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.