Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.Abbreviations. Hc. Halococcus - Hf. Haloferax - Hr. Halorubrum - Hb. Halobacterium An erratum to this article can be found at     

Identification of acetyl phosphate as the product of clostridial glycine reductase: Evidence for an acyl enzyme intermediate

It has been reported [Tanaka, H., & Stadtman, T. C. (1979) J. Biol. Chem. 254, 447-452] that glycine reductase from Clostridium sticklandii catalyzes the reaction glycine + ADP + P(i) + 2(e)- - acetate + ATP + NH(4)+. Glycine reductase consists of three proteins, designated A, B, and C. Only A has been purified to homogeneity. A dithiol serves as an electron donor. We find that ADP is not essential for the reaction and that in its absence acetyl phosphate is formed. Upon further purification of components B and C, an acetate kinase activity can be separated from both proteins. This observation establishes that acetate kinase activity is not an intrinsic property of glycine reductase, and therefore the reaction catalyzed by glycine reductase is glycine + P(i) + 2(e)- - acetyl phosphate + NH(4)+. Experiments with [(14)C]glycine and unlabeled acetate show that free acetate is not a precursor of acetyl phosphate. When glycine labeled with l8(O) is converted to product, l8(O) is lost. The l 8 (O) content of unreacted glycine remains unchanged after approximately 50% is converted to product. We propose that an acyl enzyme, most probably an acetyl enzyme,is an intermediate in the reaction and that the acetyl enzyme reacts with P(i) to form acetyl phosphate. A mechanism is proposed for the formation of the acetyl enzyme.     

Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay

As part of our attempt to map the impact of acetyl phosphate (acetyl approximately P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl approximately P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl approximately P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl approximately P produced no capsule under normally permissive conditions, while those that accumulated acetyl approximately P synthesized capsule under conditions previously considered to be non-permissive. Acetyl approximately P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl approximately P-deficient cells. Similarly, acetyl approximately P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl approximately P-sensitive activation of capsular biosynthesis and acetyl approximately P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl approximately P) in the transition of motile, planktonic individuals into sessile biofilm communities.     

Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase.     

Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.     

Histone acetylation in chicken erythrocytes. Rates of deacetylation in immature and mature red blood cells.

We analysed the rates of histone deacetylation in chicken mature and immature red blood cells. A multiplicity of deacetylation rates was observed for the histones and these rates may be subdivided into two major categories based on the extent of histone acetylation. In one set of experiments, cells were labelled with [3H]acetate in the presence of the deacetylase inhibitor n-butyrate, thereby accumulating radiolabel in the hyperacetylated forms of the histone. These hyperacetylated forms are deacetylated rapidly. [3H]Acetate-labelled tetra-acetylated H4 (H4Ac4) in mature cells was deacetylated with an initial half-life (t1/2) of approximately 5 min (time required for the removal of one-half of the labelled acetyl groups). In immature cells, all [3H]acetate-labelled H4Ac4 was deacetylated with a t1/2 of approximately 5 min. Erythrocytes were also labelled with [3H]acetate for extended periods in the absence of the deacetylase inhibitor. During this period, radiolabel accumulated predominantly in the mono- and di-acetylated forms of the histone. Using this protocol, the rate of deacetylation of H4Ac1 was observed to be approximately 145 min for mature cells, and approximately 90 min for immature cells, demonstrating that the less extensively acetylated histone is deacetylated slowly. These results are discussed in the context of the rates of histone acetylation in chicken red blood cells described in the companion paper [Zhang & Nelson (1988) Biochem. J. 250, 233-240].     

Ferredoxin-Dependent Conversion of Acetaldehyde to Acetate and H2 in Extracts of S Organism

S organism, an anaerobic gram-negative rod, which is one of two bacterial species isolated from the culture known as "Methanobacillus omelianskii," ferments ethanol to acetate and H(2). The present study shows that extracts of this organism contain ferredoxin and produce acetate from acetaldehyde via aldehyde: ferredoxin oxidoreductase activity. Electrons generated in the reaction are given off as H(2) by a previously demonstrated ferredoxin-linked hydrogenase system. Extracts were shown to contain good phosphotransacetylase and acetokinase activities, but no mechanism of adenosine triphosphate generation during acetaldehyde conversion to acetate could be detected. No evidence could be obtained for coenzyme A (CoASH) or phosphate requirement or for formation of acetyl CoA or acetyl phosphate.     

Regulation of acetyl coenzyme A synthetase in Escherichia coli

Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.     

Analysis of Ter-Ter enzyme kinetic mechanisms by computer simulation of isotope exchange at chemical equilibrium: development and application of ISOTER, a personal-computer-based program

A convenient, personal-computer-based program has been developed that allows simulation of isotopic exchange kinetics at chemical equilibrium catalyzed by a three reactant-three product (TerTer) enzyme system: A + B + C integral of P + Q + R. This program, ISOTER, utilizes a rapid algebraic method to calculate the exchange rate between any reactant-product pair as a function of the substrate concentration and avoids altogether the necessity of deriving an explicit (but cumbersome and impractical) equation for exchange rate. ISOTER was used to generate model saturation patterns for 16 different TerTer kinetic mechanisms, varying different combinations of reactant-product pairs in constant ratio at equilibrium: [all substrates], [A, P], [B, Q], and [C, R], while holding the nonvaried components constant. These model studies indicate that virtually every one of these mechanisms can be distinguished from the others. In addition, ISOTER has been used to fit multiple sets of experimental data for Escherichia coli glutamine synthetase, which produced a set of rate constants consistent with the previously proposed "preferred order random" kinetic mechanism.     

A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [14C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65 degrees C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use.     

Acetyl coenzyme A biosynthesis in the chloroplast

Acetyl coenzyme A (CoA) biosynthesis in spinach chloroplasts has been investigated by following the incorporation of bicarbonate and acetate into fatty acids under a variety of conditions. Both substrates were readily incorporated into fatty acids in a light-dependent manner by intact photosynthesising chloroplasts, but when the concentrations of these substrates were adjusted to those found in vivo, i.e. 200 M acetate, 10 M bicarbonate, then acetate was found to supply carbon atoms for fatty acids biosynthesis via acetyl CoA at forty times the rate of bicarbonate. It is proposed that extra-chloroplastic free acetate is the pricipal substrate for chloroplasts acetyl CoA biosynthesis in spinach.Abbreviations ACP acyl carrierprotein - CoASH coenzyme A     

Voice cells in the primate temporal lobe

Communication signals are important for social interactions and survival and are thought to receive specialized processing in the visual and auditory systems. Whereas the neural processing of faces by face clusters and face cells has been repeatedly studied [1-5], less is known about the neural representation of voice content. Recent functional magnetic resonance imaging (fMRI) studies have localized voice-preferring regions in the primate temporal lobe [6, 7], but the hemodynamic response cannot directly assess neurophysiological properties. We investigated the responses of neurons in an fMRI-identified voice cluster in awake monkeys, and here we provide the first systematic evidence for voice cells. "Voice cells" were identified, in analogy to "face cells," as neurons responding at least 2-fold stronger to conspecific voices than to "nonvoice" sounds or heterospecific voices. Importantly, whereas face clusters are thought to contain high proportions of face cells [4] responding broadly to many faces [1, 2, 4, 5, 8-10], we found that voice clusters contain moderate proportions of voice cells. Furthermore, individual voice cells exhibit high stimulus selectivity. The results reveal the neurophysiological bases for fMRI-defined voice clusters in the primate brain and highlight potential differences in how the auditory and?visual systems generate selective representations of communication signals.     

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.     

Occurrence of microbodies in the green algaBracteacoccus cinnabarinus grown heterotrophically

Summary A comparative study was made of the ultrastructure and abundance of microbodies in the green algaBracteacoccus cinnabarinus grown photoautotrophically and heterotrophically on a conventional culture medium containing sodium acetate, potassium acetate and glucose. Several changes were observed in the cells maintained under these conditions. Most noticeably, cells grown on acetate in both light and dark were packed with lipid bodies. Microbodies were found to be closely appressed to the lipid bodies in cells grown heterotrophically in the dark on sodium acetate and potassium acetate. The average number of microbody profiles per cell was in general threefold greater in cells grown on sodium acetate than those grown on potassium acetate. No microbodies were observed in cells maintained photoautotrophically on the three carbon sources or in cells maintained photoautotrophically on Bristol's inorganic medium alone. Cytochemical staining with 3,3-diaminobenzidine indicated the presence of catalase in the microbodies. The presence of microbodies suggests that the organelle may be performing functions similar to glyoxysomes in higher plants, namely the net conversion to succinate of acetyl CoA derived from lipid degredation. It is also apparent thatBracteacoccus can grow well as a heterotroph in the dark when acetate is included in the culture medium as a source of carbon.     

Environmental constraints on the inter-genus variation in the scaling relationship between leaf nitrogen and phosphorus concentrations

环境因子对叶氮、磷含量异速生长关系的属水平差异的影响 植物叶片中氮(N)、磷(P)含量的异速生长关系表明了植物对这两种元素的相对投入。而，现有的研究很少关注这一关系在分类单元之间的差异及其成因。本研究基于来自全国1733个样地，属于46个木本被子植物属的2483个叶片样品，利用异速生长方程([N] = α[P]^β)分别计算了各属的叶氮、磷含量异速生长指数(β_L)。然后利用谱系路径分析检验了这些属的气候和土壤生态位条件如何影响属间的β_L的差异。生活在贫磷土壤中的属更可能表现出更高的β_L，即相对于氮而言更强的磷积累，这可能表明了植物对磷限制的抵抗倾向。此外，各属的β_L与相对应的土壤氮、磷含量异速生长指数(β_S)正相关，这可能表明了叶养分的变化受制于作为来源的土壤养分的变化。最后，包括温度和湿度在内的气候因子不会直接影响β_L的属间变化，但可能通过调节土壤养分水平发挥间接的作用。谱系关系不会影响各属β_L随环境梯度的变化。这些结果揭示了植物对氮、磷摄取的权衡关系可能受属生态位，特别是土壤生态位的影响，表明了β_L可以作为一项反映植物养分利用特征如何响应生态位差异的功能属性。     

The ratio of acetate‐to‐glucose oxidation in astrocytes from a single 13C NMR spectrum of cerebral cortex

The ¹³C‐labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of ¹³C‐enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α‐ketoglutarate into glutamate in neurons, and incorporation of α‐ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single ¹³C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate‐to‐glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2‐¹³C]acetate and [1,6‐¹³C]glucose, and proton decoupled ¹³C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that ¹³C‐labeled acetate and glucose contributed approximately equally to acetyl‐CoA (0.96) in astrocytes. As this method relies on a single ¹³C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions.

     

Population density and regulation of cell division in 3T3 cells. I. Inorganic phosphate levels, uptake and release.

Triggering mechanisms for initiating density dependent inhibition of cell division in 3T3 cell monolayers are activated approximately two to three population doublings prior to cessation of cell division at monolayer confluency. This activation occurs at a critical contact cell density of approximately 8 X 10(3) cells/cm2. During this period there are selective controls on transport and storage of required low molecular weight nutrients. A possible correlation between orthophosphate and rates of cell division has been investigated. We have demonstrated a relationship between cellular concentrations of orthophosphate and initiation of density dependent inhibition of cell division. Prior to critical intercellular contact, the [Pi] in 3T3 is 10 mM. During critical contact, this concentration is quickly reduced to approximately 2 mM and remains at this concentration to confluency. Similar alterations do not occur in Py 3T3 cells, which maintain a concentration of approximately 2 mM Pi regardless of cell density. After confluent 3T3 cells are released from inhibition of cell division the [Pi] must increase several-fold before DNA synthesis commences. These are physiological changes in 3T3 cellular [Pi] as a function of cell density, and cannot be attributed to nutrient depletion, altered transport of Pi into the cell, increased [ATP], or increased [PPi] levels. The controlled modulation of [Pi] may regulate glycolysis and coordinate counter-ion changes (Ca++) may regulate mitochondrial activity.     

Acetyl-CoA provision and the acetyl group deficit at the onset of contraction in ischemic canine skeletal muscle

We examined the effects of increasing acetylcarnitine and acetyl-CoA availability at rest, independent of pyruvate dehydrogenase complex (PDC) activation, on energy production and tension development during the rest-to-work transition in canine skeletal muscle. We aimed to elucidate whether the lag in PDC-derived acetyl-CoA delivery toward the TCA cycle at the onset of exercise can be overcome by increasing acetyl group availability independently of PDC activation or is intimately dependent on PDC-derived acetyl-CoA. Gracilis muscle pretreated with saline or sodium acetate (360 mg/kg body mass) (both n = 6) was sampled repeatedly during 5 min of ischemic contraction. Acetate increased acetylcarnitine and acetyl-CoA availability (both P < 0.01) above control at rest and throughout contraction (P < 0.05), independently of differences in resting PDC activation between treatments. Acetate reduced oxygen-independent ATP resynthesis approximately 40% (P < 0.05) during the first minute of contraction. No difference in oxygen-independent ATP resynthesis existed between treatments from 1 to 3 min of contraction; however, energy production via this route increased approximately 25% (P < 0.05) above control in the acetate-treated group during the final 2 min of contraction. Tension development was approximately 20% greater after 5-min contraction after acetate treatment than in control (P < 0.05). In conclusion, at the immediate onset of contraction, when PDC was largely inactive, increasing cellular acetyl group availability overcame inertia in mitochondrial ATP regeneration. However, after the first minute, when PDC was near maximally activated in both groups, it appears that PDC-derived acetyl-CoA, rather than increased cellular acetyl group availability per se, dictated mitochondrial ATP resynthesis.