cDNA clones for 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase were isolated from rat liver expression libraries in λgt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full‐length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.

Three distinct clones encoding full-length 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) were characterized from a rat liver cDNA library. Clone 22c was 1859 bp long and coded for the 470 amino acids of the bifunctional subunit of the liver homodimer. This polypeptide is phosphorylated on serine 32 by cyclic-AMP-dependent protein kinase. Clone 4c (2681 bp) had a coding region identical to that of clone 22c but it included a putative intron of 959 bp. In clone 5c (1750 bp), the sequence upstream from amino acid 33 differed from that in clone 22c and coded for a unique N-terminal portion of 10 amino acids.Poly(A)-rich RNA from rat tissues was hybridized with cDNA probes corresponding to the unique N-terminal portions of clones 22c and 5c. Dot and Northern blots showed signals indicative of three distinct PFK-2/FBPase-2 mRNAs. There were a 6.8-kb mRNA typical of cardiac tissue, a 2.1-kb mRNA typical of liver, corresponding to clone 22c, and a 1.9-kb mRNA typical of skeletal muscle, corresponding to clone 5c. Primer extension analysis showed that clones 22c and 5c were nearly complete since their respective 5'-untranslated sequences were at most 96/97 bp and 44 bp shorter than the corresponding mRNAs. These data provide a molecular basis for the existence of PFK-2/FBPase-2 isozymes.Fructose 2,6-bisphosphate is a potent stimulator of 6-phosphofructo-1-kinase, identified in all eukaryotic tissues. It controls glycolysis by integrating hormonal and metabolic signals. Some of these act through 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2), which, respectively, catalyze the synthesis and degradation of fructose 2,6-bisphosphate. In liver, both activities are borne by a single protein composed of two identical subunits of M , 55000, each of which contains the two catalytic sites. Differences between tissues in the PFK-2/FBPase-2 ratio, in the kinetic and antigenic properties of these enzymes, and in their sensitivity to sulfhydryl group reagents and phosphorylation by protein kinases suggest the existence of several PFK-2/ FBPase-2 isozymes. For instance, phosphorylation by cyclic-AMP-dependent protein kinase inactivates PFK-2 and activates FBPase-2 in liver but not in muscle. Protein kinase C phosphorylates heart but not liver PFK-2/FBPase-2. Heart PFK-2 has little associated FBPase-2 activity (for reviews, see [I, 21).

The synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis, are catalyzed by 6-phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46), respectively. In liver, these two activities belong to separate domains of the same 470-residue polypeptide. Various mRNAs have been described for this bifunctional enzyme, which is controlled by hormonal and metabolic signals. To understand the origin and regulation of these mRNAs, we have characterized rat genomic clones encoding the liver isozyme, which is regulated by cAMP-dependent protein kinase, and the muscle isozyme, which is not. We describe here a 55-kilobase gene that encodes these isozymes by alternative splicing from two promoters. Each of the putative promoters was sequenced over about 3 kilobases and found to include nucleotide motifs for binding regulatory factors. The two isozymes share the same 13 exons and differ only by the first exon that, in the liver but not in the muscle isozyme, contains the serine phosphorylated by cAMPdependent protein kinase. The gene was assigned to the X chromosome. An analysis ofthe exon limits of6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in relation to its functional domains and to its similarity with other proteins plus its G+C content at the third codon position suggests that this gene originates from several fusion events.Fructose 2,6-bisphosphate is the most potent allosteric stimulator of 6-phosphofructo-1-kinase (EC 2.7.1.11), a key enzyme of glycolysis. The synthesis of fructose 2,6-bisphosphate is catalyzed by 6-phosphofructo-2-kinase (PFK-2; EC 2.7.1.105) and its degradation is catalyzed by fructose-2,6-bisphosphatase (FBPase-2; EC 3.1.3.46). In liver, these two activities belong to separate domains of each subunit (470 amino acids) of the same homodimeric protein. This bifunctional enzyme integrates a number of hormonal and metabolic signals including cAMP. In fibroblasts, PFK-2 activity is stimulated by growth factors, tumor promoters, and tyrosinespecific oncogenic protein kinases (1, 2).Biochemical and immunologic data suggest the existence of distinct PFK-2/FBPase-2 isozymes. Using PFK-2/ FBPase-2 cDNA probes from rat liver (4), we have identified a 2.1-kilobase (kb) mRNA coding for the liver (L) isozyme, a 1.9-kb mRNA coding for the skeletal muscle (M) isozyme, and a 6.8-kb mRNA presumably coding for the heart isozyme (5). The L and M isozymes have a common sequence of438 amino acids and differ only at the N terminus. In the L isozyme, the divergent sequence is 32 residues long and includes the serine (Ser-32) phosphorylated by cAMP-dependent protein kinase. In the M isozyme, the N terminus is 10 residues long and is unrelated to the unique part of the L isozyme. Expression of the L, but not the M, isozyme of PFK-2/FBPase-2 is controlled by diet and insulin (6, 7). This gene is 55 kb long and contains 15 exons, 13 of which are common to the two isozymes. Part of this work has been presented in the form of an abstract (9). MATERIALS...

In rat liver, the activity of 6-phosphofructo-2-kinase (PFK-2) decreases upon starvation and in diabetes. Cyclic AMPdependent phosphorylation of the enzyme is not sufficient to account for this decrease. PFK-2 content was therefore measured by immunotitration and relative PFK-2 mRNA levels were determined by hybridization with cDNA probes. The data are compatible with a posttranscriptional mechanism of regulation that involves decreased translational efficiency of PFK-2 mRNA.and (or) increased turnover of the PFK-2 protein.

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.