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)

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.
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PMID:A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts. 244 24

Myocardial ischemia is characterized by the liberation of adenosine and by complement-mediated inflammation. We have reported that amidated C3, formed when ammonia (NH3) disrupts the thiolester bond of C3, serves as an alternative pathway convertase, generates C5b-9, and stimulates phagocytic oxidative metabolism. We investigated whether the deamination of adenosine by adenosine deaminase in hematopoietic cells might liberate sufficient ammonia to form amidated C3 and thereby trigger complement-mediated inflammation at ischemic sites. In the presence of 4 mM adenosine, NH3 production per erythrocyte (RBC) was equal to that per neutrophil (PMN) (3.3 X 10(-15) mol/cell per h). Because RBC outnumber PMN in normal blood by a thousandfold, RBC are the major source of NH3 production in the presence of adenosine. NH3 production derived only from the deamination of adenosine by the enzyme adenosine deaminase and was abolished by 0.4 microM 2'-deoxycoformycin, a specific inhibitor of adenosine deaminase. When purified human C3 was incubated with 5 X 10(8) human RBC in the presence of adenosine, disruption of the C3 thiolester increased more than twofold over that measured in C3 incubated with buffer, or in C3 incubated with RBC (P less than 0.05). The formation of amidated C3 was abolished by the preincubation of RBC with 2'-deoxycoformycin (P less than 0.001). Amidated C3 elicited statistically significant release of superoxide, myeloperoxidase, and lactoferrin from PMN. Thus, the formation of amidated C3 by RBC deamination of adenosine triggers a cascade of complement-mediated inflammatory reactions.
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PMID:The erythrocyte as instigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase. 278 75

A rapid, simple, quantitative and sensitive assay for the determination of 5'-nucleotidase has been developed. The method can be applied to both soluble and membrane bound forms of the leukocyte enzyme. Enzyme activity is determined by colorimetric estimation of NH3 released from adenosine, the product of 5'-nucleotidase activity in the presence of adenosine deaminase. The assay may be performed in microtitre plates and read with an automatic multiscan spectrophotometer. Thus it can be applied to a large number of samples for routine medical and research purposes.
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PMID:A colorimetric assay for the determination of 5'-nucleotidase activity. 303 4

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.
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PMID:Radioenzymatic determination of adenosine. 312 68

An assay of adenosine deaminase activity in pleural effusions is described. For the continuous determination of adenosine deaminase, the liberated ammonia is estimated by coupling the liberated NH3 with 2-oxoglutarate. The reaction is followed by the decrease of NADH absorbance at 340 nm. The assay was optimized for a Hitachi 705 analyser, with respect to pH, adenosine concentration and glutamate dehydrogenase activity. The assay is linear to an adenosine deaminase catalytic concentration of 110 U/l. Elevated adenosine deaminase activities are found in pleural effusions of patients with tuberculosis, empyema and mesothelioma. Although elevated adenosine deaminase activity in pleural effusion is not pathognomonic for tuberculosis, it may be valuable as a first screening parameter.
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PMID:A continuous method for the estimation of adenosine deaminase catalytic concentration in pleural effusions with a Hitachi 705 discrete analyser. 406 16

The present work describes an assay which is highly specific for ribose-5-phosphate. The method is based on the following three-stage enzymatic conversion: (1) ribose 5-phosphate in equilibrium ribose 1-phosphate (phosphopentomutase); (2) ribose 1-phosphate + adenine in equilibrium adenosine + Pi (adenosine phosphorylase); (3) adenosine + H2O----inosine + NH3 (adenosine deaminase). Ribose 5-phosphate may be determined either directly following the change in absorbance at 265 nm associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity of inosine formed from [8-14C]adenine, after chromatographic separation of the nucleoside on polyethyleneimine-cellulose. The spectrophotometric assay was used to follow ribose 5-phosphate formation and ribose 1-phosphate consumption catalyzed by phosphopentomutase. Further, the ability of alkaline phosphatase, 5'-nucleotidase and crude extract of Bacillus cereus cells to act on ribose 5-phosphate was tested. The radioenzymatic assay was proved useful in determining the levels of ribose 5-phosphate in rat tissues.
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PMID:Spectrophotometric and radioenzymatic determination of ribose-5-phosphate. 653 May 7

AMP deaminase (AMPD) catalyzes the hydrolytic deamination of AMP to IMP and NH3. This activity is represented throughout mammalian tissues and cells by at least three isoforms. Human AMPD cDNAs have been cloned and sequenced, leading to predictions that each isoform contains distinct amino-ends (N-terminal regions) in contrast to their highly conserved carboxyl-ends (C-terminal regions). Wild type, truncated, and chimeric human AMPD1 (isoform M) and AMPD2 (isoform L) cDNAs were expressed and the resultant activities partially characterized as a means to examine the role of divergent N-terminal regions in these polypeptides (residues 1-262 and 1-258 of isoforms M and L, respectively) on isoform-specific catalytic properties. Similar to activities purified from human tissues, in the presence of monovalent cation, wild type isoform M displayed hyperbolic kinetics in the presence and absence of ATP, whereas wild type isoform L exhibited allosteric activation in the presence of this nucleotide effector. Expression of both a chimeric M (5'-AMPD1)/L (3'-AMPD2) construct and one in which the N-terminal region of isoform L was deleted produced activities that were also allosterically regulated by ATP. However, no AMPD activity was detectable following expression of either a chimeric L (5'-AMPD2)/M (3'-AMPD1) construct or one in which the N-terminal region of isoform M had been deleted. The N-terminal region also affected the relative ability of each recombinant AMPD activity to deaminate substrate analogs modified in either the sugar or the phosphate, but not in the purine base, moieties of AMP. These combined data show (i) that isoform M, but not isoform L, absolutely requires its N-terminal region for proper function, (ii) that the C-terminal region of isoform L is responsible for allosteric activation by ATP, (iii) an effect of the N-terminal region on substrate-enzyme interaction, a contention that is discussed in context with available information regarding the related purine catabolic activity, adenosine deaminase.
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PMID:Divergent N-terminal regions in AMP deaminase and isoform-specific catalytic properties of the enzyme. 764 62

A new kinetic method for the determination of serum adenosine deaminase (EC 3.5.4.4) is described, with adenosine as the substrate and nucleoside phosphorylase and xanthine oxidase as the reaction enzymes. Inosine is produced, which is converted to hypoxanthine. The hypoxanthine is oxidized to xanthine, which is further oxidized to uric acid. In these two reactions, blue 2,6-dichlorophenolindophenol is reduced to a colorless compound and the decrease in color is measured spectrophotometrically at 606 nm. The assay was automated by using a Cobas Mira analyzer. The automated assay had a CV of < 7%, and the calibration curve was linear from 10 to 120 U/L. The assay correlates well with an established method, based on detection of liberated NH3 with Berthelot's reaction. The reference interval (mean +/- 2 SD) was 14-34 U/L (mean 24 U/L, n = 84). The enzymatic method described is easily automated and seems to be suitable for the routine determination of adenosine deaminase in serum.
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PMID:Kinetic determination of serum adenosine deaminase. 840 5

Adenosine 5'-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) J. Biol Chem. 265, 4420-4426]. The enzyme contains 0.98 +/- 0.17 zinc atom per subunit. The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue. On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn(2+)-activated water molecule to attack C6 of AMP with the displacement of NH3. The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4. This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Taylor, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 +/- 0.2 and 7.7 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic mechanism of yeast adenosine 5'-monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects. 850 99

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3 , the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.
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PMID:A rapid enzymatic assay for high-throughput screening of adenosine-producing strains. 2558 Aug 42


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