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.).
D000241:Adenosine; D000619:Aminohydrolases / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D004592:Electrophoresis, Starch Gel; D004912:Erythrocytes / Q000201:enzymology*; D005826:Genetics, Medical; D006358:Hot Temperature; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014462:Ultrafiltration
D007527:Isoenzymes; D000619:Aminohydrolases; D000241:Adenosine