Occurrence and preservation of ATP in Antarctic rocks and its implications in biomass determinations

The low temperature and moisture availability in Antarctic rocks would seem to preclude the existence of any sizeable algal biomass. The very high levels of ATP associated with endplithic algal communities have led to speculation that the measured ATP largely represents accumulated exogenous material as well as living biomass. A method was developed to selectively hydrolyze free, extracellular ATP without affecting intracellular ATP. Use of this technique on Antarctic rock samples has led to the conclusion that the ATP found in endolithic communities exclusively reflects living material and not preserved, free ATP.

From these measurements we concluded that the ATP biomass in a gram of rock is equivalent to that found in more temperate and favorable environments.     

The energy budget of Tetrahymena and the material fluxes into and out of the adenylate pool

The material budget of the adenylate pool deals with all processes which physically establish and maintain this pool, while the energy budget is concerned with the intracompartmental ATP recycling. Both budgets were analysed in Tetrahymena thermophila exposed to various energy and material demands. Some of the general conclusions are: at a maximum growth rate the overall ATP consumption during one cell cycle is 10(-10) mol ATP; the contribution of osmoregulation and ciliary motion to the budget is about 1% each; at zero net growth, energy is consumed because of a continuous recycling of matter between the monomer and the polymer compartment. The rate of ATP production is about 1000-fold greater than the rate of adenylate monomer influx. The residence time of adenylate monomers within the pool is about 30 min, but for ATP molecules it is only 2 sec.     

Responses of adenine nucleotides in germinating soybean embryonic axes to exogenously applied adenine and adenosine

The ATP content of soybean (Glycine max [L.] Merr. cv. Kent) axes incubated for 3 hours in 1 mm solutions of adenine and adenosine increased over 100% and 75%, respectively, over axes incubated in water. The increase in ATP was primarily due to the conversion of these purines to nucleotides via the nucleotide salvage pathway. The ATP formed was in a metabolically active pool because label from adenine was incorporated into acid-insoluble material. Adenine also increased the levels of GTP, UTP, and CTP, but not to the extent of the ATP level.     

固定化细胞法在5'-三磷酸腺苷合成中的应用

从生物催化活性细胞、细胞的固定化形式、固定化细胞反应器3个方面对固定化细胞法合成5’-三磷酸腺苷(ATP)的研究情况作了扼要综述。分析了我国ATP生产的工业化现状,并对今后的研究和开发提出了建议。     

Optimization of glycolysis: new discussions

The concept of glycolysis optimization, first presented in Waddell (Biochemical Education 25 (1997) 204–205) and Heinrich et al. (Eur. J. Biochem. 243 (1997) 191–201), is reiterated and clarified in response to questions by Christoffersen et al. (Biochemical Education 26 (1998) 290–291). Indeed, increasing the ATP yield above 2ATP/glucose would lead to a decrease in the ATP production rate and a lower flux of material toward lactate.     

Observations on an ATP-sensitive protein system from the plasmodia of a myxomycete

1. Extracts of the plasmodia of the myxomycete, Physarum polycephalum, exhibit reversible decreases in viscosity in response to the addition of ATP under appropriate conditions. The protoplasm material prepared by extraction with KCl solution can apparently exist in either a high or a low viscosity state. As prepared, it is in the low viscosity condition. Rapid and extensive increases in viscosity of the extract are brought about by addition of AMP, inorganic phosphate, or, under certain conditions, of ATP. Only after the high viscosity state has been attained does addition of appropriate quantities of ATP cause a reversible decrease in viscosity. 2. The active principle of crude plasmodial extracts may be concentrated by fractional precipitation with ammonium sulfate and is found in the fraction precipitated between 30 and 40 per cent saturation. This material possesses a higher viscosity than does the original crude extract and is apparently in the high viscosity state since the addition of ATP causes an immediate reversible decrease in viscosity. 3. The ATP-sensitive fraction of myxomycete plasmodia possesses a viscosity which is dependent upon its previous thermal treatment. Extracts incubated at 0° for a period of a few hours increase greatly in viscosity when they are returned to 24.5°. This increased viscosity is structural in nature, is destroyed by mechanical agitation of the solution, and may be reversibly destroyed by addition of ATP. 4. It is suggested that the ATP-responsive protein of myxomycete plasmodia may be related to sol-gel transformations which have been observed in intact plasmodia and may participate in the protoplasmic streaming of the intact organism. This suggestion is based upon the following facts: (a) the protoplasmic streaming of myxomycete plasmodia is increased by microinjection of ATP; (b) the gel portion of the cytoplasm at the site of the microinjection of ATP is extensively converted to the sol state. The changes in structure of the intact cytoplasm are thus similar in nature to the changes exhibited in response to ATP by the purified ATP-sensitive protein. 5. The ATP-sensitive protein of myxomycete plasmodia appears to undergo reversible aggregation to form a high viscosity state. The function of ATP is to break down the aggregates thus formed. Since a specific ATPase activity is associated with the purified material, added ATP is gradually destroyed and recovery of viscosity attends the spontaneous reconstitution of aggregates.     

Use of adenosine 5'-triphosphate as an indicator of the microbiota biomass in rumen contents

A number of techniques were tested for their efficiency in extracting adenosine 5'-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H(2)SO(4), using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 mug of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in "cell-free" rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 mug of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.     

The responses of osteoblasts to fluid shear stress depend on substrate chemistries

Natural bone tissue receives chemical and mechanical stimuli in physiological environment. The effects of material chemistry alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the synergistic influences of material chemistry and flow shear stress (FSS) on biological functions of osteoblasts. Self-assembled monolayers (SAMs) on glass slides with functional groups of OH, CH₃, and NH₂ were employed to provide various material chemistries, while FSS (12 dynes/cm²) was produced by a parallel-plate fluid flow system. Material chemistry alone had no obvious effects on the expressions of ATP, nitric oxide (NO), and prostaglandin E₂ (PGE₂), whereas FSS stimuli alone increased the production of those items. When both material chemistry and FSS were loaded, cell proliferation and the expressions of ATP, NO and PGE₂ were highly dependent on the material chemistry. Examination of the focal adhesion (FA) formation and F-actin organization of osteoblasts before FSS exposure indicates that the FA formation and F-actin organization followed similar chemistry-dependence. The inhibition of FAs and/or disruption of F-actins eliminated the material dependence of FSS-induced ATP, PGE₂ and NO release. A possible mechanism is proposed: material chemistry controls the F-actin organization and FA formation of osteoblasts, which further modulates FSS-induced cellular responses.     

The isolation of myxomyosin,an ATP-sensitive protein from the plasmodium of a myxomycete

1. A procedure has been developed for the preparation of an active concentrate from the myxomycete, Physarum polycephalum. This concentrate responds with a lowered viscosity to the addition of small amounts of ATP. The preparation recovers in viscosity, and the process may be repeated. 2. In the most active concentrates, 75 per cent of the non-dialyzable material moves as a single boundary both in the descending limb in electrophoresis and in the ultracentrifuge. It contains about 10 per cent ribonucleic acid, which is at least in part reversibly bound to the protein. 3. The active material has been designated myxomyosin because of its origin and its similarity to actomyosin in ATP response.     

Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I.

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.     

Use of Adenosine 5′-Triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents

A number of techniques were tested for their efficiency in extracting adenosine 5′-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H₂SO₄, using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 μg of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in “cell-free” rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 μg of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Are growth rates of Escherichia coli in batch cultures limited by respiration?

Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.     

Externalization of membrane-bound activities during sheep reticulocyte maturation is temperature and ATP dependent

During the maturation of sheep reticulocytes in vitro, there is release of material that can be pelleted from the cell-free incubation medium by centrifugation at 100,000 X g. This pellet contains activities that are derived from both the plasma membrane and lysosomes. No evidence was obtained for the presence of mitochondrial activities or cytosolic enzyme activities. The release of these activities is ATP and temperature dependent, since reduction of either results in a greater retention of the activities by the cells and a lesser amount in the 100,000 X g pellet. The pelleted material is vesicular in nature, and the production and (or) release of the material are reduced upon ATP depletion or lowering of the temperature. It is concluded that the externalization of specific membrane components is a normal metabolic process that occurs during reticulocyte maturation and represents a means by which reticulocytes shed specific types of membrane-associated functions that are known to decrease during reticulocytes maturation.     

Phosphorylation of ATP citrate lyase in response to glucagon.

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.     

Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network

A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (<27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. (c) 1996 John Wiley & Sons, Inc.     

A dynein ATPase inhibitor isolated from a commercial ATP preparation.

Preparations of ATP from equine muscle contained an inhibitor of dynein Mg2+-activated ATPase. The inhibitory material was separated from the ATP by molecular sieve filtration. The several molecular species of dynein extracted from three different axonemal sources were all inhibited; myosin ATPase was not. With increasing amounts of inhibitor the inhibition did not go to completion but reached a plateau when the rate had been reduced to 1/5 the uninhibited rate. A plot of 1/[S] against 1/v at several inhibitor concentrations yielded parallel lines. There was little inhibition of dynein ATPase when Mg2+ was replaced by Ca2+. The inhibitor appeared slightly smaller in molecular size than ATP, had anionic character, and was not adsorbed to charcoal.     

Synthesis of an oligonucleotide inhibitor of protein synthesis in rabbit reticulocyte lysates analogous to that formed in extracts from interferon-treated cells.

A heat-stable, low-molecular-weight inhibitor of protein synthesis is formed on incubation of haemin-supplemented rabbit reticulocyte lysates with ATP and double-stranded RNA (dsRNA). It inhibits the translation of both added encephalomyocarditis virus RNA (EMC RNA) and endogeneous messenger RNA in reticulocyte lysates and mouse L-cell extracts. The enzyme responsible for the synthesis of the inhibitor binds to dsRNA and can be purified on a column of poly(I).poly (C) bound to an inert support. The highly purified enzyme in its stable column-bound state can be conveniently employed to synthesise the inhibitor and to label it with [3H]ATP, or [alpha-32P]ATP or [gamma-32P]ATP as substrate. The radioactive inhibitor synthesised in this way with material from rabbit reticulocyte lysates shows the same spectrum of resistance and sensitivity to alkali and a variety of enzymes as corresponding material similarly synthesised with extracts from interferon-treated mouse L-cells. The inhibitors from the two systems have comparable absorbance spectra, are chromatographically and electrophoretically indistinguishable and are apparently identical in specific activity in the inhibition of protein synthesis in the cell-free system. The inhibitor is also formed on inhibition of protein synthesis by dsRNA in reticulocyte lysates. On comparison of the spectrum of polypeptide products synthesised in response to EMC RNA in the reticulocyte lysate, the effects of the inhibitor or dsRNA were similar: a distinctly different effect was obtained with the haemin-controlled repressor, a known inhibitor of initiation. The significance of these results with respect to the mechanism of action of the inhibitor and its role in the inhibition observed in response to dsRNA is discussed.     

An ATP-stimulated factor that enhances the nuclear binding of "activated" receptor-glucocorticoid complex

A macromolecular material that enhances the translocation, or binding, of already "activated" receptor-glucocorticoid complex to nuclei in the presence of 5 mM ATP was separated from the cytosol of rat liver by DEAE-cellulose column chromatography with about 0.025 M NaCl. The molecular weight of the material was about 93,000 +/- 4,900, as determined by agarose gel filtration. After incubation at 60 degrees C for 15 min, this material still had activity to increase the nuclear binding, but on boiling for 15 min it lost its activity.     

Balance of production and consumption of ATP in ammonium-starved Saccharomyces cerevisiae

To establish a balance between the ATP produced in catabolism and the ATP consumed in net biosynthesis of cellular components the energy metabolism of Saccharomyces cerevisiae utilizing glucose in the absence of a nitrogen source (resting cells) was studied. The following results were obtained. (i) Cell number and biomass increased 2- and 2.5-fold, respectively, during the first 8 h of ammonium starvation. After this period, both values remained constant. (ii) The rate of sugar consumption and ATP production decreased with the duration of starvation to about 20% of the original in 24 h. (iii) About 60% of the sugar consumed was fermented to ethanol and about 10% assimilated as cellular material. Of the assimilated sugar, as much as 80% was accumulated as carbohydrate. (iv) Only 15% of the total ATP produced in catabolism seems to be consumed in net biosynthesis and maintenance of intracellular pH. The fate of the remaining 85% is unknown.