Extraction Of ATP Research Articles

Validation of a rapid, large-scale assay to quantify ATP concentration in spermatozoa

Quantification of ATP content in spermatozoa is a useful assay for evaluating sperm function; however, most detection methodology relies on assessing single samples. We have developed and validated a highly repeatable assay that permits simultaneous measurement of up to 78 samples. A key feature of this assay includes combination of a phosphatase inhibition and ATP extraction step that permits maximal detection of ATP and sample storage at −20 °C prior to assay. The assay was validated for spermatozoa from three different species, including turkey, rooster and boar. The sensitivity of the assay differed between avian and mammalian spermatozoa, with 2.5 × 10 6 spermatozoa being the lowest number of turkey and rooster spermatozoa that could be assayed compared to 2.5 × 10 5 boar spermatozoa. Concentrations of ATP in fresh turkey semen ranged from 2.14 to 15.6 nmol/10 9 spermatozoa; similarly, freshly collected rooster semen contained from 2.16 to 21.4 nmol ATP/10 9 spermatozoa. Evaluation of turkey semen that had been stored at 4 °C for 24 h revealed a decline in ATP concentrations (2.35 ± 0.34 nmol ATP/10 9 spermatozoa). Likewise, cryopreserved rooster spermatozoa contained lower concentrations of ATP (0.05 ± 0.01 nmol ATP/10 9 spermatozoa) than non-stored spermatozoa. Boar spermatozoa contained similar concentrations of ATP, whether fresh (74.2 ± 8.1 pmol ATP/10 6 spermatozoa), stored for 1 day (77.0 ± 8.1 pmol ATP/10 6 spermatozoa) or 5 days (81.96 ± 8.1 pmol ATP/10 6 spermatozoa). For all three species, assay variation was low (inter-assay, 0.66–1.9% CV; intra-assay, 1.3% CV).

Detection of fungi and control of disinfection by ATP-bioluminescence assay

Mould contamination in the indoor climate has been recognised in recent years as a major concern. In addition to damaging works of art, moulds also can be health hazards. Nowadays, the conventional method to detect and identify such micro-organisms is to put them in culture. Unfortunately, such a method is laborious and time-consuming. Bioluminescence, more particularly ATP methodology, has been widely applied in domains such as medicine, food quality control, environmental protection and pharmacology. Its success on bacteria and yeast has been demonstrated. Many commercial reagent kits are available and we tested some of them. Our early experiments have shown that the use of these kits did not give satisfactory results on mould, reasons being that the provided extraction buffer does not allow a proper extraction of ATP from the fungi’s cells. Therefore new reliable, simple and rapid extraction strategies specific to fungi are required. We compared 40 types of extraction protocols. Extraction with 90% of DMSO in Tris-acetate buffer at 100°C seems to be the best. The viability of cells is estimated by the adenylate energy charge (EC), which corresponds to the equilibrium value of the metabolic energy between the three forms of adenine-containing nucleotides (AT, ADP and AMP) . We applied our method on well-known species such as Aspergillus flavus, Aspergillus niger, Neosartorya fischeri, Fusarium solani, Eurotium chevalieri, Penicillium chrysogenum and Chaetomium globosum. The results presented here are related to species taken both individually and in a mixture. In order to demonstrate and validate our method, we treated each species with steam and ethylene oxide. The results show that the EC of viable cells is greater than 0.80; the EC of killed cells treated with steam is less than 0.50. We observed that the behaviour of cells treated with ethylene oxide is complex. For certain strains, the value of the EC varies between 0.5 and 0.8 according to the disinfection centre. So, our method can be used to rapidly monitor the efficiency of a process of disinfection with ethylene oxide. It has also been used on documents featuring suspicious stains. The results have been compared to conventional methods. It showed that the ATP technique provides a very simple and fast method.

Enhanced Firefly Bioluminescence Assay of ATP in the Presence of ATP Extractants by Using Diethylaminoethyl-Dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml−1 of E. coli.

Estimation of ATP in soil: extraction methods and calculation of extraction efficiency

Methodological problems still exist when soil ATP is estimated using a specific extractant in different laboratories or when different extractants are compared in one laboratory. I hypothesised that this may possibly be caused by uncertainties inherent in addition of an ATP internal standard. Therefore, the reliability of this method was tested using 14 agricultural soils. I chose the TCA/HPO42−/paraquat extractant with sonification (Jenkinson and Oades, 1979) to test whether the recovery of an ATP standard added to the extractant represents the true loss of ATP during extraction process. For this purpose soils were repeatedly extracted with fresh extractant, until the amount of ATP reached the limit of detection in the luciferin–luciferase system. This was reached at the latest after six extraction steps. The summed ATP contents obtained in the six extracts were compared with the amount of ATP as estimated by the conventional way of a single extraction and correction for recovery of an added ATP standard. It was found that the latter procedure considerably underestimated the amount of soil ATP, because the recovery of standards underestimated losses. The ATP contents in the extracts 2–6 during the six-fold extraction procedure were by a factor of 2–9 higher than indicated by losses of added ATP standards after a single extraction. The amount of standard added to the extraxtant did not influence its recovery. It was shown that the application of fresh extractant is the decisive step for obtaining higher ATP values by multiple extraction and not a prolonged sonification. Since a repeated extraction of a soil sample is too time- and labour-consuming for practical application, a more convenient and efficient technique was required. Extraction with dimethylsufoxide (DMSO), Na3PO4 (pH 11.7) and NRB® as proposed by Bai et al. (1988) was found to be rapid and efficient. This extractant gave nearly identical ATP contents of soils as did the repeated extraction with the TCA/HPO42−/paraquat extractant. A useful indicator of ATP extraction efficiency is the biomass C–ATP ratio which was 208–217 for all soils and both methods. This good agreement between both methods, and the use of non toxic chemicals encourage me to propose the DMSO based extraction procedure as a suitable technique to estimate soil ATP in agricultural soils.

Assessment of ionic quenching on soil ATP bioluminescence reaction

Co-extracted ions with ATP from soils may interfere with ATP luciferin–luciferase luminescence reaction when ATP is assayed. The effects were investigated in a typical concentration range of cations and anions potentially extractable in soils. A commercial ATP assay product (Sigma Chemical Co.) was used. Significant quenching is evidenced from a concentration of 0.10 mM with Cu 2+ and Zn 2+, and 1.00 mM with Ca 2+. The order of quenching at 1.00 mM was: Cu 2+>Zn 2+>Ca 2+=Na +=Mg 2+, while Mg 2+=Mn 2+, both Ca 2+ and Na +>Mn 2+. The quenching was found to be much more severe with selected special heavy metal cations with quenching in the order: Ti 3+>Hg 2+>Cr 3+. Because cation quenching can be alleviated by addition of EDTA, three forms of EDTA (Mg, Na and acid EDTA) were tested for their suitability for the assay. The Mg-EDTA was found superior to the other two. Presence of PO 4 3− at concentrations of 0.01 and 0.05 mM, and NO 3 − at 0.01 and 0.10 mM, significantly enhanced ATP light emission (8–13%). However, SO 4 2− at similar concentrations significantly decreased light emission. The quenching by CO 3 2− and Cl − was only observed at high concentrations (3.20 mM and up). The order of quenching for the anions at a concentration of 6.4 mM was: PO 4 3−>CO 3 2−>SO 4 2−>NO 3 −>Cl −. Enhanced or depressed light emission induced by ions would produce significant over or underestimation of soil ATP. While addition of Mg-EDTA may alleviate cation quenching, the interference from anions may require the ATP assay standards be prepared in a solution of similar chemical composition to that in soil ATP extracts.

Improved extraction and assay of mycobacterial ATP for rapid drug susceptibility testing.

The firefly luciferase assay of ATP is a rapid and convenient technique for monitoring growth of mycobacteria. The time needed to obtain a drug susceptibility pattern can be reduced to less than 1 week as compared to 4 weeks with conventional methods. The ATP assay is simple and reliable. However, the extraction of bacterial ATP is not a trivial problem. Lysing the cells will immediately activate ATP-degrading enzyme systems. The extractant must therefore lyse the cells and simultaneously inactivate ATP-degrading enzyme systems. Only by comparing the ATP yields obtained with different extractants we will know something about the intracellular ATP level. In the present study various extractants were compared for the extraction of ATP from Mycobacterium bovis (BCG) cultures. Dodecyl trimethyl ammonium bromide (DTAB) in Tris-buffer with EDTA resulted at 100 degrees C in an ATP yield that was approximately twice as high as the same buffer without DTAB. The optimum temperature was 80-100 degrees C. With the optimized extraction procedure the coefficient of variation for the entire assay of ATP in BCG cultures was 5%. The analytical interference from DTAB with the firefly reaction was obviated by neutralization with alpha-cyclodextrin, making it possible to increase the sensitivity by assaying 0.5 mL rather than 0.01 mL extract.

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.

Identification of Escherichia coli clinical isolates producing macrolide 2′-phosphotransferase by a highly sensitive detection method

Macrolide is inactivated with ATP plus crude extract of Escherichia coli producing macrolide 2′-phosphotransferase (MPH(2′)), but not by living cells. Therefore, a convenient method for detection of MPH(2′) using intact cells is needed. In this report, we determine that the modified lysozyme-DNase-RNase (LDR) method (named ELDR method) is at least one hundred times more sensitive for the detection of MPH(2′) activity than the LDR method and, in addition, highly sensitive for the detection of aminoglycoside-modifying enzymes. Therefore, three new MPH(2′)-producing strains were found in clinically isolated E. coli in Japan in 1997 by this method. It suggests that MPH(2′)-producing E. coli have been spread in Japanese clinical fields.

Identification of Escherichia coli clinical isolates producing macrolide 2'-phosphotransferase by a highly sensitive detection method.

Macrolide is inactivated with ATP plus crude extract of Escherichia coli producing macrolide 2'-phosphotransferase (MPH(2')), but not by living cells. Therefore, a convenient method for detection of MPH(2') using intact cells is needed. In this report, we determine that the modified lysozyme-DNase-RNase (LDR) method (named ELDR method) is at least one hundred times more sensitive for the detection of MPH(2') activity than the LDR method and, in addition, highly sensitive for the detection of aminoglycoside-modifying enzymes. Therefore, three new MPH(2')-producing strains were found in clinically isolated E. coli in Japan in 1997 by this method. It suggests that MPH(2')-producing E. coli have been spread in Japanese clinical fields.

Hepatic heterogeneity in the response to ATP studied in the bivascularly perfused rat liver.

The zonation of the purinergic action of ATP in the hepatic parenchyma was investigated in the bivascularly perfused rat liver by means of anterograde and retrograde perfusion. Livers from fed rats were used, and ATP was infused according to four different experimental protocols: (A) anterograde perfusion and ATP infusion via the portal vein; (B) anterograde perfusion and ATP via the hepatic artery; (C) retrograde perfusion and ATP via the hepatic vein; (D) retrograde perfusion and ATP via the hepatic artery. The following metabolic parameters were measured: glucose release, lactate production and oxygen consumption. The hemodynamic effects were evaluated by measuring the sinusoidal mean transit times by means of the indicator-dilution technique. ATP was infused during 20 min at four different rates (between 0.06-0.77 micromol min[-1] g liver[-1]; 20-200 microM) in each of the four experimental protocols. The results that were obtained allow several conclusions with respect to the localization of the effects of ATP along the hepatic acini: (1) In retrograde perfusion the sinusoidal mean transit times were approximately twice those observed in anterograde perfusion. ATP increased the sinusoidal mean transit times only in retrograde perfusion (protocols C and D). The effect was more pronounced with protocol D. These results allow the conclusion that the responsive vasoconstrictive elements are localized in a pre-sinusoidal region; (2) All hepatic cells, periportal as well as perivenous, were able to metabolize ATP, so that concentration gradients were generated with all experimental protocols. Extraction of ATP was more pronounced in retrograde perfusion, an observation that can be attributed, partly at least, to the longer sinusoidal transit times. In anterograde perfusion, the extraction of ATP was time-dependent, a phenomenon that cannot be satisfactorily explained with the available data; (3) ATP produced a transient initial inhibition of oxygen uptake when protocols A and B were employed. These protocols are the only ones in which the cells situated shortly after the intrasinusoidal confluence of the portal vein and the hepatic artery were effectively supplied with ATP. The decrease in oxygen consumption was more pronounced at low ATP infusions when protocol B was employed. These observations allow the conclusion that the former phenomenon is localized mainly in cells situated shortly after the intrasinusoidal confluence of the portal vein and hepatic artery. Oxygen consumption in all other cells, especially the proximal periportal ones, is increased by ATP; (4) In agreement with previous data found in the literature, glycogenolysis stimulation by ATP was more pronounced in the periportal region. The cells that respond more intensively are not the proximal periportal ones, but those situated in the region of the intrasinusoidal confluence of the portal vein and the hepatic artery.

Batch Analysis of the ATP Content of Bovine Sperm, Oocytes, and Early Embryos Using a Scintillation Counter to Measure the Chemiluminescence Produced by the Luciferin–Luciferase Reaction

This report describes an approach to using a commercial luciferin–luciferase kit and a scintillation counter for batch analysis of the ATP content of bovine sperm, oocytes and embryos. After ATP extraction, the luciferin–luciferase assay mixture was added to as many as 48 samples at one time, and the tubes counted once, and then again, in reverse order. The square-root of the geometric mean of the two counts for a tube was directly proportional to the ATP content. The measured ATP content was directly proportional to the number of sperm, oocytes, or blastocysts in a sample. The assay is sensitive enough to measure the ATP content of a single oocyte or blastocyst, or approximately 1000 sperm. The assay does not require the complicated and expensive automated sampling and mixing apparatus that is required for batch analysis using luminometer counting. This approach may significantly simplify the determination of the ATP content of large numbers of samples.

Adenosine 5′-triphosphate measurements in soils containing recently added glucose

A transient ATPase was produced when a grassland soil was incubated with glucose (5000 μg glucose C g −1 soil) and inorganic N. The activity of this enzyme reached a maximum after about 2 days, declining to ambient levels by 10 days. Its formation was strongly repressed by the addition of inorganic phosphate during the incubation with glucose and N. This ATPase was surprisingly resistant to denaturation in the trichloracetic acid-based reagent we use to extract ATP from soil, although it had almost completely disappeared 30 s into extraction. In determining ATP in soil with the TCA-based extractant, a spike of ATP is added to correct for losses of ATP during extraction. These losses can be by hydrolysis (enzymatic or chemical) during extraction, by adsorption on the soil colloids and by quenching during measurement of ATP by the luciferin-luciferase reaction. An ATP spike added at the beginning of the extraction of a soil recently amended with glucose was much more completely broken down than a spike added 30 s into the extraction. Presumably the glucose-induced ATPase was active at the beginning of extraction but almost completely denatured by 30 s. In contrast, the extraction of ATP from the soil microbial biomass did not appear to be influenced by the ATPase, presumably because this ATP was released during extraction and was therefore not all present at the start, unlike spike ATP. Most measurements of soil ATP using TCA-based extractants will not be influenced by this ATPase because it is only active in soils that have recently received substrate. However, even in such soils, valid ATP measurements can be obtained by taking simple precautions — adding the spike after 30 s or cooling the soil before extraction.

Interaction of long-chain choline derivatives with firefly luciferase and their use as reagents for the extraction of intracellular ATP from microorganisms

Various long-chain choline derivatives, differing in the length of the hydrocarbon backbone, have been synthesized. The influence of these compounds on the activity of firefly luciferase has been studied. Whereas C 4–C 10 derivatives do not influence enzyme activity, C 12–C 18 derivatives inactivate luciferase rapidly and irreversibly. It is suggested that interaction between the enzyme and these compounds has a hydrophobic nature. Phospholipids stabilize luciferase against the inactivation by these derivatives. It was shown that long-chain choline derivatives with a backbone of 10 carbon atoms and more can be used for ATP extraction and further bioluminescent microassay in the presence of phospholipid additives as the agents which stabilize luciferase.

Improvements in the extraction of bacterial ATP from soil with field application

An improved procedure for the preparation of an extractant and a demonstration of field applicability of that extractant for extracting microbial ATP from soil samples are reported. Precipitation difficulties that occurred during application of the six-component extractant developed in this laboratory [Webster et al. (1984) Soil Biol. Biochem., 16, 335] have been eliminated by division of the extractant into two mixes. Separate additions of phosphoric acid and/or EDTA at the time of extraction prevented the unwanted precipitation. Although a precipitate formed in the completed extractant, it did not affect ATP extraction from soil samples. A substitute detergent for Lubrol (which is no longer readily available) was selected; it is polyoxyethylene 10 lauryl ether and it functioned as well as Lubrol in the extraction of ATP from soil. Extraction procedures done using either a Brinkmann Polytron and wrist-action shaker (in the laboratory procedure) or a Waring blendor (for the field procedure) extracted equivalent amounts of ATP from soil. A detailed protocol is described that works well in the field. The procedure was made field-applicable by using a portable generator, desktop high speed centrifuge, and a normal laboratory luminometer.

Regulation of calmodulin binding to the ATP extractable 110 kDa protein (myosin I) from chicken duodenal brush border by 1,25-(OH) 2D 3

In earlier studies we observed that the active vitamin D metabolite 1,25-(OH) 2D 3 increased the calmodulin content of purified duodenal brush-border membrane vesicles where it bound principally to the 110 kDa protein myosin I. In this study we further evaluated the regulation of calmodulin binding to ATP releasable myosin I. Whole brush borders (BB) or purified brush-border membrane vesicles (BBMV) were prepared from duodena of vitamin D-deficient rachitic chicks treated 12–18 h before killing with either 625 pmol 1,25-(OH) 2D 3 or vehicle. The ATP extractable myosin I from BB resulted in an 1.6-fold increase of calmodulin binding to the 110 kDa band after treatment with 1,25-(OH) 2D 3. In contrast to BB, ATP extraction of myosin I from purified BBMV required alamethicin for ATP entry. As for BB extracts, calmodulin binding to the 110 kDa band in BBMV extracts was also increased about 2.4-fold by 1,25-(OH) 2D 3. It was concluded that both intact BB and purified BBMV showed the same type of increase in calmodulin binding to ATP releasable myosin I by 1,25-(OH) 2D 3. To see whether 1,25-(OH) 2D 3 increased the intrinsic affinity of calmodulin binding to myosin I, the ATP extractable myosin I from BB was purified from rachitic chicks treated with 1,25-(OH) 2D 3 or vehicle. In contrast to ATP extracts of BB or BBMV, calmodulin binding to the purified myosin I was not different between preparations from 1,25-(OH) 2D 3- or vehicle-treated chicks. We conclude that 1,25-(OH) 2D 3 does not change the affinity of calmodulin binding to myosin I but increases the amount of myosin I in the membrane or alters its ATP releasability. It was further investigated whether phosphorylation is involved in these 1,25-(OH) 2D 3 dependent posttranslational changes of myosin I. Phosphorylation of brush-border membrane proteins in vivo was performed by incubation of [ 32P]P i in the lumen of a ligated duodenal loop in situ for 15 min. Brush-border membrane proteins were phosphorylated in vitro by incubating BB or BBMV with [γ- 32P]ATP for 1 min. Incubation experiments in vivo and in vitro in fact resulted in phosphorylation of several proteins including 110 kDa proteins. However, there was no specific effect of 1,25-(OH) 2D 3 on phosphorylation of 110 kDa proteins. We conclude that the effects of 1,25-(OH) 2D 3 on protein phosphorylation are minimal and not likely to explain 1,25-(OH) 2D 3 stimulated calmodulin binding to ATP extractable brush-border membrane myosin I and 1,25-(OH) 2D 3 stimulated changes of calcium uptake across the brush-border membrane.

Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle

A caldesmon kinase activity was detected in an ATP extract of the myofibril-like pellet from sheep aorta. The enzyme was purified 745-fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon- h. Ser-73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.

Isolation of kinesin from rat liver.

Kinesin is a translocator protein which mediates microtubule-based transport of organelle and vesicles. Microtubules support major roles in some intracellular transport systems in a variety of cells. However, it is not known which proteins comprise the translocator in the liver 9 In this study, we determined the molecular weight and motility of hepatic kinesin. The translocator protein fraction, isolated from rat liver by a modification of Vale's method, consisted of many kind of protein species as identified by SDS-PAGE. The most prominent of these was a ll6kDa protein. The ll6kDa polypeptide had property of forming a high affinity complex with microtubules in the presence of nonhydrolyzable ATP analog, adenylyl imidodiphosphate(AMP-PNP). The ll6kDa polypeptide released from the microtubules with ATP reacts with monoclonal antibodies to sea urchin and chicken brain kinesin heavy chain (Fig.A,B). Gliding of reconstituted microtubules in the presence of ATP on the ll6kDa polypeptide treated with glass plate was visualized with DIC-video enhanced A B microscopy. The mean i ~ 3 ~ 5 6 7 s i 2 3 4 5 6 7 s v oywa0000 1 IIi o u I,, 9 116K-.,QKHC 116-97.~indicated that the ,~_ ~ == ll6kDa polypeptide ,s~,r~ 4~from rat liver was ~ __~i__ kinesin heavy chain. Figure: Electrophoretic and immunoblotting pattern obtained in the of the preparation of kinesin from rat liver extract. A: SDA-PAGE with silver staining. B: Immunoblotting using sea urchin kinesin mAb. Lanel: Supernatant after 150000xg centrifugation. 2: Microtubule containing pellet. 3: Supernatant after wash in buffer containing ATP. 4: Pellet after ATP extraction. 5: Supernatant after wash in buffer containing GTP. 6: Pellet after GTP extraction 9 7: Supernatant after wash in buffer containing ATP and AMP-PNP. 8: Pellet after ATP and AMP-PNP extraction.

Dissociation of complexes between 70 kDa stress proteins and presecretory proteins is facilitated by a cytosolic factor

Members of the 70 kDa stress protein family were shown previously to facilitate the posttranslational translocation of presecretory proteins into the endoplasmic reticulum and protein precursors into mitochondria. To identify proteins that interact with 70 kDa stress proteins during the early steps of posttranslational translocation, polyclonal antibodies were raised against purified yeast cytosolic stress proteins. They were used to immunoprecipitate complexes between 70 kDa stress proteins and a radiolabeled presecretory protein, prepro-alpha-factor, that was translated in vitro. Complexes between prepro-alpha-factor and 70 kDa stress proteins were stable, but could be dissociated in the presence of ATP and crude cytosolic extracts from yeast. These results are consistent with the idea that 70 kDa stress proteins act as molecular chaperones in translocation by binding to precursor proteins before or during their passage across membranes.

Detection of bacterial ATP by reversed flow-injection analysis with luminescence detection

Flow-injection analysis with luminometric detection was used to detect trace concentrations of ATP released from bacteria. Luciferin and luciferase reagents were injected into a flowing stream of aqueous ATP of bacterial extract. A liquid scintillation counter was used as the detector. Experimental variables were studied and 120 samples per hour could be analysed. In pure solution, ATP was detected at the 10 −14 M level. Because of incomplete extraction of the cellular ATP and the quenching effects of co-extracted materials, the limit of detection for the bacterial extracts was much poorer at ca. 10 3–10 4 cells ml −1, corresponding to ca. 10 −11–10 −10 M ATP.

Chemotaxis by Octopus maya Voss et Solis in a Y-maze

Chemotaxis by Octopus maya Voss et Solis ( n = 20) to chemicals introduced at a distance (teloreception) was measured in a Y-maze. The trials ( n = 195) were conducted in a 40-1 Y-maze system with chemicals entering only one arm of the Y-maze. Octopuses were placed into the base compartment near the convergence of the Y-maze arms and allowed to acclimate to the Y-maze (10 min). A chemical was added to one of the Y-maze arms and octopus movements were recorded (10 min) on videotape. The videotape provided information on the arm first penetrated, the sequence of the penetrations, the number of penetrations into each arm, the duration of time spent in each arm and the base and the mean time spent in each arm per penetration. Eight chemicals (ATP, AMP, alanine, betaine, glutamic acid, glycine, proline and taurine), two crude extracts (crab and shrimp) and the control (no chemical) were introduced at random to only one arm of the Y-maze and each octopus was tested for all chemicals and the control. The final concentration of the chemicals was 10 −4 M except ATP at 10 −5 M and AMP at 10 −5 M, while the crude extracts were prepared by homogenizing crab or shrimp tissue to a final concentration of 5 × 10 −5 g · 1 −1. Control and chemically exposed octopuses were very active while in the Y-maze, penetrating each arm multiple times. Chemotaxis was demonstrated for octopuses exposed to proline, ATP and crab extract since these octopuses most often penetrated the arm receiving chemical first. Increases in the total amount of activity (number of penetrations into arms and total time spent in arms) were correlated to exposure to proline, alanine, ATP and crab extract; these chemicals functioned as excitants. Betaine and taurine were correlated with lower activity; these chemicals functioned as arrestants. Differences in the number of penetrations per arm and the total duration spent in each arm were affected by both the chemical used and individual octopuses. The sequence of penetrations was not affected by different chemicals. As a result, chemotaxis by octopuses was associated most strongly with first exposure to a chemical, causing initial movement by the octopus into the arm receiving the attractant; octopuses probably habituated to the presence of the chemicals thereafter.