Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.4 (adenosine deaminase)
5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.
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PMID:Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line. 390 13

The present study was undertaken to demonstrate and characterize potentiation of ventricular overdrive suppression by adenosine. To substantiate that adenosine has an enhanced effect on overdrive suppression, it would be necessary to demonstrate that adenosine increases pause duration independent of slowing spontaneous pre-drive rate. In isolated perfused guinea pig hearts with surgically induced complete atrioventricular block, the effect of adenosine (2-20 microM) on pause duration was compared to two alternative means of slowing the pre-drive rate, i.e., hypothermia (28.0 degrees C to 34.0 degrees C) and cesium chloride (0.3-1.0 mM). The slope value of the linear regression line describing the relationship between pre-drive cycle length and pause duration for adenosine (15.8) was significantly greater than control (1.7), hypothermia (1.7), and cesium chloride (5.4). The competitive adenosine antagonist, aminophylline (60 microM), when infused at the initiation of overdrive during adenosine administration, significantly reduced the effect of adenosine on pause duration by 72.9 +/- 4.2% (mean +/- SEM). The reduction in pause duration by aminophylline was specific for adenosine and did not occur under control conditions or during cesium chloride administration. During hypoxia, aminophylline and adenosine deaminase, when infused at the initiation of overdrive, caused 72.3 +/- 5.6 and 63.3 +/- 6.1% reductions in pause duration, respectively. Endogenous adenosine levels rose significantly with hypoxia (1,687 +/- 202 vs. 36 +/- 4 pmol/min per g during normoxia) and increased significantly further during hypoxic overdrive (3,004 +/- 323 pmol/min per g). In isolated guinea pig Purkinje fibers (n = 4), adenosine (20 microM) increased pause duration by 73.6 +/- 9.9% while only minimally affecting the pre-drive cycle length (7.6 +/- 3.8%). These fibers, when stimulated at 1.5 Hz, also displayed an adenosine-induced reduction in action potential duration at 90% repolarization (16 +/- 2 msec). In addition, we demonstrated that adenosine had an enhanced effect on pause duration in the presence of ouabain (1 microM)-induced attenuation of overdrive suppression. Thus, in isolated Purkinje fibers, it is unlikely that the potentiating effect of adenosine on pause duration, which is independent of its chronotropic effect, is mediated via an enhancement of sodium potassium adenosine triphosphatase pump activity. The effect of adenosine is likely to be secondary to a direct action on outward potassium conductance.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of adenosine on ventricular overdrive suppression in isolated guinea pig hearts and Purkinje fibers. 404 82

A study was made to develop a new method for the production of flavine-adenine dinucleotide (FAD) from riboflavine and adenine by a mutant of Sarcina lutea deficient in the enzyme adenosine deaminase. It was found that this strain could convert exogenously supplemented riboflavine to extracellular FAD. The yields of FAD were increased by addition of D-cycloserine in the culture medium. The culture conditions for FAD production were investigated under the addition of D-cycloserine, and increased production of FAD was observed with the addition of an appropriate amount of thiamine, acetate, and sodium ion. The yield of 0.7 g/liter was obtained in the optimal culture in 5 days. Accumulated FAD was readily isolated by adsorption chromatography and ion-exchange chromatography in a 70% yield.
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PMID:Production of flavine-adenine dinucleotide from riboflavine by a mutant of Sarcina lutea. 482 82

The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.
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PMID:New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. 613 22

The adenosine deaminase-binding protein has previously been localized to the cell surface of human fibroblasts (Andy, R. J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925). In this study we examine the biosynthesis of binding protein in human fibroblasts, human hepatoma HepG2 cells, and a human kidney tumor cell line. Binding protein immunoprecipitated from radioiodinated detergent-extracted fibroblast membranes has a molecular weight of 120,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional band of Mr 100,000 is also present which we believe is a result of proteolysis of the 120,000 band. Purified soluble kidney binding protein has an Mr of 112,000. Binding protein from fibroblasts pulse-labeled with [35S]methionine for 15 min migrates as a 110-kDa band on sodium dodecyl sulfate-polyacrylamide gels. Within 30-60 min of chase, the intensity of the 110-kDa band is diminished, and a 120-kDa band has appeared. Binding protein reaches the cell surface of fibroblasts within 30-60 min of chase. The same results are obtained with the other cell lines studied. Thus, binding protein is initially synthesized as a precursor of 110 kDa which chases into a 120-kDa mature form. The shift of 10 kDa is probably due to processing of its oligosaccharide chains since soluble kidney-binding protein contains 7-9 complex N-linked chains. Upon endoglycosidase H treatment, the 110,000 precursor shifts to a Mr of 89,000 while the 120,000 mature band shifts to 115,000, consistent with the presence of 7-9 high mannose chains on the precursor and 1-2 high mannose chains on the mature form. These results and the presence of complex N-linked chains on binding protein were confirmed by lectin affinity chromatography of glycopeptides derived from [2-3H]mannose-labeled binding protein. Analysis of [6-3H]glucosamine-labeled binding protein indicates the presence of 1 sialic acid residue per chain.
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PMID:Biosynthesis of the adenosine deaminase-binding protein in human fibroblasts and hepatoma cells. 614 21

The action of endogeneous adenosine on isolated hamster brown adipocytes was examined. Adenosine production from brown adipocytes was measured after labeling of the intracellular nucleotide pool with [3H]adenine. Accumulation of [3H]adenosine in the incubation medium was maximum after 5 min of incubation and was still present after 20 min. When adenosine accumulation was prevented by addition of adenosine deaminase, the stimulatory effects of isoproterenol on oxygen uptake, lipolysis, and adenosine 3',5'-cyclic monophosphate (cAMP) generation were enhanced. However, basal rates of lipolysis and oxygen consumption and levels of cAMP were not affected on addition of adenosine deaminase. A similar potentiation of isoproterenol responses was produced by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine, present at a concentration (10 microM) which did not change basal levels of respiration or lipolysis. Addition of the adenosine analogue 2-chloroadenosine antagonized isoproterenol-stimulated respiration and lipolysis and prevented potentiation of isoproterenol responses with 3-isobutyl-1-methylxanthine. To localize the site of adenosine action, activity of adenylate cyclase in membrane preparations from brown adipocytes was measured. Isoproterenol-stimulated adenylate cyclase activity was partially inhibited by 2-chloroadenosine in a GTP-dependent manner. Addition of Na+ enhanced the inhibitory effect of 2-chloroadenosine, and 3-isobutyl-1-methylxanthine blocked it. The calculated 50% effective dose for 2-chloroadenosine inhibition was between 10 and 15 nM. These data suggest that adenosine produced by brown adipocytes is an endogenous regulator of respiration in these cells acting at the level of the adenylate cyclase enzyme.
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PMID:Role of adenosine as an endogenous regulator of respiration in hamster brown adipocytes. 619 83

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.
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PMID:Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes. 624 64

Adenosine is known to have effects on electrophysiologic parameters of the sinus node, AV node and atrium and to antagonize isoproterenol-induced increased inotropy in the ventricle. However, the effects on cardiac Purkinje fibers are not well established. Therefore, the purpose of the present experiments was to examine the effects of adenosine alone and adenosine on isoproterenol-treated canine cardiac Purkinje fibers. Microelectrode techniques were used to record transmembrane action potentials. Adenosine alone (10(-7) to 10(-4) M) produced no effects on action potential characteristics of paced fibers. Adenosine in concentrations of 10(-7), 10(-6), 10(-5) and 10(-4) M produced a 15, 24, 44* and 72*% attenuation of isoproterenol (10(-6) M)-induced action potential duration shortening, respectively (*P less than .001). In 7 of 7 fibers depolarized with 22 mM K+, adenosine (10(-4) M) ablated calcium-dependent action potentials restored with isoproterenol (10(-6) M). These effects were antagonized by theophylline (5 X 10(-5) M) and adenosine deaminase (1 U/ml). Action potential shortening due to superfusion of high calcium Tyrode's solution and calcium-dependent action potentials generated in Na+ free-high Ca++ Tyrode's solution were not antagonized by adenosine. Adenosine (10(-5) M) produced a negative chronotropic effect, increasing escape intervals from 2669 +/- 647 to 3702 +/- 717** msec in control fibers and from 1864 +/- 329 to 2658 +/- 399** msec in tyramine (10(-4) M)-treated fibers (**P less than .05), but failed to produce a negative chronotropic response in fibers pretreated with propranolol (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Adenosine attenuation of the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers. 632 81

The uptake of adenosine in brush border vesicles of the proximal tubule of the rat kidney has been studied with a filtration technique. The initial rate of uptake was almost 6 times greater in the presence of NaCl than in the presence of KCl. The stimulatory effect of Na+ was strictly dependent on a gradient of Na+ (out greater than in). The time course of uptake showed an overshoot with a maximum at 20 s with a gradient of NaCl, but not with KCl. Inosine and 5'-AMP were produced from adenosine within the vesicles. In the presence of an inhibitor or adenosine deaminase adenosine was not significantly metabolized during the first 20 s of uptake. Thus, kinetic parameters of transport could be studied in the absence of interferences with metabolism. A Km of 1.1 microM and a Vmax of 232 pmol X min-1 X mg protein-1 were calculated for the Na+ gradient-dependent transport. The dependency on a Na+ gradient, the capacity for uphill transport and the high affinity for adenosine situate this transport system apart from the mechanisms of transport of nucleosides described so far. It may be relevant in regard to the role of adenosine in the regulation of glomerular filtration.
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PMID:Sodium gradient-energized concentrative transport of adenosine in renal brush border vesicles. 647 65

Incubation medium, as previously described (J Histochem Cytochem 27:774, 1979), was used to demonstrate the presence of adenylate cyclase (AC) in myocardium. NaF and ouabain were used to inhibit adenosine triphosphatases (ATP) and NaF and isoproterenol were used as activators of AC. The inhibitory effect of adenosine on AC was blocked by the addition of adenosine deaminase. The addition of tetramisol blocked the influence of the alkaline phosphatases on adenylyl imidodiphosphate hydrolysis. The use of these substances resulted in specific precipitation localized in junctional sarcoplasmic reticulum and sarcolemma. The reaction product was dramatically intensified after activation of AC by NaF or isoproterenol. Preincubation in 10-100 mM of propranolol, for 30 min, blocked AC stimulation by isoproterenol and prevented the appearance of the specific precipitate. The localization of specific precipitate in junctional sarcoplasmic reticulum and subsarcolemmal cisternae corresponds to the localization of Na+, K+ ATPase and may reflect the similar role that AC and Na+, K+ ATPase play in calcium release from sarcoplasmic reticulum of internal and peripheral couplings.
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PMID:Cytochemical studies of myocardial adenylate cyclase after its activation and inhibition. 669 May 96


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