Determination of isozyme-specific mRNAs of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in glutamate obese rats by RNA probes.

To study the possible alterations in the metabolism of fructose 2,6-bisphosphate in glutamate induced obese rats, the mRNA levels of different isozymes coding for the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/F2,6Pase) were determined in various rat tissues obtained from fed and starved animals. For Northern analysis and dot blotting radioactively labeled RNA probes were synthesized by in vitro transcription. A modified ligase-free subcloning method was applied to obtain the plasmid vectors containing specific fragments of the cDNAs coding for the muscle, liver and heart isozymes of PFK-2/F2,6Pase. In liver but not in heart and skeletal muscle of glutamate obese rats the content of mRNA of PFK-2/F2,6Pase is significantly lower than in normal rats. As in normal rats short-term starvation has no effect on mRNA levels of the bifunctional enzyme. These results are compared with the maximum activities of PFK-2 and F2,6Pase determined in cell-free extracts prepared from the three organs.