Estimation of pyruvate,phosphate dikinase activity in maize leaf tissue by (phosphoenolpyruvate plus pyrophosphate)-dependent phosphorylation of AMP

Crude extracts of maize leaf tissue catalysed the phosphorylation of AMP by ³²PPi in the presence of phosphoenolpyruvate (PEP). The reaction was enhanced by F^? and NH₄⁺. The optimum concentrations of AMP, PEP and PPi were 0.3, 10 and 1 mM, respectively. Under these conditions, ca75% of the AMP phosphorylated by ³²PPi was present as ATP and ca25 % as ADP. The activity was reversibly cold labile. The specific activity of crude extracts in the presence of F^? was proportional to enzyme concentration only at protein concentrations < 25,μg/ml. Partially purified pyruvate, phosphate dikinase (PPD) from maize leaf quantitatively phosphorylated AMP to ATP in a (PEP plus PPi)-dependent reaction with the concomitant production of 0.9 mol of pyruvate per mol of AMP phosphorylated. It was concluded that (PEP plus PPi)-dependent phosphorylation of AMP provides a reliable method for estimating PPD activity in crude extracts of maize. Crude maize extracts also catalysed ³²Pi-ATP and ³²PPi-ATP exchange but these activities were not specific for PPD.     

α-l-Rhamnosidase Activity in Culture Filtrate of Corticium rolfsii Enzymic Activity at Low pH

A phosphorylation system for formation of ATP from AMP by Zymolyase-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was developed as an ATP production process. This system was shown to be an energy conversion system, from a reduced C₁ -compound to ATP through reduction of NAD⁺ and oxidative phosphorylation but not substrate level phosphorylation, together with phosphorylation of AMP to ADP.

Reaction conditions for the ATP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pressure, and oxygen supply. Under the optimal conditions, 26 mM (13 g/liter) and 8.5 dim (4g/liter) of ATP were produced with methanol and formate as C₁ -substrate, respectively.     

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h^?1 ml^?1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h^?1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h^?1 ml^?1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.     

Further studies on the properties of the rabbit reticulocyte adenosine 3',5'-cyclic monophosphate-dependent protein kinase I

The properties of cyclic AMP-dependent protein kinase I isolated from rabbit reticulocytes were further investigated. The enzyme catalyzes the phosphorylation of histone in the presence of ATP and Mg²⁺ and this reaction is stimulated by cyclic AMP. The pH optimum of the reaction was between 8.5 and 9.0, when assayed in the presence of cyclic AMP. No distinct pH optimum was observed in the absence of the cyclic nucleotide. The K_m values for ATP appeared to be very similar whether it was determined in the presence (K_m = 1.7 × 10⁻⁴m) or absence (K_m = 2.5 × 10⁻⁴m) of cyclic AMP. The rate of heat inactivation of the catalytic activity and the cyclic AMP binding activity of kinase I were found to be dependent on the presence of Mg²⁺, ATP, and/or cyclic AMP. In the presence of cyclic AMP, the rate of inactivation of the catalytic activity of kinase I at 53 ° was accelerated. On the other hand, the cyclic AMP binding activity appeared to be protected from heat inactivation by the cyclic nucleotide. When both ATP and Mg²⁺ were present in the heating mixture, no loss of catalytic and binding activities of kinase I were observed even up to 8 min of heating at 53 °. The cyclic AMP binding activity of kinase I was almost completely inhibited by mercuric acetate at a concentration of 1 mm, while the loss in catalytic activity was only 50%. These results substantiate our previous observation that kinase I contains two nonidentical subunits, a catalytic subunit and a cyclic AMP binding subunit.     

Correlations among the responses of suspensions of Dictyostelium Discoideum to pulses of 3′,5′-cyclic AMP: Transient turbidity decrease,the cyclic AMP signal,and variation in adenine mononucleotide levels

Aggregation-competent myxamoebae of the cellular slime mold Dictyostellium discoideum are known to exhibit two responses to extracellular pulses of 3′5′-cyclic AMP: an immediate chemotactic movement; and a delayed generation of intracellular cyclic AMP which is subsequently released into the medium. The mechanism of the latter, the so-called signalling response, may depend on alterations in intracellular metabolite levels and is the subject of this communication.Myxamoebae of the wild-type strain NC-4 of D. discoideum were suspended in an aerated, stirred 17 mM potassium phosphate buffer. pH 6.0, at a concentration of approx. 6 · 10^?7 cells/ml (8%, v/v) at 25°C and were pulsed with 1. 10^?8—1 · 10^?7 M cyclic AMP at 10–20-min intervals for periods of 3–5 h over incubation of 4–9 h. Suspensions were monitored continuously for transient turbidity decreases following the cyclic AMP pulses as an indication of the magnitude and duration of the cellular response to cyclic AMP. When the pattern of turbidity decrease indicated that a signalling response had developed, samples were withdrawn at 10–15-s intervals from the suspension, inactivated with perchloric acid, and analyzed for cyclic AMP, ATP, ADP, AMP, pyruvate, and glucose 6-phosphate. In separate experiments, steady-state oxygen tension was monitored along with turbidity to detect possible changes in respiratory rate.The following consistent patterns were observed after the added cyclic AMP pulse: a transient increase in the ADP level which reaches maximum between 0.7 and 1.7 min; transient decreases in ATP and pyruvate which concide with and approximately equal the magnitude of the increase in ADP; a later increase in glucose 6-phosphate which reaches maximum approx. 2 min after the ADP     

H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips

The relationship between changes in H⁺ flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H⁺ efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H⁺-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H⁺ extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H⁺ secretion under the conditions of this investigation.     

Protein kinases in brown adipose tissue of developing rats I. GTP as nucleotide substrate for histone phosphorylation

100 000 × g soluble extracts from interscapular brown adipose tissue catalyzed the transfer of the terminal phosphoryl group from GTP to histone. Maximal velocity was achieved only with both cyclic AMP and ATP present. The cyclic AMP dose-response curve was the same as for the ATP-utilizing enzyme, with maximum stimulation at 0.5 μM. ATP (1–100 μM) increased the rate of histone phosphorylation with GTP as the radioactive substrate. Higher concentrations had a dilution effect similar to that of GTP on the ATP-utilizing enzyme. Similar effects were observed with ADP and AMP. The apparent K_m values for histone were the same with both GTP and ATP as nucleotide substrates. The effects of pH, purified beef muscle kinase inhibitor and of NaCl were also the same. Maximum velocities of histone phosphorylation from ATP and those from GTP were almost the same in brown fat of all age groups tested. Separated on histone-Sepharose, the GTP-utilizing activity was absolutely dependent on the re-addition of the ATP-utilizing enzyme (a linear relationship with a slope of approx. 0.95). An extremely active nucleotide phosphotransferase activity was found in the same subcellular fraction. The rate of equilibration of the γ-³² P between GTP and ATP could account for all the histone phosphorylation with [γ-³² P] GTP. It is concluded that, in spite of the presence of nucleotide phosphotransferase and ATP-protein kinase activities, a direct transfer from GTP to a protein substrate cannot be excluded. Also, histone may not be the natural protein acceptor for GTP-linked phosphorylation.     

Measurement of adenine nucleotides in plasma

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A ‘stop solution’ containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg⁺² and Ca⁺² that are co‐factors for many ATPases. Stop solution and blood were mixed using a two‐syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75–7.95) and Mg²⁺ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0–15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright © 2003 John Wiley & Sons, Ltd.     

Production of cyclic AMP from extracellular ATP by intact LM cells

Intact LM cells, a line of cultured mouse fibroblasts, exhibited and adenylate cyclase (APT pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence exogenous [α-³²]ATP which was 20–30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E₁ stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an endenylate cyclase activity whic is accessible to extracellular ATP.     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

AMP promotes oxygen consumption and ATP synthesis in heart mitochondria through the adenylate kinase reaction: an NMR spectroscopy and polarography study

Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP using ³¹P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led to a 25% increase in ATP synthesis as measured by the increase in βATP signal. More importantly, we show that AMP can support ATP synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ, α and β signals and the increase of P_i. Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance of γ, α and β ATP signals, in conjunction with a decrease in P_i, which is being used for ATP synthesis. Polarographic studies showed Mg²⁺ dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with ³¹P NMR spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd. SIGNIFICANCE OF RESEARCH PARAGRAPH The data generated in the present study indicate that ³¹P NMR spectroscopy can effectively be used to study the adenylate kinase reaction under a variety of conditions. This is important because understanding of adenylate kinase function and/or malfunction is essential to understanding its role in health and disease. The data obtained with ³¹P NMR were confirmed by polarographic studies, which further strengthens the robustness of the NMR findings. In summary, ³¹P NMR spectroscopy provides a sensitive tool to study adenylate kinase activity in different physiological and pathophysiological conditions, including but not exclusive of, cancer, ischemic injury, hemolytic anemia and neurological problems such as sensorineural deafness.     

Alteration of 1,2-diacylglycerol content in ischemic and reperfused heart

Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN₃ were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase.A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05–0.8 µM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates.The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.Abbreviations Ap₅A P¹ - P⁵-di(adenosine-5) Pentaphosphate - ATP-DPH ATP Diphosphohydrolase - DCCD N,N Dicyclohexycarbodiimide - Fru-P₂ase Fructose 1,6-biphosphatase - SDS Sodium Dodecyl Sulfate - TLC Thin Layer Chromatography     

Highly efficient production of ATP by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

Improvement of efficiency of ATP production from adenosine with sorbitol-treated cells of Candida boidinii (Kloeckera sp.) no. 2201 was investigatedOrthophosphate, pyrophosphate and divalent metal ions inhibited the deamination of AMP to IMP, a major by-product. No hypoxanthine nucleotidic compound was accumulated by addition of coformycin. By successive feeding of K₂HPO₄ to maintain the phosphorus concentration at over 100 mM, the conversion rate from adenosine to ATP was improved to 70%. Simultaneous feeding of K₂HPO₄ and adenosine resulted in the accumulation of 100 mM ATP (50.7 g/l) after 28 h of incubation and the increase of IMP without decrease of ATP for 48 h.ATP production was further prolonged until 60 h of incubation and 198 mM ATP (100 g/l) was accumulated at the conversion rate of 77.4% by control of pH in the range of 6.5 to 6.8 during the reaction.     

l-serine production from methanol and glycine with an immobilized methylotroph

l-Serine production from methanol and glycine was attempted using immobilized resting cells of a methylotroph, Protomonas extorquens NR 1, under automatically controlled conditions. A Ca-alginate system was selected. The conditions for l-serine formation were optimized at 30°C. A concentration of glycine 100 g·l⁻¹ which was the optimum concentration for l-serine production by free resting cells was used in the reaction mixture. The optimum concentrations of methanol and dissolved oxygen were 20 g·l⁻¹ and 5 ppm, respectively. Under the optimum conditions, 11.3 g·l⁻¹ of l-serine was produced within 36 h. The selectivities (mole of l-serine/mole of substrate consumed) of l-serine from methanol and glycine were 4.5% and 95.1%, respectively. The size of gel beads affected the l-serine formation rate. The initial rate of l-serine formation decreased with an increase in the size of beads. However, the l-serine formation rate increased at elevated concentrations of dissolved oxygen, even with large sized beads. This result implies that the oxygen diffusion inside the gel beads limited the l-serine formation rate. The observed effectiveness factor of the immobilized cells could be estimated by the theoretical effectiveness factor of the zero-order reaction with respect to the dissolved oxygen.Repeated use was not feasible without reactivation of the immobilized cells. Reusability was examined by reactivation of the immobilized resting cells in appropriate media for 12 h. The reactivated immobilized resting cells were used again in the next cycle. By this procedure, several cycles of l-serine formation were made possible.     

Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity

In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many ³²P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-³²P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-³²P] phosphate from [γ-³²P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]₀ e^?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [³²P] phosphate from [γ-³²P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.     

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [¹⁴C]pyruvate to haemoglobin and the rate of efflux of [¹⁴C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent K_m and V_max for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.     

Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [¹⁴C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [¹⁴C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.     

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1–0.9 h^?1) and initial glucose concentrations (20–60 g l^?1). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h^?1 dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml^?1 and 9,450 IU ml^?1 h^?1, respectively) with the combination of a 0.9-h^?1 dilution rate and a 40-g l^?1 initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.     

Adenylate kinase bound to the envelope membranes of spinach chloroplasts.

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K_m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10^?4m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP^?. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.     

Enzymatic production of α-dehydrobiotin from biotin

The enzymatic production of α-dehydrobiotin (α-DHB), an antibiotic, from biotinyl-CoA using acyl-CoA oxidase and from biotin using a coupling system of biotinyl-CoA synthetase and acyl-CoA oxidase was developed. Acyl-CoA oxidase was found to show activity for biotinyl-CoA. K_m and V_max values of acyl-CoA oxidase for biotinyl-CoA were 75 μM and 3.92 μmol min⁻¹ mg⁻¹, respectively. Optimum reaction conditions for the α-DHB production from biotin were examined. The maximum production of α-DHB (4.29 μmol ml⁻¹) was obtained, when the reaction was carried out at 30°C for 36 h in a mixture consisting of 100 mM potassium phosphate buffer (pH 8.0), 20 mM biotin, 20 mM ATP, 60 mM CoA, 20 mM MgCl₂, 2 units of biotinyl-CoA synthetase, 90 units of acyl-CoA oxidase and 25 units of catalase in a total volume of 0.6 ml under aerobic conditions. The product was purified from 14 ml of the reaction mixture and 10 mg of crystals with white needle form were obtained. From NMR, mass spectra and other physical analyses, this compound was identified as (+)-trans-α-DHB.