STRUCTURE AND EXPRESSION OF THE CDNA FOR THE C ISOZYME OF PHOSPHOFRUCTO-1-KINASE FROM RABBIT BRAIN

Rabbit brain contains three phosphofructo-1-kinase (PFK) isozymic subu nits designated A, B, and C. The primary structures of the first of th ese two isozyme types have been determined previously. The isozyme C o f rabbit brain was isolated by immunoaffinity chromatography and subje cted to proteolytic and chemical digestion. A large number of peptides were sequenced, the total number of amino acids identified being equa l to about 80% of the total structure. The sequence of the cDNA derive d from brain mRNA for C isozyme was determined from polymerase chain r eaction fragments synthesized using oligonucleotides designed on the b asis of the peptide sequences. The deduced size of the C isozyme was 8 6,371 Da, slightly larger than PFKs described previously. The amino ac id sequence identity with the rabbit A isozyme was 68.9% and a range o f identity to other sequenced mammalian PFKs was 67-69%. Using these d ata plus previously published data on chemical modification, assignmen ts of the 6 organic ligand binding sites of PFK were inferred. The ful l-length cDNA was cloned into and expressed in Escherichia coli. Phosp hofructokinase C was purified to homogeneity from the bacterial extrac ts.