GENETIC AND BIOCHEMICAL CHARACTERISTICS OF THREE DIFFERENT TYPES OF MUTANTS OF CHINESE HAMSTER OVARY CELLS AFFECTED IN ADENOSINE KINASE: 76

Abstract

In CHO cells, three different types of mutants affected in purine nucleoside salvage pathway enzyme adenosine kinase (AK) have been isolated. One class of mutants obtained at high frequency using various adenosine analogs (Class A) exhibit high degree of cross resistance to various C- as well as N- adenosine analogs. These mutants show greatly reduced phosphorylation of all adenosine analogs and cell extracts from these contain no measurable activity of AK. The second type of mutants (Class B) obtained using formycin A (a C- adenosine analog) exhibit increased cross resistance to various C- nucleosides (viz. 9-deazaade-nosine, pyrazofurin, etc.),but their sensitivity towards various N- adenosine analogs was found to be unaltered. Cell extracts from these mutants also contained no measurable activity of AK. Reduced phosphorylation of C- adenosine analogs but not N- adenosine analogs by Class B mutants provides strong evidence that they contain a novel genetic lesion affecting AK that specifically affects the phosphorylation of C- nucleosides. The third type of mutants (Class C) selected using inosine analog formycin B, exhibit cross resistance and reduced phosphorylation of both C- as well as N- adenosine analogs. However, their degree of resistance was lower in comparison to the Class A mutants. Cell extracts from the Class C mutants contained between 50-100% of AK activity, but its affinity towards adenosine analogs was found to be altered in comparison to the wild-type enzyme.