Extraction Of ATP Research Articles

Bioluminescent assay for measurement of bacterial attachment to polyethylene

A bioluminescent method for measuring the number of bacteria attached to synthetic polymers was developed with strains of S. epidermidis and S. aureus. The method was optimized with respect to extraction of bacterial ATP and time of incubation of synthetic polymer with bacterial strains. The attachment to polyethylene was a rapid process and a measurable amount of attached bacteria was obtained within 5 min but a standard assay required 1 h incubation to reach a constant amount of attached cells. Longer incubation did not result in an increase in the number of adhering organisms. At <10 10 CFU/ml the number of attached bacteria increased with the concentration of bacteria, while at ⩾10 10 CFU/ml the number of attached bacteria became independent of the concentration. The maximum number of attached bacteria was 2.5 × 10 7 cells/cm 2 of polyethylene. The bioluminescent method for measuring bacterial attachment correlated well with the widely used microscopic technique. Furthermore, this method was rapid, simple, time saving and more convenient than the microscopic technique and suitable for automation. In contrast to the microscopic technique a bioluminescent method for determining bacterial attachment can also be applied to a great variety of materials having irregular surfaces such as catheters and wound dressings.

Energy sources in fully aerobic rest-work transitions: a new role for glycolysis

Rate of O2 consumption (VO2), intracellular PO2, lactate extraction, and tissue contents of phosphocreatine, creatine, ATP, lactate, and pyruvate were measured during rest-work transitions in dog gracilis muscles. Samples were taken at rest and after 5, 10, 15, 30, 60, and 180 s of twitch contraction at 4/s. There was no anoxia at any time or location [companion paper, Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H914-H921, 1985]. Energy was supplied by a continuous utilization of phosphocreatine stores, a slow rise in VO2, and two distinct bursts of glycolysis. Glycolytic rate was independent of tissue PO2 and VO2 and was poorly correlated with phosphocreatine under the aerobic conditions of our experiments. The glycolytic bursts served as a source of ATP, and the bulk of the lactate formed remained in the tissue. Lactate efflux was a small fraction of tissue lactate; it depended on blood flow but not on tissue lactate content. At steady state a small glycolytic flux maintained the high tissue lactate mainly by matching rates of pyruvate production and oxidation. We propose that this flux and the high tissue lactate concentration buffer cytosolic redox and/or substrate supply in support of the aerobic functions of mitochondria during exercise.

Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin.

Microvilli isolated from intestinal epithelial cells contain a cytoskeletal Mr 110,000 polypeptide complexed with calmodulin (110K-CM) that is believed to link the microfilament core bundle laterally to the plasma membrane. Previous work has shown that physiological levels of ATP can partially solubilize the 110K-CM complex from isolated microvillus cytoskeletons or isolated microvilli. However, once extracted, the 110K-CM complex has been found to be difficult to maintain stably soluble in aqueous buffers. This is due to the presence of an endogenous ATPase (approximately 100 nmol Pi/min per mg at 37 degrees C) in microvillus cytoskeletal preparations that depletes the ATP with subsequent precipitation of 110K-CM. Addition of ATP to such precipitates resolubilizes 110K-CM. Inclusion of an ATP regenerating system in the solubilization of 110K-CM from cytoskeletons, or membrane-bound brush borders, increases the amount of 110K-CM solubilized. Solubilization of 110K-CM from microvillus cytoskeletons was found to require a divalent cation (Mg2+, Mn2+, or Co2+, but not Zn2+) and a nucleoside triphosphate (ATP, GTP, CTP, or ITP). ADP did not solubilize 110K-CM, but could partially inhibit ATP-dependent solubilization. Solubilized 110K was phosphorylated during extraction of microvillus cores with [gamma-32P]ATP, but this was unrelated to the solubilization of 110K-CM as the endogenous kinase was specific for ATP, whereas the solubilization was not. The 110K-CM was purified using ATP extraction of brush border cytoskeletons in the presence of an ATP regenerating system, gel filtration of the solubilized extract, an ATP depletion step to specifically precipitate 110K-CM with F-actin, and resolubilization followed by phosphocellulose chromatography. The purified complex was stably soluble in aqueous buffers both in the presence and absence of ATP. It bound almost quantitatively to F-actin in the absence of ATP, and showed nucleotide solubilization characteristics from F-actin similar to that found for solubilization of 110K-CM from microvillus cores. At low ATP levels, the binding to F-actin was increased in the presence of ADP. These results suggest that the purified complex has been isolated in a native form. The data confirm and extend the studies of Howe and Mooseker (1983, J. Cell Biol., 97:974-985) using a partially purified preparation of 110K-CM and further emphasize that 110K-CM is a stably water soluble complex and not an integral membrane protein.

Biochemical evidence for the presence of an actin protein in Tetrahymena pyriformis.

A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyriformis GL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecyl sulphate (SDS)/10% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of 47 500 (47.5 X 10(3) Mr). Partial proteolysis of this band with Staphylococcus aureus V-8 protease followed by electrophoresis revealed a pattern of peptides in which at least four peptides were similar to those observed after digestion of rabbit skeletal muscle actin. The 47.5 X 10(3) Mr protein appeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited by leupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column and a distinct peak was eluted with 3 M-guanidine . HCl; the DNase I-binding protein appeared as a distinct band on SDS/PAGE with an apparent molecular weight of 47.5 X 10(3) Mr. In the absence of leupeptin, the DNase I-binding protein appeared as a broad 34 X 10(3) Mr band on gels. Both the ATP extract and the DNase I-binding protein showed reactivity with commercially available antiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linked immunosorbance assay (ELISA). Immuno-blotting studies and affinity purification of this antiserum showed that the recognition was not specific to the 47.5 X 10(3) Mr protein. However, using affinity-purified anti-actin antibodies raised against denatured actin from chick smooth muscle, recognition of the 47.5 X 10(3) Mr protein and a 34 X 10(3) Mr protein was shown. In negatively stained preparations from an ATP extract after two cycles of polymerization and depolymerization there were filaments, 8-12 nm diameter, which did not decorate with subfragment S-1 of myosin, but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicated an intensely stained 54 X 10(3) Mr band. It is suggested that, in vitro, Tetrahymena intermediate filaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahymena there is a protein that resembles actin in its extractability, molecular weight, peptide pattern after partial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However, this protein did not assemble into actin filaments in crude extracts.

Atp-measurements in soil: A combination between the TCA and NRB ® extraction methods

The extraction of ATP from soils with the TCA- and NRB ®-methods shows qualitative and quantitative differences. A combination of both methods results in a higher yield of extracted ATP and in a solution of low optical quenching.

Establishment of denitrification capacity in soil: Effects of carbon, nitrate and moisture

The effects of moisture, NO 3 −1 concentration and C addition on changes in denitrification capacity and total microbial biomass in a clay loam soil were investigated. Denitrification capacity was evaluated with an anaerobic slurry technique. Total microbial biomass was measured by CHC1 3 fumigation and by extraction of microbial ATP. The results indicate that denitrification capacity and total microbial biomass were increased only by the C addition; differences in NO 3 −1 concentration and moisture had no effect in this agricultural soil. The increase in denitrification capacity could be attributed solely to microbial growth, since the ratio of denitrification capacity to total microbial biomass remained constant and the increased respiration from the C amendment did not increase anaerobiosis. The results also show that denitrifiers compete as effectively for added C as do other heterotrophs.

Determination of Microbial Cell Numbers in Subsurface Samples

ABSTRACTGround‐water pollution by organic compounds has become a major environmental concern. Because the transport and fate of the organic pollutants may be influenced by microorganisms present in subsurface material, reliable measurements of the number of organisms in subsurface samples and their metabolic activity are needed. A special drilling rig and aseptic procedures have been developed by the Robert S. Kerr Environmental Research Laboratory of the United States Environmental Protection Agency to yield uncontaminated subsurface samples. The number of bacteria in subsurface samples has been determined by microscopic counting after acridine orange staining; the proportion of cells capable of respiration was determined by INT reduction. An independent measure of metabolic activity was obtained by measuring ATP extracted from the samples. A procedure and extradant for the extraction of ATP from subsurface material have been developed. The extractant contains reagents to reduce the loss of the extracted ATP. Subsurface samples from Oklahoma and Texas contain 106‐107 cells per g of subsurface material (depths of 2–9 m). Both methods show that usually between 1 and 10% of the cells were metabolically active. Thus, significant numbers of metabolically active bacteria exist in subsurface material with the potential to modify pollutants.

The action of 2-deoxy-D-glucose on the incorporation of glucose into (1----3)-beta-glucan in stationary phase cultures of Candida albicans.

2-Deoxy-D-glucose added to cultures of Candida albicans in the stationary phase of growth inhibited the incorporation of glucose into the (1----3)-beta-glucan fraction of the organisms. In the presence of ATP and cell extracts it was converted to 2-deoxy-D-glucose phosphate and when UTP was also present, material with the electrophoretic properties of UDP-2-deoxy-D-glucose was formed. In similar conditions glucose formed glucose phosphates, UDP-glucose and other products. Evidence was obtained that the analogue, after conversion to a phosphate derivative, was an inhibitor of phosphoglucomutase. When C. albicans was grown in the presence of 2-deoxy-D-glucose for 24 h, analogue residues became incorporated into the (1----3)-beta-glucan fraction and the subsequent rate of incorporation of glucose into that fraction was enhanced. The rate of turnover of glucose in this beta-glucan fraction was greater than in controls. Pretreatment of cultures with beta-glucanase, or incubation under conditions known to stimulate endogenous beta-glucanases, increased the subsequent rate of glucose incorporation and this increase was enhanced by growth in the presence of 2-deoxy-D-glucose. The analogue thus had the effect of altering the stability and glucose-acceptor function of (1----3)-beta-glucan chains. This could affect the properties of the polymer network leading to the known effect of the analogue in delaying the onset of phenotypic resistance to amphotericin methyl ester in stationary phase cultures of C. albicans.

Extraction of Algal ATP and Interpretation of Measurements

We compared ATP samples collected by direct injection with those collected by filtration from exponential and P-limited algal cultures, and from summer populations of several lakes. The direct injection method yielded higher amounts of measurable ATP than did filtration on 2.5- and 4.5-cm diameter membrane filters. ATP lost to the filtrate was negligible. Variation in ATP content with increasing nutrient deficiency was measured on six cultures. ATP/C decreased with N deficiency and to a greater degree with P deficiency. Large changes of ATP/C usually occurred with little change in viability of the cells. Short-term (1 h) nutrient enrichments generally did not change the ATP/C ratio. Phosphorus deficiency tended to increase the chlorophyll/ATP ratio and N deficiency to decrease it. We found that the short- and long-term effects of alkaline phosphatase on ATP extracts from P-sufficient and P-deficient Scenedesmus quadricauda were negligible. There appeared to be little effect on frozen ATP samples stored up to 60 d. Any short-term effects were similarly negligible, since temperature and the addition of P did not affect the rate of ATP loss. The use of 0.33 mol H3PO4∙L−1 as an extractant proved to be completely unreliable.

Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs.

Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein.

Firefly luciferase ATP assay as a screening method for bacteriuria

A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding greater than 10(5) CFU/ml and specimens yielding less than 10(5) CFU/ml. A level of 13.5 nM ATP was chosen to define the limit between negative and positive results. At this discriminatory level, 92% of specimens yielding greater than 10(5) CFU/ml and 88% of specimens yielding less than 10(5) CFU/ml were correctly classified with the luciferase method, whereas corresponding figures for the nitrite test were 55 and 99%, respectively. Of the 12% false luciferase positives, 20% were shown to contain greater than 10(5) CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 10(5) to 10(6).

ATP as a short term bioassay response parameter for nitrogen deficiency in Algae

The effects of nitrate and ammonium additions on the ATP contents of eight axenically cultured species of nitrogen deficient algae were studied to determine the reliability of short term (4 h) ATP responses in identifying nitrogen deficiency in algal populations. Addition of either nitrate or ammonium to nitrogen deficient cultures significantly increased cellular ATP content (20–30%) in all bioassays. The magnitude of the response was not affected by the source of nitrogen used. Increases in ATP content were also observed following the addition of ammonium to natural assemblages of phytoplankton. The magnitude of the response declined when samples were concentrated onto membrane filters prior to the extraction of ATP.

Adenoviral protein-primed initiation of DNA chains in vitro.

The initiation of DNA chains by the 80-kilodalton form of the adenovirus terminal protein has been studied. This protein, which can be covalently linked to dCMP, is isolated complexed to a 140-kilodalton protein possessing DNA polymerase activity. In the presence of adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex requires the addition of ATP and nuclear extract from uninfected cells in addition to Mg2+ and dCTP. When single-stranded DNA is used in place of the adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex occurs in the absence of ATP and nuclear extract. In the presence of the four dNTPs, the complex yields DNA chains of various sizes between 100 and 300 nucleotides. The products formed with bacteriophage phi X174 single-stranded circular DNA as the template are site specific, predominantly derived from the sequences between nucleotides 2363 and 2977 and between nucleotides 3760 and 4206. These small dNA chains are blocked at their 5' ends with the 80-kilodalton protein but possess free 3'-OH ends that are susceptible to degradation by exonuclease III and can be elongated to replicative form II products with DNA polymerase I of Escherichia coli or eukaryotic DNA polymerase beta preparations. A protein priming model explaining the different requirements for initiation with adenovirus DNA-protein and with phi X174 DNA is presented.

Continuous flow method for extraction and bioluminescence assay of ATP in baker's yeast

The intracellular ATP of baker's yeast (Saccharomyces cerevisiae) was measured using the bioluminescent firefly luciferase assay. Benzalkonium chloride and trichloro-acetic acid served in the experiments as extracting agents and optimal conditions for the extraction and assay of the intracellular ATP are reported. Using the results obtained from manually performed experiments two continuous flow systems were designed for the measurement of ATP in yeast cells during cell growth. Good correlation between the amount of cellular ATP and cell growth was found during the exponential growth phase.

Bioluminescent Determination of ATP in Ischemic Rat Heart

Abstract Two different ATP extraction methods, one using perchloirc acid and the other boiling Tris bufer, for ATP determinations in myocardial samples werer investigated and compared. A bioluminescent assay was used to quantify the extracted ATP. In spite of some drawbacks, eprchloric acid extraction is zix times more efficient thatn boiling Tris buffer extraction for releasing ATP from myocardial tissue. The perchloric acid extraction was used to determine ATP depletion in the endocardium of isolated ischemic rat hearts.

植物プランクトンのＡＴＰの測定方法に関する検討

Measuring method of phytoplankton ATP was experimentally investigated using a suspension of Scenedesmus dimorphus. Boiling glycine buffer including Mg-EDTA (O.O1M and 0.005M respectively, and pH 10.0) was found to be more suitable as an ATP extractant and also as a buffer solution to prepare ATP standard solution than boiling Tris buffer (0.025M, pH 7.75). ATP was stable in glycine buffer inculding Mg-EDTA for sixty-eight days at any selected storage temperature, whereas ATP was hydrolyzed in Tris buffer to some extent depending upon storage temperature and storage period. The optimum condition for ATP extraction from phytoplankton using glycine buffer including Mg-EDTA was identified as follows; heating system: test tube-block heater, extraction temperature (block heater temperature): 120°C, and extraction period: one minute. The results of the application study of the method proposed above are also described.

Critical problems with extraction of ATP for bioluminescence assay of plankton biomass

Recovery of ATP by boiling tris extraction was 90–95 percent greater in 1 liter grab samples than in concentrated net samples. ATP losses were attributed to insulating effects promoted by accumulation of detritus on filters. A series of extractions over a concentration range of whole or size-segregated plankters and cultured algae was made to determine volume of water to be filtered for optimum extraction efficiency. Accuracy of ATP assays was optimized by: (i) using large diameter (i.e. 47 mm) acetate filters; (2) limiting sample volume filtered to 50 ml when particulate organic carbon (POC) exceeded 0.4 mg l−1; and (3) performing extractions in boiling tris maintained initially on a laboratory hot plate at 400°C as opposed to hot water bath at 100°C.

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

AbstractThe importance of having a rapid method for determining the viable biomass in activated‐sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out‐of‐coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated‐sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed‐liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.

Influence of cation content on adenosine triphosphate determinations in soil

Adenosine triphosphate in soil is measured by the luciferin-luciferase bioluminescence method. Cation exchange in the ATP extraction procedure is used to reduce the content of, e.g., Fe, Al, and Cations in extracts. The efficiency of the cation exchange is determined by measuring the concentration of Fe by X-ray fluorescence spectrometry. The time dependence of the light emission from the bioluminescence process is investigated, and a method for correlating the ATP content and the light emission is proposed.

A Firefly Bioluminescence ATP Assay Method for Rapid Detection and Enumeration of Brewery Microorganisms

A rapid, sensitive firefly bioluminescence ATP assay using a liquid scintillation counter for measurement of assay light output is described. The use of partially purified luciferase and synthetic D-luciferin in this assay permits the detection of as little as 10−16 moles ATP and as few as 10 yeast cells. The assay light output is directly proportional to the quantity of ATP from at least 10 to 104 fmol and is on scale throughout this range. The method developed for detection and enumeration of viable microorganisms, which requires only a few minutes per sample, involves extraction of the cellular ATP with DMSO at room temperature and estimation of the ATP by the bioluminescence assay—for dilute suspensions, prior concentration and isolation of the cells by membrane filtration is necessary. The ATP contents of a selection of yeast and bacteria determined using this technique are reported. A linear relation between quantity of ATP and number of yeast cells is demonstrated, and the changes in intracellular yeast ATP pools during fermentation of wort by ale and lager yeast are presented. A number of other possible applications of this ATP assay in brewing research, development, and microbiological quality control are evaluated and/or discussed.