Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats.

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6.) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, Pi and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10--20% of its maximum capacity.     

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.     

Intracellular distribution of fumarase in rat skeletal muscle

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11–19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.     

The effect of spermine on the interaction of AMP deaminase with threonine dehydratase activity in yeast

Evidence suggesting that AMP deaminase (EC 3.5.4.6) is responsible for the stimulation of threonine dehydratase (EC 4.2.1.16) activity in situ is presented using yeast cells which have been rendered permeable. The addition of polyamine, an activator of AMP deaminase, resulted in the increase in ammonia concentration, which can stimulate the activity of yeast threonine dehydratase. Polyamine may regulate the synthesis of isoleucine and valine, and of the intermediates of citric acid cycle through the activation of AMP deaminase-threonine dehydratase system as a ‘cascade system’ in yeast.     

固态发酵生产腺苷酸脱氨酶

对多株曲霉产腺苷酸脱氨酶的性能进行了比较,发现米曲霉3.800(Aspergillus oryzae)产酶水平较高。该菌株固态发酵产酶的适宜培养基为：以麸皮为主原料,蔗糖2％,鱼粉2％,（NH4）2SO4 0．1％,柠檬酸钠0．2％,MgSO4 0.05%,吐温－80 0．1％,含水量50％。最佳的培养条件为：250mL三角瓶装20g培养基,在28－30℃培养60h。在优化条件下,培养物酶活可达到1543．48u/g鲜曲。     

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.     

miRNAS in normal and diseased skeletal muscle

The last 20 years have witnessed major advances in the understanding of muscle diseases and significant inroads are being made to treat muscular dystrophy. However, no curative therapy is currently available for any of the muscular dystrophies, despite the immense progress made using several approaches and only palliative and symptomatic treatment is available for patients. The discovery of miRNAs as new and important regulators of gene expression is expected to broaden our biological understanding of the regulatory mechanism in muscle by adding another dimension of regulation to the diversity and complexity of gene-regulatory networks. As important regulators of muscle development, unravelling the regulatory circuits involved may be challenging, given that a single miRNA can regulate the expression of many mRNA targets. Although the identification of the regulatory targets of miRNAs in muscle is a challenge, it will be critical for placing them in genetic pathways and biological contexts. Therefore, combining informatics, biochemical and genetic approaches will not only expected to reveal the elucidation of the miRNA regulatory network in skeletal muscle and to bring a better knowledge on muscle tissue regulation but will also raise new opportunities for therapeutic intervention in muscular dystrophies by identifying candidate miRNAs as potential targets for clinical application.     

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Mg²⁺-selective microelectrodes have been used to measure the intracellular free Mg²⁺ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg²⁺ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg²⁺. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than ?82 mV, the intracellular free Mg²⁺ concentration was 3.8±0.41 (S.E.) mM (n=58) at 22°C. No significant change in the intracellular free Mg²⁺ was observed following extensive (approx. 6 h) incubation in Mg²⁺-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg²⁺]_i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na⁺-free solutions. In depolarized muscle fibers (?23±2.7 mV) treated with 100 mM K⁺, the myoplasmic [Mg²⁺] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg²⁺. These results point out that [Mg²⁺]_i is not modified under these three different experimental conditions.     

The regulatory role of spermine and fatty acid in the interaction of AMP deaminase with phosphofructokinase

The role of fatty acid and polyamine in the interaction of AMP deaminase (EC 3.5.4.6)-ammonium system with glycolysis was investigated using permeabilized yeast cells. (1) The addition of fatty acid inhibited the activity of AMP deaminase in situ, resulting in a decrease in the total adenylate pool depletion, and in the recovery of the adenylate energy charge. (2) The addition of fatty acid resulted in an indirect decrease in the activity of phosphofructokinase (EC 2.7.1.11) through a reduced level of ammonium ion; fatty acid itself did not inhibit phosphofructokinase activity in the presence of excess ammonium ion. (3) Spermine protected AMP deaminase from inhibition by fatty acid: the increased ammonium level enhanced phosphofructokinase activity, glycolytic flux and the recovery of the energy charge. In contrast, alkali metals, which are also activators of AMP deaminase had little effect on the inhibition of the enzyme. The inhibition of glycolysis by fatty acid and its reversal by polyamine can be accounted for by the changes in ammonium ion through the action of AMP deaminase-ammonium system, and the physiological relevance is discussed.     

Comparative enzymology of AMP deaminase,adenylate kinase,and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15-to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution. Lower skeletal muscle activities in anurans may simply represent the contribution of tonic muscle fiber bundles containing low levels of adenosine monophosphate deaminase, but the explanation for the extremely high adenosine monophosphate deaminase levels in heart ventricular muscle is not apparent.Abbreviations AK adenylate kinase - AMP adenosine monophosphate - AMPD, AMP deaminase - CPK creatine (phospho)kinase - EHNA erythro-9-(2-hydroxy-3-nonyl)-adenine-HCl     

Compartmentation of high-energy phosphates in resting and working rat skeletal muscle

The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

Purification and characterization of adenosine deaminase from camel skeletal muscle

Adenosine deaminase was purified (780-fold) from skeletal muscle of camel (Camelus Dormedarius) to homogeneity level by using DEAE Sephadex chromatography, ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The enzyme appeared to be monomeric with subunit molecular weight of 43kDa and isoelectric point of 4.85. The enzyme showed specificity for adenosine and exhibited Michaelis-Menten Kinetics with kappa(cat) of 1112.41 min(-1) and K(m) of 14.7 microM at pH 7.5. The pH and temperature optima for enzyme activity were 7-7.5 and 25 degrees C, respectively. Free energy (DeltaG*), enthalpy (DeltaH*) and entropy (DeltaS*) of activation for denaturation of adenosine deaminase at 50 degrees C were 88.94, 99.65 kJmol(-1) and 33.16 Jmol(-1), respectively. The purified enzyme had half-lives of 636 and 61 min at 25 and 50 degrees C, respectively. The activation energy for catalysis of camel skeletal muscle adenosine deaminase was 9.13 kJmol(-1). Free energy (DeltaG#), enthalpy (DeltaH#) and entropy (DeltaS#) of activation for hydrolysis of adenosine deaminase at 25 degrees C were 50.35, 6.65 kJmol(-1) and -146.62 Jmol(-1), respectively. Purine riboside inhibited the enzyme competitively with K(i) of 16 microM.     

Isolation and kinetic properties of 5′-nucleotidase from guinea-pig skeletal muscle

5′-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5′-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg²⁺. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The K_i values for ADP and ATP are 0.036 and 0.28 μM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.     

Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms

Summary— In this study, using a modified electrophoretic technique, we have defined in the mouse the myosin heavy-chain composition of both newborn and adult skeletal and cardiac muscles. Using this high resolution technique it was possible to detect modifications in the myosin heavy-chain expression in both cardiac and skeletal muscles of desmin knock-out mice.     

EMBRYONIC FACTOR 1 encodes an AMP deaminase and is essential for the zygote to embryo transition in Arabidopsis

Fusion of the egg and the sperm cells in plants produces a zygote that develops into an embryo. Screening of ethyl methanesulfonate-mutagenized populations of Arabidopsis led to the identification of EMBRYONIC FACTOR 1 (FAC1), a locus that gives a zygote-lethal phenotype when mutated. The FAC1 gene was identified by positional cloning and confirmed by a genetic complementation test against a T-DNA insertion allele. It encodes an AMP deaminase (AMPD) that is known in human and yeast to convert AMP to IMP to maintain the energy potential. Expression of FAC1 in a yeast AMPD mutant after removal of its N-terminal putative transmembrane domain complemented the mutant phenotype, suggesting a functional conservancy but a structural divergence through evolution. Although a low level of FAC1 expression was observed in all organs tested, using a reporter construct we observed a significantly increased FAC1 expression in the zygote, early embryo and endosperm. Furthermore, during somatic embryogenesis, a high level of FAC1 expression was observed in developing embryos including putative embryogenic cells. FAC1, therefore, represents one of the earliest expressed genes known in plants. It may act through AMP depletion to provide sufficient energy for the zygote to proceed through development.     

用GAP启动子在Pichia pastoris GS115中组成型表达鼠灰链霉菌腺苷酸脱氨酶

【目的】构建产AMP脱氨酶的重组毕赤酵母(Pichia pastoris GS115)菌株,并初步优化其发酵条件。【方法】以鼠灰链霉菌(Streptomyces murinus)基因组为模板PCR扩增获得腺苷酸脱氨酶基因AMPD,以pGAP9K为载体构建重组表达质粒pGAP9K-AMPD并通过电转化法转入Pichia pastoris GS115,筛选转化子对其酶活进行测定,并初步优化其发酵条件。【结果】构建了毕赤酵母重组菌,通过分光光度法测定,显示重组菌有明显的酶活;初步优化发酵条件为:该重组菌最适发酵培养基为:甘油2%,蛋白胨2%,酵母膏1%,KH2PO40.5%,MgSO4·7H2O0.05%,pH 6.0;发酵条件为:接种龄24 h,转接量3%,30°C﹑200 r/min培养96 h,取发酵上清液测定酶活,重组菌腺苷酸脱氨酶酶活达到2 230±60 U/mL。【结论】构建了一株产AMP脱氨酶活性较高的重组毕赤酵母菌株,并通过优化发酵条件使其酶活达到2 230±60 U/mL。为AMP脱氨酶工业化生产奠定了一定的基础。     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.