Distribution of orphan metabolic activities

A significant fraction (30-40%) of known metabolic activities is currently orphan. Although orphan activities have been biochemically characterized, we do not know a single gene responsible for these reactions in any organism. The problem of orphan activities represents one of the major challenges of modern biochemistry. We analyze the distribution of orphans across biochemical space, through years of enzymatic characterization, and by biological organisms. We find that orphan metabolic activities have been accumulating for many decades. They are widely distributed across enzymatic functional space and metabolic network neighborhoods. Although orphans are relatively more abundant in less studied species, over half of orphan reactions have been experimentally characterized in more than one organism. Shrinking the space of orphan activities will likely require a close collaboration between computational and experimental laboratories.     

Chitotriosidase,a prematurely orphan enzyme

Human Chitotriosidase (CHIT) is a member of the chitinase family and is synthesized by activated macrophages. Recently, a genetic polymorphism was found to be responsible for the common deficiency in CHIT activity, frequently encountered in different population. We analyzed the CHIT gene in some ethnic groups from Mediterranean and frican area, to evaluate whether the CHIT gene polymorphism correlates with the changes in environmental features and the disappearance of parasitic diseases. We evaluate the plasma CHIT activity and analyzed, by PCR, the Chit gene polymorphism in 100 Sicilian, 107 Sardinian and 99 Sub-Saharan subjects. We found an heterozygote frequency for a duplication of 24 base pair in exon 10 of 44,54% in Sicily and 32,71 % in Sardinia, whereas homozygous chit deficient were 5,45 % and 3,73%, respectively. On the contrary in Burkina Faso, a mesoendemic regio nforPlasmodium falciparum malaria and other intestinal parasites, a low incidence of CHIT mutation was found (heterozygous 2%) and any subject was homozygous for CHIT deficiency. Our results suggest that in sub-Saharan population the intact CHIT gene seems essential for sustaining resistance against chitin-coated parasitic disease, whereas the presence of CHIT gene polymorphism in Mediterranean population could be the result of a recent positive selection due to improved environmental conditions, which makes prematurely orphan this enzyme.     

Rescue of the orphan enzyme isoguanine deaminase

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 μM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 μM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.     

Predicting genes for orphan metabolic activities using phylogenetic profiles

Homology-based methods fail to assign genes to many metabolic activities present in sequenced organisms. To suggest genes for these orphan activities we developed a novel method that efficiently combines local structure of a metabolic network with phylogenetic profiles. We validated our method using known metabolic genes in Saccharomyces cerevisiae and Escherichia coli. We show that our method should be easily transferable to other organisms, and that it is robust to errors in incomplete metabolic networks.     

Elucidation of signaling and functional activities of an orphan GPCR, GPR81

GPR81 is an orphan G protein-coupled receptor (GPCR) that has a high degree of homology to the nicotinic acid receptor GPR109A. GPR81 expression is highly enriched and specific in adipocytes. However, the function and signaling properties of GPR81 are unknown because of the lack of natural or synthetic ligands. Using chimeric G proteins that convert Gi-coupled receptors to Gq-mediated inositol phosphate (IP) accumulation, we show that GPR81 can constitutively increase IP accumulation in HEK293 cells and suggest that GPR81 couples to the Gi signaling pathway. We also constructed a chimeric receptor that expresses the extracellular domains of cysteinyl leukotriene 2 receptor (CysLT2R) and the intracellular domains of GPR81. We show that the CysLT2R ligand, leukotriene D(4) (LTD4), is able to activate this chimeric receptor through activation of the Gi pathway. In addition, LTD4 is able to inhibit lipolysis in adipocytes expressing this chimeric receptor. These results suggest that GPR81 couples to the Gi signaling pathway and that activation of the receptor may regulate adipocyte function and metabolism. Hence, targeting GPR81 may lead to the development of a novel and effective therapy for dyslipidemia and a better side effect profile than nicotinic acid.     

A comparison of ATP-degrading enzyme activities in rat incisor odontoblasts.

In active odontoblasts from the rat incisor, used as a model system for biologic calcification, two distinguishable enzyme activities capable of degrading adenosine monophosphate (ATP) exist. Once can be inhibited ny 1-tetramisole, (+/-)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1B) THIAZOLE HYDROCHLORIDE (Levamisol) and (+/-)-6(m-bromophenyl)-5.6-dehydroimidazo (2.1-b) thiazole oxalate (R823) and is probably identical with nonspecific alkaline phosphatase (EC 3.1.3.1). The activity of the other enzyme, named Ca2+-ATPase, is dependent on the presence of Ca2+ or Mg2+ and is activated by these ions. The pH optimum of Ca2+-ATPase is 9.8. The Ca2+-ATPase is unaffected by Levamisole, R 8231, ouabain, ruthenium red, Na+ and K+ ions. Maximal activity was found against ATP, whereas adenosine diphosphate, guanosine triphosphate, inosine triphosphate and adensoine monophosphate were hydrolysed at lower rate. It may be speculated that the Ca2+-ATPase is concerned with the transmembranous transport of Ca2+ ions to the mineralization front.     

Oxygenate fuels for diesel engines: A survey of world-wide activities

The various possible categories of non-petroleum internal combustion engine fuels are reviewed briefly. The oxygenate fuels (i.e. those containing oxygen in chemical combination) are considered in greater detail. The main types are methanol, ethanol, vegetable oils and mono-esters derived from vegetable oils. The problems involved in applying these fuels to compression-ignition engines are considered.The organisations involved in engine and/or service tests on oxygenate fuels for these engines are listed and a short summary of the nature and extent of the work is given in each case. More than 100 organisations are listed and references are provided in most cases to their published work. Summary tables, showing the activities in each country, are included.The survey covers developments known to the authors by mid-July 1982. Additional organisations and activities, on which information has subsequently become available, are listed in the Appendix.     

Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases

Alpha-synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of alpha-synuclein has not yet been known. Here we have shown that alpha-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of alpha-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with alpha-synuclein. Our results indicate that alpha-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.     

A rapid method for the determination of proteolytic activities of enzyme preparations

A method has been described for the determination of proteolytic activities of enzyme preparations using casein as substrate. The rate of digestion is proportional to the enzyme concentration used. This relationship is utilized as a measure of the enzyme activity. One unit of activity is defined as the amount which is required to digest casein in 15 minutes at 37.5 degrees C. so that 50 per cent of the protein in 1 ml. of 0.25 per cent solution is not precipitable by trichloroacetic acid. This method has been used to determine the activity of enzymes from different sources and also used to follow the rate of activation of enzymes.     

Concanavalin A increases glyoxalase enzyme activities in polymorphonuclear leukocytes and lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.     

几种荞麦的抗氧化酶活性研究

测定了荞麦属六种十个居群的SOD、CAT、POD和ASP活性。结果表明在栽培荞麦中,苦荞的抗氧化酶活性高于甜荞;野生荞麦中差异很大,抗氧化酶活性高的有金荞和贵州云台山大野荞。     

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.