Heterodimeric deoxynucleoside kinases of Lactobacillus acidophilus R-26: functional assignment of subunits using limited proteolysis controlled by end-product inhibitors.
Heterodimeric quaternary structures for two enzyme complexes from Lactobacillus acidophilus R-26 exhibiting deoxycytidine kinase/deoxyadenosine kinase (I) and deoxyguanosine kinase/deoxyadenosine kinase(II) activities  have been proven by the following steps: (1) separation of each complex into two components on SDS-PAGE at pH 6.6; (2) N-terminal amino acid sequencing of each component; (3) functional assignment of each component by differential limited proteolysis. The third step was facilitated by the finding that the binding of a specific end-product inhibitor dNTP, to each kinase active site makes the corresponding kinase subunit resistant to trypsin, while leaving the heterologous kinase subunit susceptible to proteolysis. Analysis on SDS-PAGE has revealed only two fragments (15.8 and 11.0 kDa) following proteolysis of dCyd kinase/dAdo kinase (I) with trypsin in the presence of dATP. This may indicate that the kinase polypeptide chain (27.2 kDa) not protected by dNTP is cut by trypsin at a single specific site, with concomitant loss of activity. Thus, this work presents a unique approach to the clarification of structure and function of enzymes composed of heterologous subunits.
Ikeda
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Department of Biochemistry, Ohio State University, Columbus 43210.
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004589:Electrophoresis, Disc; D007700:Kinetics; D007779:Lactobacillus acidophilus / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D014357:Trypsin