Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Evaluation of adenosine deaminase assay for analyzing t-lymphocyte density in vitro

Summary The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.     

Adenine nucleotide metabolism in relation to purine enzymes in liver,erythrocytes and cultured fibroblasts

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hypoxanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5′-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5′-nucleotidase present in this tissue interferred with the formation of LaCl₃ salts. The lanthanum and the thin-layer chromatographic methods agreed with-in 10%.Liver cell sap had the highest activities of all purine enzymes except for 5′-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erythrocytes were devoid of 5′-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue.Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5′-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control of adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte > liver > fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase

Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed lambda(max) of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl-p-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.     

A rapid radiometric assay for dihydrofolate reductase

A rapid radiochemical procedure for the measurement of dihydrofolate reductase activity is described. The method employs separation of the radiolabeled substrate from the products of the reaction by precipitation with acetic acid and zinc sulfate. This method permits rapid processing of samples and climinates time-consumlng column chromatography techniques. Good agreement of results is obtained when the radioactive method is compared to the spectrophotometric assay method.     

A micromethod for estimation of adenosine deaminase and adenosine nucleosidase with modified cellulose nitrate membranes

Modified cellulose nitrate membrane strips were applied in a new chromatographic procedure for rapid and sensitive estimation of adenosine deaminase (EC 3.5.4.4) and adenosine nucleosidase (EC 3.2.2.7). In this method the enzymes serve each other as reagents. The products of their subsequent action are adenine and inosine, well separable on membrane strips, thanks to the different adsorptive affinities of these two compounds to the cellulose nitrate membranes. Employing adenine-labeled adenosine, microgram amounts of wet biological material may be used for estimation of the enzymes. The method has been applied to routine estimations of these two enzymes in various biological materials and examples are presented. A simple method is described for preparative purification and stabilization of adenosine nucleosidase of barley leaves used as reagent for adenosine deaminase assay.     

Radiochemical assays for adenylate kinase and AMP deaminase using polyethyleneimine-cellulose thin layers

Simple and specific radiochemical asays for adenylate kinase and AMP deaminase activities in crude tissue extracts are described. The radioactive substrate (AMP) is separated from the radioactive product (ADP or IMP) by chromatography on polyethyleneimine-cellulose thin layers. A rapid modification of the adenylate kinase assay is described in which samples of the reaction mixture are transferred directly to the polyethyleneimine-cellulose thin layer.     

Human adenosine deaminase. Purification and subunit structure.

Human erythrocyte adenosine deaminase has been purified approximately 800,000-fold to apparent homogeneity using antibody affinity chromatography. The enzyme was shown to be a single polypeptide chain with an estimated molecular weight of approximately 38,000. The three electrophoretic forms of erythrocyte adenosine deaminase purified simultaneously by this technique were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Several properties of the highly purified adenosine deaminase including pH optimum, Km for substrate, Ki for product, Stokes radius, sedimentation coefficient, and apparent substrate specificity were identical with the properties observed with an impure preparation of the enzyme.     

Radioactive-electrophoretic assay of adenosine 5'-triphosphate sulfurylase activity in crude extracts with sulfate or selenate as a substrate

An assay method for ATP sulfurylase is presented which employs Na2(35)SO4 as a substrate and measures the production of labeled adenosine 5'-phosphosulfate and 3'-phosphoadenosine 5'-phosphosulfate by low-voltage, hanging paper strip electrophoresis. The method is applicable to crude bacterial or mammalian extracts and accurately measures picomole amounts of product(s). Na2(75SeO4 can also be employed as a substrate, if the unstable radioactive product, adenosine 5'-phosphoselenate, is converted to elemental 75Se degrees by inclusion of reduced glutathione in the reaction mixture. The same paper strip electrophoretic technique can then be used to separate 75Se degrees from the radiolabeled substrate. The method also has utility for measuring any direct reduction by crude microbial extracts of radioactive selenate to selenite, independent of ATP sulfurylase.     

Purification of Adenosine Deaminase from Chicken-Egg Yolk by Affinity Column Chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Adenosine deaminase of cultured brain cells

Two types of adenosine deaminase (EC 3.5.4.4) were found in cultured cells of central-nervous-system origin. The predominant and more active enzyme was obtained in soluble form from the cytosol of mouse neuroblastoma (N-18), neonatal hamster astrocytes (NN), human oligodendroglioma (HOL) and human astrocytoma (Cox Clone). Particulate adenosine deaminase was probably associated with the plasma membrane. When radioactive adenosine was added to superfusates of monolayer cultures it was rapidly converted into inosine and hypoxanthine. The metabolic conversion required adenosine uptake by the cells, a probable transition through the intracellular ATP pool(s) and a rapid excretion into the superfusate of the catabolic products. We discuss the evidence that points to adenosine and its derivatives as neurohumoral modulators of central-nervous-system function.     

Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells.

A novel assay for 5'-nucleotidase is described in which 1,N6-etheno-AMP is converted into ethenoadenosine. The product ethenoadenosine is neither a substrate for nor an inhibitor of adenosine deaminase. Ethenoadenosine appears to have little effect at adenosine receptors on adipose-tissue cells.     

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.     

Isolation of mutant adenosine deaminase by coformycin affinity chromatography

Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.     

Cyclic AMP-Generating Systems in Cell-Free Preparations from Guinea Pig Cerebral Cortex: Loss of Adenosine and Amine Responsiveness Due to Low Levels of Endogenous Adenosine

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine-labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP-generating systems. It is proposed that, in particulate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or coupling of receptors to cyclic AMP-generating systems.     

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaryotic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing paired-ion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formycin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent Km of 18.2 microM and an apparent Vmax of 32.4 mmol min-1 mg-1 were observed for the activity. A purine-nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent Km of 236 microM and Vmax of 2.78 mumol min-1 mg-1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent Ki value of 136 microM was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a Km value for GTP, IMP, and aspartic acid of 23, 34, and 714 microM, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have Km and Vmax values for hypoxanthine of 55.5 microM and 34.3 nmol-1 min-1 mg-1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subtraction method for the high-performance liquid chromatographic measurement of plasma adenosine

The measurement of plasma adenosine with traditional high-performance liquid chromatographic techniques is difficult because of its nanomolar concentration, its short half-life in blood, and because of the difficulty in isolating adenosine from interfering peaks in the chromatogram. To prevent loss of adenosine in the blood sample, a “stop solution” is used to prevent enzymatic degradation and cellular uptake. Peak-shifting techniques on fractionated samples to measure adenosine derivatives have been used in the past to avoid interfering peaks in the chromatogram. A new method has been developed by which nanomolar levels of plasma adenosine can be accurately measured despite co-eluting peaks in the chromatogram. In this method, plasma samples are collected with a stop solution, processed, and divided. Adenosine deaminase is added to part of the sample to form a blank. A computer program subtracts the blank chromatogram from the paired unknown, and the result is compared to adenosine standards prepared from the blank and subtracted in a similar fashion. With this subtraction method, the overall recovery of physiological concentrations of adenosine was 89% from dog blood, and the average coefficient of variation was 12%. In summary, the subtraction method of plasma adenosine measurement offers good recovery, reproducibility, and the ability to quantify low levels of adenosine despite interfering peaks in the chromatogram.     

Theoretical study of antagonists and inhibitors of mammalian adenosine deaminase. II. Isomeric aza-deazaanalogues of adenosine

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of the electrostatic potential around their van der Waals molecular surface were calculated using the ab initio STO-3G method. The conformational studies were carried out by the MM+ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.     

Theoretical Study of Antagonists and Inhibitors of Mammalian Adenosine Deaminase: II. Isomeric Aza-deazaanalogues of Adenosine

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of electrostatic potential around their van der Waals molecular surface were calculated using the ab initioSTO-3G method. The conformational studies were carried out by the MM⁺ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.