Inhibition of tubifex pyruvate kinase by storage phosphagens and adenosine triphosphate

• 1.1. Within the first 6–12 hr of experimental anaerobiosis the concentration of Tubifex phosphagens, phospholombricine and phosphoarginine, decrease to 10–30% of the values under aerobic conditions (7.5 and 3. μM/g dry wt).
• 2.2. High concentrations of both phosphagens (5–25 mM) inhibit Tubifex pyruvate kinase (PK) activity, the inhibitory mechanisms seem to be different. The inhibition is not mediated by the corresponding phosphagen kinases. Inhibition by phosphoarginine obviously requires an additional factor, which chemical nature is still unknown.
• 3.3. Certain phosphocreatine preparations also inhibit Tubifex PK. This inhibition, however, is likely due to contaminants than to phosphocreatine.
• 4.4. Mg-ATP demonstrates the strongest inhibitory effect on PK activity with a mixed competitive inhibition vs PEP and a non competitive vs ADP.

     

Studies on phosphagen synthesis by mitochondrial preparations

Mitochondrial preparations from muscles of a crab (Cancer pagurus), two fish (Trachurus trachurus and Scyliorhinus canicula) and a bird (Columba livia) are able to synthesise, through ATP, the phosphagen related to that species. This indicates the presence of a bound phosphagen kinase. Addition of creatine kinase and creatine to crab mitochondria results in the synthesis of phosphocreatine. Similarly, the addition of arginine kinase and arginine to mitochondrial preparations from the fish and bird results in the synthesis of phosphoarginine. In the crab, the mitochondrial form of arginine kinase released by sonication had the same kinetic affinity constants and electrophoretic mobility and could not be distinguished immunologically from the cytosolic form. The close similarity of bound and cytosolic forms of arginine kinase in this crustacean suggests that the two forms have not evolved separately as has creatine kinase in the mammal.     

Study of phosphagens in two trematodes

Extracts from the adults of two trematodes Fasciola gigantica and Paramphistomum explanatum have been shown to contain three phosphagens, phosphoarginine, phosphocreatine, and phosphoglycocyamine. The three phosphokinases involved in the transphosphorylation of these phosphagens in both worms were inhibited by EDTA and mercuric ions while iodoacetate had no effect.     

The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp,Crangon crangon

Summary The mobilization of glycogen and phosphoarginine during work and their resynthesis during periods of recovery were investigated in abdominal muscles of the shrimpCrangon crangon. All parameters, metabolite levels as well as glycogen phosphorylase (EC 2.4.1.1) and synthase (EC 2.4.1.11) activities were determined in each individual shrimp investigated. At the onset of work both glycogen and phosphoarginine were degraded with the rate of phosphoarginine utilization being more than 80-fold faster than glycogen. After exhaustive work phosphoarginine stores were replenished within 30 min and seemed to exceed the resting level thereafter. In contrast, glycogen was not resynthesized immediately after work, but was further degraded during recovery leading to the accumulation of lactate. Only when the phosphagen level had reached the resting level did glycogenolysis shift to its resynthesis. The shift is characterized by: (1) a change in the mass action ratio of phosphoglucomutase from values below the equilibrium constant to values above the constant, (2) a dramatic decrease in the ratio fructose 1,6-bisphosphate/fructose 6-phosphate indicating phosphofructokinase inhibition, (3) an increase in the glucose concentration, and (4) an increase in the proportion of glycogen synthase I. The inactivation of glycogen phosphorylase by dephosphorylation during recovery was 2.4-fold. 36±8% (n=5) of total activity remained in the phosphorylated form. It is proposed that this part of the enzyme was inactivated by the drop in inorganic phosphate level due to the restoration of phosphoarginine.     

Estimation of effective concentrations of ATP-regenerating enzymes in cilia of Paramecium caudatum

The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP‐regenerating enzymes, we attempted to reconstitute certain ATP‐regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high‐energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 mg/ml creatine kinase and 0.4 mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP‐regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 μM based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 μM. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.     

Measurement of an intracellular pH rise after fertilization in crab eggs using 31P-NMR

The effect of fertilization upon the intracellular pH, pH_i, in crab ovulated eggs was examined by ³¹P-NMR. The pH_i values were obtained from the chemical shift differences between the phosphoarginine PA resonance and the inorganic phosphate P_i resonance. The detection of the P_i peak was accomplished by Hahn spin-echo experiments in order to cancel the broad signal arising from phosphoproteins which overlaps the P_i signal. The average pH_i of the unfertilized unactivated eggs was 6.55 and a rise of 0.12 pH unit occurred after fertilization.     

Arginine kinase in Phytomonas, a trypanosomatid parasite of plants

Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer.     

Sustained isometric contraction of skeletal muscle depleted of phosphocreatine

To evaluate the need for phosphocreatine as an energy reservoir to sustain isometric contraction of skeletal muscle, rats were depleted of phosphocreatine by feeding β-GPA (β-guanidinopropionate) as 1% of the diet. In the place of phosphocreatine, β-GPAP (phosphorylated β-GPA) accumulated to concentrations of 20–25 μmoles/g wet weight of muscle. Although the maximum isometric tension produced by the soleus was always less than that produced by the plantaris muscle, the maximum for either muscle was not significantly affected by feeding β-GPA. The endurance of experimental soleus muscles was prolonged, however. These muscles held 70% of their maximum isometric tension for 106 ± 40 seconds (mean ± SD, n = 4) whereas the value for five control muscles was 43 ± 18 seconds. With fatiguing, isometric contractions of control plantaris and soleus muscles, phosphocreatine concentrations decreased by 68–70%; in experimental muscles, the β-GPA concentration decreased less than 12%. This difference in phosphagen consumption demonstrates that skeletal muscle can sustain fatiguing, isometric contractions without using large amounts of phosphocreatine or a substitute phosphagen as an energy reservoir. Phosphocreatine hydrolysis during muscle contraction normally may serve some other purpose.     

Functional expression of phosphagen kinase systems confers resistance to transient stresses in Saccharomyces cerevisiae by buffering the ATP pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.     

A 31P nuclear magnetic resonance study of the hydrothermal vent tube worm Riftia pachyptila.

31P nuclear magnetic resonance (NMR) was used to study the major phosphorylated compounds visible in perchloric extracts of three body regions of the vestimentiferan worm Riftia pachyptila: winged vestimentum, trunk and segmented posterior opisthosome. Two phosphagens (PGs) were present in vestimentum and opisthosome. The major resonance corresponded to those of phosphoarginine and phosphotaurocyamine, which cannot be discriminated on 31P NMR spectra. We have identified four distinct phosphodiesters (PDEs) in these tissues: glycerophosphorylethanolamine (GPE), serine ethanolamine phosphodiester (SEP), glycero-phosphorylcholine (GPC) and threonine ethanolamine phosphodiester (TEP). Three phosphonates or derivates (PAs) were observed in the three body regions. The minor one was identified as 2-aminoethyl phosphonate (2-AEP). The phosphorus profile of the trunk was appreciably different: one additional resonance in the PDE region and only one phosphagen peak were observed.     

Changes in phosphometabolites and intracellular pH in the tail muscle of the prawnPalaemon serratus as shown by in vivo31P-NMR

Summary ³¹P NMR spectra were recorded from tail muscles of the prawnPalaemon serratus, at rest, after exhaustive work and during subsequent recovery. At rest, the spectra indicated concentrations of phosphoarginine and ATP in good agreement with those obtained from resting fast skeletal muscles in mammals, which are characterized by a high phosphocreatine/P_i ratio. Following exhaustive work, phosphoarginine dropped by ca. 60% and ATP by 20%, while inorganic phosphate increased by 160%. The increase in inorganic phosphate immediately after contractions and in the first minutes of recovery corresponded partially to the changes in phosphoarginine and ATP. During recovery, the decrease of inorganic phosphate balanced the resynthesized phosphoarginine which was fully replenished within 30–40 min. The position of the inorganic phosphate resonance peak was used to monitor changes in intracellular pH (pH_i). The average pH_i in resting tail muscles was 7.20. After stimulation it was observed to decrease by 0.22 units. The return to pre-stimulation value was not achieved within 45 min. A NMR index (ATP+Arg-P)/(ATP+Arg-P+P_i) was calculated to characterize the extent of energetic changes caused by exercise.     

Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and related compounds

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 microg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by (31)P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.     

Distribution of fluorescently labeled actin in living sea urchin eggs during early development

Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.     

Intracellular pH controls protein synthesis rate in the sea urchine egg and early embryo.

Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artificially by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.     

pH measurement in human skeletal muscle samples: effect of phosphagen hydrolysis

Measurements of muscle pH (pHm) with the homogenate technique are routinely made when extensive phosphagen hydrolysis has occurred. Upon exposure of the homogenate to 37 degrees C in the pH meter, phosphocreatine and ATP were rapidly degraded to 35 and 60% of control concentrations after 30 s. Attempts at chemically arresting this hydrolysis were unsuccessful. Therefore we examined the significance of phosphagen hydrolysis on pHm measurement in human biopsies taken at rest and following intense electrical stimulation. To accomplish this, pHm was measured at 0 degree C, where extensive hydrolysis did not occur. On the same homogenate, pHm was measured at 0 degree C with phosphagens and at 0 and 37 degrees C after phosphagen hydrolysis. The effect of phosphagen hydrolysis on pHm at 0 degrees C was used to estimate this effect at 37 degrees C. In resting samples, phosphagen hydrolysis produced a nonsignificant acidification of 0.008 pH units and, in electrically stimulated samples, a nonsignificant alkalinization of 0.033 units. Measurements of homogenate PCO2 suggested that most of the CO2 remained in the sample during pHm measurement at 37 degrees C. The present work substantiates the use of the homogenate technique as an accurate and practical method for the estimation of intracellular pH in resting and exercise human muscle samples.     

Repeated inseminations required for natural fertility in a wild bird population

In most bird species, pairs copulate many times before egg laying. The exact function of repeated inseminations (i.e. successful copulations) is unknown, but several suggestions have been made. We tested the hypothesis that repeated inseminations are required to ensure fertilization of eggs, by using an experimental method where free-ranging male collared flycatchers (Ficedula albicollis) were prevented from inseminating their mates. We show that egg fertility was lower when females had not copulated during the studied part of their fertile period. By counting sperm on the inner perivitelline layer of eggs, we estimated that a minimum of 86 sperm must reach the site of fertilization to ensure average fertility. Using the timing of inseminations and the numbers of sperm on successive eggs, we show that repeated copulations are necessary to achieve an average rate of fertilization of a single clutch. Our results thus provide evidence that repeated inseminations function to ensure fertilization success. We discuss possible constraints on sperm production and utilization that may have contributed to this pattern.     

The importance of arginine in evolution

The special chemistry and metabolism of arginine has been considered in relation to the evolution of metabolic and structural features of animals. Arginine and compounds derived from it act in muscle as a major reserve of ATP and as a regulatory sink for phosphate. The metabolism of arginine in less complex animals has been extended to produce urea, firstly as an osmotic regulator and subsequently as a means for terrestrial animals to excrete surplus nitrogen. The ornithine component of arginine is metabolised to form important polyamines and also proline. It is also argued that the change from phosphoarginine to phosphocreatine was a permissive step in the development of the vertebrates which are rich in connective tissue.     

Origins and Evolution of Pathways of Anaerobic Metabolism in the Animal Kingdom

Energetic characteristics and functional roles define two maintypes of anaerobicpathways in the animal kingdom: high efficiency/lowrates of energy production pathways geared to anoxia survival(aspartate-succinate and glucose-succinate pathways), and lowefficiency?/high rates of energy production pathways gearedto maintaining or increasing metabolic activity (multiple opinepathways and lactate pathway). The aspartate-succinate and opinepathways require both amino acids and carbohydrate as substrates,whereas the glucose-succinate and lactate pathways are dependenton carbohydrate only. Phylogenetic, functional and chemicalconsiderations indicate an evolutionary progression from aminoacid-linked to carbohydrate-based anaerobic pathways. The tauropineand strombine pathwaysare possibly the most ancient opine pathwaysso far discovered, and the octopine pathway the most advanced.The roles of the aspartate-succinate and opine pathways mayoriginally have been not too dissimilar. A hierarchy of "ratesof energy production pathways" of phosphagen > lactate >octopine > other opine pathways is proposed, which definesmuch of their phylogenetic selection and how they are used.The different properties of phosphocreatine compared to otherphosphagens is indicated to have been a key factor in the emergenceof vertebrates     

Expressions of the prefertilization polar axis in sea urchin eggs

The distribution of pigment granules in eggs of three species of sea urchins is described with reference to developmental stage and an egg's animal-vegetal axis of organization. Polarity in unfertilized sea urchin eggs has been a debated subject; present evidence demonstrates that the animal-vegetal axis is established before fertilization. The pigment pattern in some batches of Paracentrotus eggs exhibiting the celebrated “pigment band,” originally described by Theodor Boveri, is revised and is interpreted as a comparatively precocious expression of the underlying egg polarity. “Unbanded” Paracentrotus eggs and eggs of Arbacia lixula and Arbacia punctulata can be induced to exhibit the same pigment pattern by artificial activation. The induced pigment pattern aligns with an axis defined by polar bodies and the jelly canal, which are two external markers of the animal pole which are only rarely seen. It is therefore concluded that all of these eggs possess an animal-vegetal axis before fertilization even though it usually remains unexpressed until later developmental stages. Polarized changes in pigmentation are consistent with the following general mechanism: A change is triggered in the cortex of the vegetal pole; the change is programmed for a time which corresponds to the fourth mitotic division, even though mitosis itself is not involved; activation at fertilization initiates the “clock” in most cases, although in “banded” Paracentrotus eggs the “clock” is apparently started before ovulation; only the vegetal hemisphere's pigment is affected by the change. The nature of the underlying axis which defines animal and vegetal poles is discussed. Aspects of the axis have been tentatively traced back to the primary oocyte stage, but its fundamental nature remains unknown.     

Creatine ethyl ester: a new substrate for creatine kinase

The creatine kinase/phosphocreatine system plays a key role in cell energy buffering and transport, particularly in cells with high or fluctuating energy requirements, like neurons, i.e. it participates in the energetic metabolism of the brain. Creatine depletion causes several nervous system diseases, alleviated by phosphagen supplementation. Often, the supplementation contains both creatine and creatine ethyl ester, known to improve the effect of creatine through an unknown mechanism. In this work we showed that purified creatine kinase is able to phosphorilate the creatine ethyl ester. The K(m) and V(max) values, as well as temperature and pH optima were determined. Conversion of the creatine ethyl ester into its phosphorylated derivative, sheds light on the role of the creatine ethyl ester as an energy source in supplementation for selected individuals.