The role of adenosine monophosphate nucleosidase in the regulation of adenine nucleotide levels in Azotobacter vinelandii during aerobic-anaerobic transitions

When cultures of Azotobacter vinelandii are made anaerobic the adenylate pool size remains constant or increases slightly while the adenylate energy charge decreases. Under these conditions, cell growth stops but the cells remain viable for at least 5 h with the decreased energy charge. The changes in the adenylate pool during the aerobic-anaerobic transition include: the formation of adenylates as a result of RNA degradation; the degradation of a portion of the excess AMP to form hypoxanthine by the sequential actions of AMP nucleosidase and adenine deaminase; an increase in the total adenylate pool which is stabilized at approximately 1.5 times the level in growing cells; and stabilization of the adenylate energy charge at a value near 0.3. The degradation of AMP is regulated by AMP nucleosidase, an allosteric enzyme which is activated by MgATP^2? and inhibited by P_i. The in vivo activity of AMP nucleosidase was estimated by measuring the rate of hypoxanthine formation in the culture or by measuring the activity of purified enzyme at the concentrations of AMP, ATP, and P_i found in the cells. The maximum estimated in vivo rate of AMP degradation was less than 3% of the catalytic capacity of AMP nucleosidase. Thus ample activity is present for rapid adjustments of the AMP levels in these cells. Expression of AMP nucleosidase catalytic activity is tightly controlled since high constant concentrations of intracellular AMP can be maintained for extended time periods at low adenylate energy charge values. Under these conditions controlled degradation of AMP can occur to maintain a constant AMP concentration.     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast DeltapH within Hevea Latex Cells

The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the “sink effect” and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast ΔpH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H⁺-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel.     

Regulation of biosynthesis of secondary metabolites

Two partly purified malate dehydrogenase (EC 1.1.1.37) isoenzymes were isolated fromStreptomyces aureofaciens. This is the first example of a non-homogeneous enzyme in actinomycetes and one of the very few cases in bacteria in general. The characteristics of the enzymatic reaction were studied for each enzyme in relation to the concentration of both substrates and cofactors and the apparent Michaelis constant was calculated. It was found that the reaction was affected by Mg²⁺ ions and that SH-groups could be specifically inhibited. The optimal pH and the influence of temperature changes were also determined. In all the parameters, one of the isoenzymes resembled mitochondrial MDH, while the other resembel the supernatant MDH described in the literature in the tissues of higher organisms. The functional relationship of the two MDH isoenzymes inStreptomyces aureofaciens is discussed.     

Cross-reactivity studies on the interaction between elongation factors Tu and Ts from Streptomyces aureofaciens and Escherichia coli in the GDP exchange reaction

The method of purification of elongation factor Ts from Streptomyces aureofaciens is described. Purified elongation factors Ts from S. aureofaciens and Escherichia coli were tested in cross-reactivity studies with elongation factors Tu from both species in a GDP exchange reaction under equilibrium and non-equilibrium conditions. Experiments have revealed that slower spontaneous release of GDP from S. aureofaciens EF-Tu is compensated for by higher affinity of homologous EF-Ts towards EF-Tu and thus the initial rates of EF-Ts catalysed GDP exchange can be kept the same in both E. coli and S. aurefaciens in vitro systems.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Phosphorylation and nitrogenase activity in isolated heterocysts from Anabaena variabilis (ATCC 29413)

Adenylate-pool composition, energy charge, and nitrogenase activity were examined in isolated heterocysts from Anabaena variabilis (ATCC 29413). ATP formation was detected as a light- or oxygen-induced increase in ATP concentration. No cofactors or substrates had to be added for photophosphorylation to occur, whereas oxidative phosphorylation was dependent on hydrogen and oxygen (Knallgas reaction). The increase in ATP concentration was reflected by a decrease in AMP concentration, accompanied by small changes in ADP levels. Thus, a regulation of the adenylate pool by a myokinase (adenylate kinase) has to be assumed. Upon dark-light transitions, the energy charge in heterocysts increased from values below 0.4 to values approaching 0.8. High energy-charge values, reached in the light only, allowed for high rates of acetylene reduction in the presence of hydrogen. The increase in the energy charge in the dark to approx. 0.64 by addition of oxygen (5% ($\text{v}\text{v}$) in the presence of hydrogen) resulted in low nitrogenase activities, generally not exceeding 1–3% of the light-induced rates. In the dark, oxygen concentrations above 10% were inhibitory to both ATP formation and acetylene reduction. Increasing light intensities led to a steep increase in energy charge followed by an increase in nitrogenase activity. Plotting enzyme activity versus energy charge, a nonlinear, asymptotic relationship was observed.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

Role of ATP-glucokinase and polyphosphate glucokinase inStreptomyces aureofaciens

The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomyceteStreptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It appears that the biosynthesis of CTC inStreptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates.     

How is glutamine synthetase I activity from Streptomyces aureofaciens regulated ?

Summary Activity of glutamine synthetase I (GSI) from Streptomyces aureofaciens increased markedly during tetracycline production phase. The purified GSI exhibited a low affinity for glutamate but high affinities for ATP and ammonium. Its Mn²⁺-dependent activity was more sensitive to feedback inhibitors than Mg²⁺-dependent activity. Both the activities were significantly stimulated by Co²⁺ but inhibited by other divalent cations. ADP was a strong inhibitor. These results suggest that GSI activity is regulated by availability of substrates, feedback inhibitors, divalent cations, and cell energy charge.     

In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts

The role of AMP deaminase reaction in the stabilization of the adenylate energy charge was investigated using permeabilized yeast cells. The addition of P_i or Zn²⁺, which inhibits AMP deaminase, remarkably retarded the depletion of total adenylate pool and the recovery of the adenylate energy charge. Polyamine, an activator of the enzyme, decreased total adenylates, resulting in the enhanced recovery of the energy charge in situ. AMP deaminase can act as a regulatory enzyme in the system that stabilizes the adenylate energy charge in yeast cells under the conditions of severe metabolic stress.     

Mutagenic treatment of chlortetracycline producer with acriflavine

Summary Acriflavine treatment of Streptomyces aureofaciens gives rise to a high proportion of mutants withloss of antibiotic production and with morphological changes. Mutants so obtained are compared with those arising after UV irradiation.     

Energy status in the fat body of Trichoplusia ni parasitized by the insect parasite Hyposoter exiguae

The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguae. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association.     

Electron-cytochemical demonstration of polyphosphates and the appropriate phosphatases in the glycocalyx ofStreptomyces aureofaciens

Electron-cytochemical stainings based on Gomori type lead precipitation techniques showed that both polyphosphates (by energy dispersive analysis of X-rays) and appropriate phosphatases were present in the capsular fibrils covering the mycelium ofStreptomyces aureofaciens, producing low quantities of chlortetracycline. In light of the known differences in polyphosphate contents and glycocalyx secretion in low- and high-producing strains, our results point to new aspects of compartmentation of actinomycetal cells with respect to secondary metabolism and to the role of microbial glycocalyx in the process.     

Changes in adenine-nucleotide content in the apical bud of Sinapis alba L. during floral transition

The total adenylate pool of the apical buds of vegetative plants of Sinapis alba L. continuously grown in short days fluctuates over a 24-h cycle with the minimum occurring at the end of the dark period. In the buds of plants induced to flower by a single long-day treatment, total adenylate pool increases above the control level 16 h after the start of the long day, resulting mainly from a rise in ATP and ADP contents. This occurs 6 h after the increase in the soluble carbohydrate content previously shown to occur in the apical buds of plants induced to flower (Bodson 1977, Planta 135, 19–23). A transient rise of the energy charge occurs 22 h after the start of the inductive long day.Abbreviations LD long day - SD short day     

The adenylate energy charge in marine phytoplantkon: The effect of temperature on the physiological state of Skeletonema costatum (Grev.) Cleve

The adenylate energy charge $\text{([ATP] +}\text{1}\text{2}\text{[ADP])}\text{[0ATP+ADP+AMP]}$ was measured in axenic batch cultures of Skeletonema costatum (Grev.) Cleve at 2°, 10°, 15°, 20°, 24° and 30°C. The results suggest that this eurythermal diatom is physiologically capable of adapting to the 28 °C range of temperature with little apparent difference in the potential energy available to the cell. In N-limited continuous cultures at 15 °C, the energy charge values were lower than those observed in batch culture by 0.2, implying nutrient stress may result in decreased intracellular chemical energy. The utilization of the adenylate energy charge as an indicator of physiological state is suggested.     

Biochemical characterization of a novel metalloendopeptidase from Streptomyces aureofaciens TH-3 with post-proline hydrolysis activity

Streptomyces aureofaciens TH-3 secretes a protease termed ‘kibilysin’, for which we showed unique substrate specificity and preference for Tyr, Pro, and Leu at the P₁ position using fluorescence energy transfer substrate (FRETS) combinatorial libraries. Using (7-methoxycoumarin-4-yl) acetyl-Lys-Pro-Leu-Gly-Leu-d-2,3-diamino propionic acid (2,4-dinitrophenyl)-Ala-Arg-NH₂, we confirmed that kibilysin digests the substrate between Pro and Leu. Its gene was cloned and sequenced. The primary structure of the enzyme showed 40, 66, and 61% identity, respectively, with those of thermolysin from Bacillus thermoproteolyticus, and metalloendopeptidases from Streptomyces cinamoneus TH-2 and S. griseus. Its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, which is a common motif of the peptidase family M4. Moreover, we succeeded in over-expression of kibilysin using Streptomyces lividans.     

Morphology of Submerged Growth of Streptomycetes as a Taxonomic Aid: I. Morphological Development of Streptomyces aureofaciens in Agitated Liquid Media

In a study of the submerged growth morphology of 145 different streptomycetes in A C Broth, the species Streptomyces aureofaciens was observed to represent an unusual type. It was characterized by profuse mycelial segmentation, transformation of disarticulated fragments of segmented mycelium into chains of thickened arthrospores, and formation of filiform appendages on the arthrosporic cells giving an insectival appearance to these elements. This unusual morphology was determined to be a consistent feature in 110 strains of S. aureofaciens studied, hence suggesting a useful taxonomic aid in identification of this species. Preliminary studies of multiple strains of many different Streptomyces species indicate that other useful taxonomic features based on morphology of submerged growth are found in a significantly large part of the genus.