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. 1973 May;133(1):117–123. doi: 10.1042/bj1330117

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

W R A Osborne 1,*, N Spencer 1
PMCID: PMC1177676  PMID: 4721618

Abstract

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 Km values for adenosine of about 30μm and Ki values of about 8μm 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.).

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akedo H., Nishihara H., Shinkai K., Komatsu K., Ishikawa S. Multiple forms of human adenosine deaminase. I. Purification and characterization of two molecular species. Biochim Biophys Acta. 1972 Jul 13;276(1):257–271. doi: 10.1016/0005-2744(72)90028-9. [DOI] [PubMed] [Google Scholar]
  2. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BRADY T. G., O'CONNELL W. A purification of adenosine deaminase from the superficial mucosa of calf intestine. Biochim Biophys Acta. 1962 Aug 13;62:216–229. doi: 10.1016/0006-3002(62)90035-5. [DOI] [PubMed] [Google Scholar]
  4. Baer H. P., Drummond G. I., Gillis J. Studies on the specificity and mechanism of action of adenosine deaminase. Arch Biochem Biophys. 1968 Jan;123(1):172–178. doi: 10.1016/0003-9861(68)90116-1. [DOI] [PubMed] [Google Scholar]
  5. Brownson C., Spencer N. Partial purification and properties of the two common inherited forms of human erythrocyte adenylate kinase. Biochem J. 1972 Dec;130(3):797–803. doi: 10.1042/bj1300797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conway E. J., Cooke R. The deaminases of adenosine and adenylic acid in blood and tissues. Biochem J. 1939 Apr;33(4):479–492. doi: 10.1042/bj0330479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cory J. G., Weinbaum G., Suhadolnik R. J. Multiple forms of calf serum adenosine deaminase. Arch Biochem Biophys. 1967 Feb;118(2):428–433. doi: 10.1016/0003-9861(67)90371-2. [DOI] [PubMed] [Google Scholar]
  8. Foster R. J., Niemann C. The Evaluation of the Kinetic Constants of Enzyme Catalyzed Reactions. Proc Natl Acad Sci U S A. 1953 Oct;39(10):999–1003. doi: 10.1073/pnas.39.10.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoagland V. D., Jr, Fisher J. R. Purification and properties of chicken duodenal adenosine deaminase. J Biol Chem. 1967 Oct 10;242(19):4341–4351. [PubMed] [Google Scholar]
  10. Luffman J. E., Harris H. A comparison of some properties of human red cell acid phosphatase in different phenotypes. Ann Hum Genet. 1967 May;30(4):387–401. doi: 10.1111/j.1469-1809.1967.tb00040.x. [DOI] [PubMed] [Google Scholar]
  11. Ma P. F., Fisher J. R. Multiple adenosine deaminases in the frog (Rana catesbeiana). Comp Biochem Physiol. 1968 Oct;27(1):105–112. doi: 10.1016/0010-406x(68)90756-1. [DOI] [PubMed] [Google Scholar]
  12. Markert C. L. The molecular basis for isozymes. Ann N Y Acad Sci. 1968 Jun 14;151(1):14–40. doi: 10.1111/j.1749-6632.1968.tb11876.x. [DOI] [PubMed] [Google Scholar]
  13. Murphy P. M., Noonan M., Collins P., Tully E., Brady T. G. Preparative isolation of the isoenzymes of adenosine deaminase from bovine mucosa by ion-exchange chromatography. Biochim Biophys Acta. 1969 Jan 7;171(1):157–166. doi: 10.1016/0005-2744(69)90115-6. [DOI] [PubMed] [Google Scholar]
  14. Murray A. W. The biological significance of purine salvage. Annu Rev Biochem. 1971;40:811–826. doi: 10.1146/annurev.bi.40.070171.004115. [DOI] [PubMed] [Google Scholar]
  15. Mustafa S. J., Tewari C. P. Latent adenosine deaminase in mouse brain. II. Purification and properties of mitochondrial and supernatant adenosine deaminases. Biochim Biophys Acta. 1970 Nov 11;220(2):326–337. doi: 10.1016/0005-2744(70)90017-3. [DOI] [PubMed] [Google Scholar]
  16. Pfrogner N. Adenosine deaminase from calf spleen. I. Purificiation. Arch Biochem Biophys. 1967 Mar;119(1):141–146. doi: 10.1016/0003-9861(67)90439-0. [DOI] [PubMed] [Google Scholar]
  17. Pfrogner N. Adenosine deaminase from calf spleen. II. Chemical and enzymological properties. Arch Biochem Biophys. 1967 Mar;119(1):147–154. doi: 10.1016/0003-9861(67)90440-7. [DOI] [PubMed] [Google Scholar]
  18. Phelan J., McEvoy F., Rooney S., Brady T. G. Structural studies on adenosine deaminase from calf intestinal mucosa. Biochim Biophys Acta. 1970 Feb 17;200(2):370–377. doi: 10.1016/0005-2795(70)90179-0. [DOI] [PubMed] [Google Scholar]
  19. Rockwell M., Maguire M. H. Studies on adenosine deaminase. I. Purification and properties of ox heart adenosine deaminase. Mol Pharmacol. 1966 Nov;2(6):574–584. [PubMed] [Google Scholar]
  20. Ronca G., Zucchelli G. Competitive inhibition of adenosine deaminase by purine and pyrimidine bases. Biochim Biophys Acta. 1968 Apr 24;159(1):203–205. doi: 10.1016/0005-2744(68)90265-9. [DOI] [PubMed] [Google Scholar]
  21. Scott E. M. Kinetic comparison of genetically different acid phosphatases of human erythrocytes. J Biol Chem. 1966 Jul 10;241(13):3049–3052. [PubMed] [Google Scholar]
  22. Vesterberg O. Isoelectric focusing of proteins in polyacrylamide gels. Biochim Biophys Acta. 1972 Jan 26;257(1):11–19. doi: 10.1016/0005-2795(72)90248-6. [DOI] [PubMed] [Google Scholar]
  23. WILKINSON G. N. Statistical estimations in enzyme kinetics. Biochem J. 1961 Aug;80:324–332. doi: 10.1042/bj0800324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. White I. N., Butterworth P. J. A comparison of the stabilities of the isoenzymes of human erythrocyte acid phosphatase (type B). Biochim Biophys Acta. 1971 Jan 19;229(1):202–207. doi: 10.1016/0005-2795(71)90334-5. [DOI] [PubMed] [Google Scholar]
  25. Wolfenden R. On the rate-determining step in the action of adenosine deaminase. Biochemistry. 1969 Jun;8(6):2409–2412. doi: 10.1021/bi00834a023. [DOI] [PubMed] [Google Scholar]

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