Effects of different expression systems on characterization of adenylate deaminase from Aspergillus oryzae.

Adenylate deaminase (AMPD) is an amino hydrolase that catalyzes the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, the effect of different hosts on the enzymatic properties of AMPD from Aspergillus oryzae GX-08 was investigated and showed that Bacillus subtilis WB600 was more suitable for producing AMPD with a higher activity of 2540 U/mL. After purification, the optimal temperature and pH of recombinant AMPD were 55 °C and pH 6.0, respectively, and its activity was significantly enhanced by 10 mM Fe3+ with an increase of 236%. More importantly, the recombinant AMPD specifically and effectively catalyzed the conversion between AMP and IMP, in which 10 mL of crude AMPD achieved a conversion ratio of 76.4% after 40 min. Therefore, B. subtilis WB600 provides a potential platform for producing AMPD with excellent catalytic ability and catalytic specificity.

Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP.

JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4-Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes.

Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes. AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis.

Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes.

OBJECTIVE: In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. METHODS: Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. RESULTS: We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066). CONCLUSION: Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation.

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.

Gene expression profiles among immature and adult reproductive castes of the termite Reticulitermes flavipes.

Array-based genomic studies were conducted with the goal of identifying immature (i.e. nymph) and adult reproductive caste-biased gene expression in the termite Reticulitermes flavipes. Using cDNA macro-arrays, we identified thirty-four nymph-biased genes falling into eight ontogenic categories. Based on gene expression profiles among diverse castes and developmental stages (determined by quantitative PCR), several important trends emerged. These findings highlight the importance of several developmental and survival-based factors among immature and adult termite reproductives, including: vitellogenesis, nutrient storage, juvenile hormone sequestration, ribosomal translational and filtering mechanisms, fatty acid biosynthesis, apoptosis inhibition, and both endogenous and symbiont cellulase-assisted nutrition. These findings are highly significant as they are the first to elucidate the molecular biology underlying termite reproductive caste differentiation and reproductive caste-specific biology. Other gene expression results are in agreement with previous findings that suggest roles for vitellogenin-like haemolymph proteins in soldier caste differentiation.

Coordinate induction of AMP deaminase in human atrium with mitochondrial DNA deletion.

Despite the heteroplasmic lower population of mitochondrial (mt) DNA deletion, mtDNA deletion is significantly related to the loss of atrial adenine nucleotides. To elucidate its mechanism, we examined the frequency of a 7.4-kb mtDNA deletion, the concentration of adenine nucleotides, and the activity of AMP catabolic enzymes in 10 human right atria obtained from cardiac surgery, using quantitative PCR, HPLC, and immunoprecipitations. The atrial concentrations of ATP, ADP, AMP, and the total adenine nucleotides were significantly lower in patients with deletion than those in patients without deletion, despite the lower frequency of their deletion. The activities of total AMP deaminase (AMPD), liver-type (AMPD 2), and heart-type isoform (AMPD 3) were significantly higher in patients with deletion than in patients without deletion, although there was no significant difference in the cytosolic 5(')-nucleotidase among them. In conclusion, mtDNA deletion coordinately induces AMP deaminase to contribute to the loss of atrial adenine nucleotides through degrading AMP excessively.

Metabolic causes of recurrent rhabdomyolysis.

OBJECTIVES: The aim of the study was to evaluate the biochemical causes of recurrent rhabdomyolysis in Finland. MATERIAL AND METHODS: We examined 22 patients with recurrent rhabdomyolysis, and 26 patients with one episode of rhabdomyolysis or other symptoms compatible with metabolic myopathy. Muscle histopathology and activities of phosphorylase (PHRL) (total and active), phosphofructokinase (PFK), carnitine palmitoyltransferase (CPT) and myoadenylate deaminase (MAD) were studied. The limit of enzyme deficiency was defined as enzyme activity less than 5% of the mean of the control subjects. RESULTS: We found 4 patients with muscle PHRL deficiency, 1 patient with PFK deficiency and 1 patient with evidence of phosphorylase kinase deficiency. One patient had Becker's muscle dystrophy, 2 patients had unspecified dystrophies, 1 patient had Miyoshi myopathy, and 1 patient had a form of mitochondrial encephalomyopathy (MELAS). CONCLUSION: Enzyme defects were found in 23% of the patients with recurrent rhabdomyolysis. Other muscle diseases, muscular dystrophies or myopathies, were detected in 18% of these patients, emphasizing the value of clinical and histopathological examination of patients with previous rhabdomyolysis.

Protective effect of adenylate deaminase (from Penicillium lanoso-viride) against acute infections in mice.

We examined the effects of the immunomodulator-adenylate deaminase (E.C. 3.5.4.6) from Penicillium lanoso-viride on experimental mice infections. Prophylactic intraperitoneal administration of adenylate deaminase (ADA) increased survival time and numbers of survivors after infection with Salmonella typhimurium, Pseudomonas aeruginosa and influenza A (H3N2) virus. Protection against influenza virus after intranasal ADA application was also observed. The influence of ADA was time and dose dependent. The most pronounced protection was obtained by administration of 3 U ADA/mice 24 h prior to infection. ADA had no antibiotic effect against these bacterial strains. Protective effects of ADA were studied in immunosuppressed mice under different regimes of treatment including cyclosporin A and trypan blue. The results indicated, that the protective effect of ADA is of a complex nature and probably depends on both T-cell and macrophage components.

Characterization of the human AMPD3 gene reveals that 5' exon useage is subject to transcriptional control by three tandem promoters and alternative splicing.

Previous work has identified multiple human AMPD3 transcripts proposed to differ by mutually exclusive alternative splicing of three exons located at, or near, the 5' end of the gene. In this study, we perform a more comprehensive evaluation of human AMPD3 gene expression. Combined Northern blot and RNase protection analyses show that alternative mRNAs are widely expressed in human tissues and cells, but at variable relative abundances. Sequencing of human genomic clones, together with human-mouse somatic cell hybrid analysis, demonstrates that the entire gene is comprised of seventeen exons spanning approx. 60 kilobases on the short arm of chromosome 11 in the region p13-pter. Together, RT-PCR and additional RNase protection analyses establish that exons 1a, 1b, and 1c are 5' terminal sequences in alternative transcripts. Transient transfection experiments show fusion constructs containing proximal flanking and 5' untranslated sequence from each of these exons are able to direct expression of a reporter luciferase gene in mammalian cell lines. These combined results reveal that AMPD3 gene expression is subject to transcriptional control by three tandem promoters. Differential regulation of the exon 1b promoter in skeletal myocytes, as compared to retinal pigment epithelial cells, is proposed to be mediated by skeletal muscle-specific basic helix-loop-helix protein/E-box interactions. Finally, an internal splice acceptor site in exon 1c is shown to be used alternatively to retain the 3' portion of this exon in mature AMPD3 transcripts initiating upstream in exon 1b.

Characterization of human AMP deaminase 2 (AMPD2) gene expression reveals alternative transcripts encoding variable N-terminal extensions of isoform L.

AMP deaminase (AMPD) is a highly regulated enzymic activity and multiple isoforms of this enzyme are coded for by a multigene family in mammalian species, including man. Isoform L (liver) is the main activity present in adult human liver and is the protein product of the AMPD2 gene, which is widely expressed in non-muscle tissues and cells. A previous report described almost the full-length cDNA sequence and part of the human AMPD2 gene and also presented Northern blot evidence for multiple transcripts in brain. This study was performed to further characterize the AMPD2 gene and its expression in human tissues. AMPD2 genomic and human cerebellum cDNA clones were isolated, sequenced and used as probes in RNase protection analyses which together demonstrated the following: (1) an intervening sequence near the 5'-end of the published AMPD2 cDNA, which affects the predicted N-terminal amino acid sequence of isoform L; (2) alternative transcripts resulting from exon shuffling at, or near, the 5'-end of the AMPD2 gene that exhibit tissue-specific patterns of relative abundance; (3) predicted usage of three different initiation codons to confer variable N-terminal extensions on isoform L polypeptides; and (4) an extension of a 3' untranslated sequence in some AMPD2 transcripts. In addition, reverse transcriptase PCR and additional RNase protection analyses were used to map the 5'-ends of two mutually-exclusive exon 1 sequences, both of which contain multiple transcription-initiation sites. These results are discussed in relation to predicted isoform L diversity across human tissues and cells.

Characterization of AMD, the AMP deaminase gene in yeast. Production of amd strain, cloning, nucleotide sequence, and properties of the protein.

The structural gene for AMP deaminase (AMD) from Saccharomyces cerevisiae has been cloned and characterized. A yeast strain deficient in AMP deaminase activity was produced and shown to be deficient in AMP deaminase protein by Western blot analysis. The gene for AMP deaminase was located in a lambda gt11 library of yeast genomic DNA, and a DNA fragment from the lambda gt11 clone was used to locate homologous DNA in a yeast genomic library in the centromeric plasmid YCp50, a yeast-Escherichia coli shuttle vector. One plasmid was selected for its ability to restore AMP catalytic activity to the deficient strain. Yeast deficient in AMP deaminase or those overproducing the enzyme grow at near normal rates. The open reading frame corresponding to AMD codes for a protein of 810 amino acids, molecular weight 93,286. The yeast AMD transcript is 3.0 +/- 0.2 kb, and the transcriptional initiation sites have been identified. Western blot analysis of extracts prepared from actively growing yeast indicates a major band at approximately 96,000 molecular weight with several bands at lower molecular weight, including 83,000. When the AMD gene is expressed in E. coli, the large Mr form of AMP deaminase is produced. These results show that the purified enzyme (Mr = 83,000) is a truncated form of the full-length translation product. No adenine nucleotide binding sites were located based on the consensus sequence from other nucleotide binding proteins. No overall homology was found between yeast AMP deaminase and E. coli AMP nucleosidase. Although their metabolic roles and regulatory mechanisms are similar, these enzymes have arisen from separate ancestral proteins.

Expression of three stage-specific transcripts of AMP deaminase during myogenesis.

AMP deaminase, a ubiquitous enzyme in eucaryotes, plays a central role in energy metabolism. In the present study, RNase protection analyses and immunoprecipitation with tissue-specific antisera were used to examine the transcripts and peptides of AMP deaminase produced during myogenesis in vivo and during myocyte differentiation in vitro. In embryonic muscle and undifferentiated myoblasts, a 3.4-kilobase (kb) transcript encoded a 78-kilodalton (kDa) AMP deaminase peptide that cross-reacted with antisera raised to the AMP deaminase isoform purified from kidney of the adult animal. In perinatal muscle and myocytes at an intermediate stage of differentiation in vitro, a 2.5-kb transcript was produced, and it encoded a 77.5-kDa AMP deaminase peptide that cross-reacted with antisera to the isoform purified from adult heart muscle. At about the time of birth, another 2.5-kb AMP deaminase transcript that encoded an 80-kDa peptide became detectable. This peptide cross-reacted with antisera to the predominant isoform purified from adult skeletal muscle.

Evidence for sequential expression of multiple AMP deaminase isoforms during skeletal muscle development.

AMP deaminase (myoadenylate deaminase; EC 3.5.4.6) is an integral part of the myofibril in skeletal muscle, and this enzyme plays an important role in energy metabolism in this tissue. We report here the identification of three AMP deaminase isoforms during skeletal muscle development in the rat. An embryonic isoform is expressed in the developing hindlimb of the rat between 7 and 14 days of gestation. This isoform is not unique to skeletal muscle or the embryo as it is also expressed in many nonmuscle tissues of the perinatal and adult rat. A perinatal isoform of AMP deaminase that is restricted to skeletal muscle is produced 4-6 days before birth and persists for 2-3 weeks of postnatal life. An adult, skeletal muscle-specific isoform of AMP deaminase appears at birth and reaches maximal levels after 3 weeks of postnatal development. We conclude from these studies there is a developmentally controlled program that leads to the sequential expression of AMP deaminase isoforms during the transition from embryonic to adult skeletal muscle.

Ammonia metabolism in exercise and fatigue: a review.

Although fatigue is a well-known phenomenon and the phrase "exercised until exhaustion" is commonly understood, there is no unequivocal agreement on the fundamental nature of the fatigue process. Ammonia was linked to the development of fatigue as early as 1922, when ammonia production was observed from stimulated nerve and the question whether there could be a relationship between ammonia production and the muscle activity was raised. The immediate source of ammonia from muscle appears to be a result of the deamination of AMP and is more apparent in fast-twitch than in slow-twitch fibers. More recently, increases in blood ammonia levels have been reported in rats after swimming and in humans after arm work, maximal cycle ergometry, and treadmill exercise. Elevated blood ammonia has also been linked to a surprising variety of functional and metabolic neurological disturbances other than exercise and fatigue, including the development of hepatic coma, convulsions from ammonia toxicity precipitated by high-pressure oxygen breathing, epileptic seizures, and decreased neuronal excitability. In addition, a number of genetic disorders (inborn errors in metabolism, or IEMs) are characterized by elevated blood ammonia concentrations. Symptoms of neural disability in all of the above conditions have been related to the concentration of ammonia in blood. Although these studies do not relate to exercise or fatigue directly, it is conceivable that our understanding of the effect of high concentrations of blood ammonia in these clinical conditions may provide valuable insight into the effect of ammonia during exercise. This paper reviews the effect of ammonia production during exercise and other conditions upon purposeful activity and the development of fatigued states.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.