Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography.
A highly efficient new affinity medium for deoxycytidine kinase, deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose), has been constructed. A dCp4-Sepharose column effects a one-step, 19,000-fold, purification to homogeneity of dCyd kinase from the ammonium sulfate fraction of Lactobacillus acidophilus R-26 extract, with 60% recovery. dCTP, a potent end-product inhibitor, is used as an eluent, and it also stabilizes the extremely labile purified enzyme. A noncompeting deoxyadenosine kinase activity accompanies the deoxycytidine kinase activity eluted. Native polyacrylamide gel electrophoresis shows a single protein band, which coincides with both deoxycytidine kinase and deoxyadenosine kinase activities at several gel concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide band of 26,000 daltons. Since the native enzyme is known to have an Mr of 50,000, it appears that the enzyme is composed of two subunits of similar size. Sequence analysis of the intact protein from the N-terminus reveals but a single amino acid species per residue up to the 17th residue; at the 18th, 21st, 26th, and 27th residue positions of the sequence, however, there appear to be two different amino acids in almost equal amounts. This may indicate that the enzyme is composed of two nonidentical subunits having the same amino acid sequence near the N-terminus. Residues 6-13 contain the highly conserved Gly-X-X-Gly-X-Gly-Lys sequence found at the active sites of kinases and other nucleotide-binding proteins.
Ikeda
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Department of Biochemistry, Ohio State University, Columbus 43210.
Swenson
R P
RP
Ives
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DH
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D007202:Indicators and Reagents; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor)
D007202:Indicators and Reagents; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases