The long range objective of this project is to gain insight into the structure and expression of the phosphofructokinase (PFK) gene from rabbit muscle in the normal and diabetic states. The information gained is expected to contribute to our understanding of the structure/function/regulation relationship of this key regulatory enzyme of carbohydrate metabolism. Our approach combines the recent techniques of recombinant DNA with those of affinity labeling and conventional methods of sequence studies.
The specific aims of the project can be summarized as follows: (1) Cloning of the PFK gene and elucidation of its base sequence by the method of Maxam and Gilbert. This part constitutes the major thrust of this proposal. Through the determination of the base sequence of the PFK cDNA, the primary structure of the enzyme itself will be deduced. (2) Structure/function/regulation relationship: This part of the project involves efforts to identify the specific amino acid residues involved in the catalytic and regulatory sites of PFK. In this area, predetermined site specific mutagenesis of the cloned cDNA of PFK will be brought about and the effects of such mutations on the kinetic and allosteric behavior of the enzyme will be determined. (3) Mode of expression of the gene(s) of PFK isozymes. The genetic mechanism of expression of the three established isozymes of PFK, namely, M for muscle, L for liver and P for platelet PFK, will be studied by using 32p labeled cDNA of PFK as a hybridization probe. (4) The mechanism of diminished phosphofructokinase activity in diabetic animals. We have reported that PFK activity in muscle from streptozotocin diabetic animals was lower than that in muscle from normal controls. The possible impairment of PFK synthesis at the translation or transcription level in the diabetic state will be studied.
