Purification of multiple forms of adenosine deaminase from rabbit intestine.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.     

Partial purification and properties of the common inherited forms of adenosine deaminase from human erythrocytes

1. The partial purification of adenosine deaminase, types 1, 2 and 2-1, from human erythrocytes is described. 2. The isoenzyme components characteristic of the three forms of the enzyme were partially resolved by chromatography on DEAE-Sephadex. 3. Gel chromatography of the various forms of the enzyme gave estimates of the molecular weights in the range 30000-35000. 4. Electrophoresis in starch gels containing increasing percentages of starch did not reveal any differences in molecular weight between the genetic variants or their isoenzyme components. 5. Analytical isoelectric-focusing experiments in polyacrylamide gels gave the following pI values for the four isoenzyme components present in type 2-1 erythrocytes: 4.70, 4.83, 4.94 and 5.06. 6. All forms of the enzyme gave K(m) values for adenosine of about 30mum and K(i) values of about 8mum for the competitive inhibitor purine riboside. 7. Reaction rates of the type 1 and 2 enzymes were measured at different temperatures. Both enzymes gave values for the energy of activation for hydrolysis of adenosine of about 33.4kJ/mol (8kcal/mol). 8. Heat inactivation of all forms of the enzyme was markedly dependent on ionic strength, the rate of inactivation increasing with increasing ionic strength. The type 1 and type 2 forms of the enzyme differed significantly in their susceptibility to heat inactivation. From the variation of rates of inactivation with temperature, values were obtained for the energies of activation for the heat inactivation of both enzymes as follows: type 1 enzyme 275.5kJ/mol (65.9kcal/mol) and type 2 enzyme 241.6kJ/mol (57.8kcal/mol.).     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Expression of a functional human adenosine deaminase in transgenic tobacco plants

An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5′ UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves.     

Structure of adenosine deaminase mRNAs from normal and adenosine deaminase-deficient human cell lines.

The structure of human adenosine deaminase mRNA from normal and mutant lymphoblasts was examined by sequence analysis of a cDNA for normal mRNA and electrophoretic analyses of DNA fragments generated by S1 endonuclease cleavage of mRNA-cDNA hybrids. The 1,533-base sequence of the cloned cDNA represents the complete mRNA sequence with the possible exception of some of the 5' untranslated region. S1 nuclease analyses of hybrids between cloned cDNA and normal adenosine deaminase mRNA confirmed that a 76-base sequence in a previously examined adenosine deaminase cDNA is an intron. S1 nuclease analyses of mRNAs from seven mutant cell lines demonstrated that four of the mutants, those in the GM-2471, GM-2756, GM-4258, and GM-2606 cells, contain small defects, such as single-base changes, that are not detectable by the S1 nuclease technique. Three of the mRNAs, those in GM-3043, GM-2294, and GM-2825A cells, do contain defects detectable with S1 nuclease. These defects differ from each other and have been mapped to specific regions of the mRNA. Some or all of these defective mRNAs are postulated to result from anomalous RNA processing.     

Heterogeneity for adenosine deaminase deficiency: Expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells.

Adenosine deaminase (ADA) could be quantitated and the isozyme pattern characterized in cultured amniotic fluid cells. In 20 amniotic fluid cell cultures the mean specific activity was 14.3 U/g protein +/- 6.7 (SD) and compared favorably with values of 14.6 U/g protein +/- 6.8 (SD) observed in 26 cultures of skin fibroblasts. In cultures of skin fibroblasts established from two obligate heterozygotes for ADA deficiency, the specific activity of ADA was 7.0 and 7.7 U/g protein. The ADA isozyme pattern that existed in cultures of amniotic fluid cells was the same as that observed in cultured skin fibroblasts. This identification of the same apparent enzyme may permit the prenatal diagnosis of that form of combined immunodeficiency disease caused by ADA deficiency. Residual enzyme activity of less than 1% and 10% of the mean of normal fibroblasts could be measured in cultured fibroblasts from two unrelated children with ADA deficiency and combined immunodeficiency disease. The tissue-specific enzyme from cultured skin fibroblasts from the child with 10% residual activity had a faster electrophoretic mobility and greater heat stability than normal ADA. This enzymatic evidence indicates that at least two mutant alleles exist at the locus for ADA which predispose to combined immunodeficiency disease when present in the homozygous state.     

The role of GTP in the regulation of two forms of AMP deaminase from chicken kidney

1. Two molecular forms of AMP deaminase have been revealed by phosphocellulose column chromatography of the chicken kidney extract. 2. The chromatographic, kinetic and regulatory properties of these two forms were similar to these of two enzyme forms previously found in the chicken liver, lizard liver and in rat small intestine. 3. GTP exerted different effect from MgGTP on the activity and kinetic parameters of both AMP deaminase I and II from chicken kidney.     

Purification of an adenosine deaminase complexing protein from human plasma.

A protein which specifically complexes with adenosine deaminase (complexing protein) has been purified to homogeneity from human plasma. This protein was compared with complexing protein isolated from human kidney. The two proteins produce electrophoretically different forms of high molecular weight adenosine deaminase when combined with the Mr = 36,000 enzyme monomer from erythrocytes. This difference may, at least in part, be due to the greater sialic acid content of complexing protein from plasma. By other criteria, including amino acid composition, total carbohydrate content, and subunit structure, the two proteins are quite similar. In addition, plasma complexing protein shows complete cross-reactivity with anti-kidney complexing protein serum. These results suggest that plasma and kidney complexing proteins are products of the same gene.     

Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.     

Isolation of cDNA clones for human adenosine deaminase

Clones encoding human adenosine deaminase (ADA) were isolated from a cDNA library made from the lymphoblastoid cell line MOLT-4. The isolation procedure was based on the selection of clones hybridizing with a radioactive probe complementary to an RNA preparation, which had been highly enriched in ADA-specific mRNA. The latter RNA preparation was obtained by size-fractionating MOLT-4 RNA and selecting fractions that were translatable into ADA. The assay for the presence of ADA in the in vitro translation products, was based on immunoprecipitation with a specific anti-ADA serum. The antiserum used was shown to precipitate a 42-kDal protein with the properties of ADA. Positive clones were further screened by means of hybrid-released in vitro translation assays. Two clones were obtained which were able to select mRNA that could be translated into a 42-kDal protein immunoprecipitable with the ADA-antiserum. By use of Southern blots containing DNA from somatic cell hybrids, one of these ADA cDNA clones was assigned to the human chromosome 20 known to contain the ADA gene.