Affinity purification of human deoxycytidine kinase: avoidance of structural and kinetic artifacts arising from limited proteolysis.
Homogeneous deoxycytidine kinase has been isolated from leukemic human T-lymphoblasts by affinity chromatography based on a multisubstrate analog, deoxycytidine 5'-adenosine 5"'-P1,P4-tetraphosphate (dCp4A). Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the Mr of the native protein, 59,300, is comparable to the value of 52,000 from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric pH was 6.1. But, enzyme isolated without protease inhibitors exhibited two fragments of Mr = 30,000 and 33,000, suggesting that proteolytic cleavage of the parental polypeptide had occurred during affinity chromatography. Both the parental and proteolyzed enzymes phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. However, the proteolyzed enzyme had an increased apparent Km for deoxycytidine. In consequence of this, a mixture of the two forms produced bimodal kinetic plots, whereas linear kinetics were displayed by each form alone.
Kim
M Y
MY
Department of Biochemistry, Ohio State University, Columbus 43210.
Ikeda
S
S
Ives
D H
DH
D002846:Chromatography, Affinity; D003842:Deoxycytidine Kinase / Q000097:blood* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D007938:Leukemia / Q000201:enzymology; D008214:Lymphocytes / Q000201:enzymology; D008970:Molecular Weight; D010770:Phosphotransferases / Q000097:blood* / Q000302:isolation & purification*; D013379:Substrate Specificity
D010770:Phosphotransferases; D003842:Deoxycytidine Kinase