did three lysine-requiring strains utilize any lysine-containing peptides tested, although cell-free extracts from the respective mutants contained the necessary peptidase activity. The absence of a transport system of relatively high affinity for these peptides is suggested as the reason for their inability to satisfy the nutritional requirements of the cells.     

Methylthiol:coenzyme M methyltransferase from Methanosarcina barkeri, an enzyme of methanogenesis from dimethylsulfide and methylmercaptopropionate.

During growth on acetate, Methanosarcina barkeri expresses catabolic enzymes for other methanogenic substrates such as monomethylamine. The range of substrates used by cells grown on acetate was further explored, and it was found that cells grown on acetate also converted dimethylsulfide (DMS) and methylmercaptopropionate (MMPA) to methane. Cells or extracts of cells grown on trimethylamine or methanol did not utilize either DMS or MMPA. During growth on acetate, cultures demethylated MMPA, producing methane and mercaptopropionate. Extracts of acetate-grown cells possessed DMS- and MMPA-dependent coenzyme M (CoM) methylation activities. The activity peaks of CoM methylation with either DMS or MMPA coeluted upon gel permeation chromatography of extracts of acetate-grown cells consistent with an apparent molecular mass of 470 kDa. A 480-kDa corrinoid protein, previously demonstrated to be a CoM methylase but otherwise of unknown physiological function, was found to methylate CoM with either DMS or MMPA. MMPA was demethylated by the purified 480-kDa CoM methylase, consuming 1 mol of CoM and producing 1 mol of mercaptopropionate. DMS was demethylated by the purified protein, consuming 1 mol of CoM and producing 1 mol of methanethiol. The methylthiol:CoM methyltransferase reaction could be initiated only with the enzyme-bound corrinoid in the methylated state. CoM could demethylate, and DMS and MMPA could remethylate, the corrinoid cofactor. The monomethylamine corrinoid protein and the A isozyme of methylcobamide:CoM methyltransferase (proteins homologous to the two subunits comprising the 480-kDa CoM methylase) did not catalyze CoM methylation with methylated thiols. These results indicate that the 480-kDa corrinoid protein functions as a CoM methylase during methanogenesis from DMS or MMPA.     

Effects of Different Substrates on Glucose Uptake and Hexokinase Activity in Euglena gracilis*

SYNOPSIS. Hexokinase levels and glucose uptake rates were measured in cells of Euglena gracilis adapted for growth on glucose, acetate, and ethanol. Glucose-grown cells had 3 times the hexokinase levels found in acetate- and ethanol-grown cells. However, glucose- and ethanol-grown cells had the same rates of glucose uptake, while acetate-grown cells accumulated only 1/8 as much glucose in the same interval.     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.     

Reversal of 2-bromoethanesulfonate inhibition of methanogenesis in Methanosarcina sp.

2-Bromoethanesulfonate (BES) inhibition of methanogenesis from methanol by resting-cell suspensions or cell extracts of Methanosarcina was reversed by coenzyme M. BES inhibition of methylcoenzyme M methylreductase activity in cell-free extracts was reversed by methylcoenzyme M but not by coenzyme M. Methanol/coenzyme M methyltransferase activity was not inhibited by 10 microM BES. Inhibition of methylreductase by BES and 3-bromopropionate was competitive with methylcoenzyme M, but inhibition by 2-bromoethanol exhibited mixed kinetics. The Ki values for the inhibitors in cell-free extracts were similar to the concentrations which inhibited intact cells. BES-resistant mutants of strain 227 were apparently permeability mutants because in vitro assays showed that mutant and parent strain methylreductases were equally sensitive to BES.     

Carbon monoxide-dependent methyl coenzyme M methylreductase in acetotrophic Methosarcina spp

Cell extracts of acetate-grown Methanosarcina strain TM-1 and Methanosarcina acetivorans both contained CH3-S-CoM methylreductase activity. The methylreductase activity was supported by CO and H2 but not by formate as electron donors. The CO-dependent activity was equivalent to the H2-dependent activity in strain TM-1 and was fivefold higher than the H2-dependent activity of M. acetivorans. When strain TM-1 was cultured on methanol, the CO-dependent activity was reduced to 5% of the activity in acetate-grown cells. Methanobacterium formicicum grown on H2-CO2 contained no CO-dependent methylreductase activity. The CO-dependent methylreductase of strain TM-1 had a pH optimum of 5.5 and a temperature optimum of 60 degrees C. The activity was stimulated by the addition of MgCl2 and ATP. Both acetate-grown strain TM-1 and acetate-grown M. acetivorans contained CO dehydrogenase activities of 9.1 and 3.8 U/mg, respectively, when assayed with methyl viologen. The CO dehydrogenase of acetate-grown cells rapidly reduced FMN and FAD, but coenzyme F420 and NADP+ were poor electron acceptors. No formate dehydrogenase was detected in either organism when grown on acetate. The results suggest that a CO-dependent CH3-S-CoM methylreductase system is involved in the pathway of the conversion of acetate to methane and that free formate is not an intermediate in the pathway.     

Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins.

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.     

Induction by Butyrate of Differentiated Properties in Cloned Murine Rhabdomyosarcoma Cells

A cell line derived from the murine rhabdomyosarcoma BW10139 (Dexter, Cancer Res. 37: 3136, 1977) was subcloned and examined with respect to growth and myogenic characteristics in the presence and absence of 1 mM butyrate. Without butyrate, these cells behave as typical transformed cells: they grow rapidly and chaotically, do not form multinucleated muscle fibers and have little or no creatine kinase activity. In the presence of 1 mM sodium butyrate or butyric acid, growth slows, cells become arranged in whorl patterns, and creatine kinase activities increase to levels comparable to those found in normal chick myoblasts immediately prior to cell fusion. The increase in creatine kinase activity is detectable within 2 h exposure to butyrate, reaches a maximum by 24 h, and the elevated level can be maintained for at least six weeks. The induction is reversible upon sequential addition, deletion, and readdition of butyrate to the culture medium. Isoenzyme analyses demonstrated that only the BB form of creatine kinease is induced; MM creatine kinase was not detected. Although formation of multinucleated cells increases after exposure to butyrate, no typical myotubes form. The results suggest that this rhabdomyosarcoma cell line can, under appropriate conditions, re-express some properties characteristic of skeletal muscle, but not the complete muscle phenotype.     

Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.