Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase: Fructose-2,6-bisphosphatase

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a M_r of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced M_r of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were K_m^{Fru 6-P} = 85 μM and Ku_m^ATP = 270 μM, and those of fructose 2,6-bisphosphatase were K_m^{Fru 2.6-pz} = 21 μM and K_i^{Fru 6-P} = 3.4 μM. The enzyme was phosphorylated by Fru-2,6[2-³²P]P₂ and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.