Rat hepatoma (HTC) cell 6‐phosphofructo‐2‐kinase differs from that in liver and can be separated from fructose‐2,6‐bisphosphatase

Abstract

6-Phosphofructo-2-kinase was purified from rat liver and hepatoma (HTC) cells. The HTC cell enzyme had kinetic properties different from those of the liver enzyme (more sensitive to inhibition by citrate and not inhibited by sn-glycerol3-phosphate) and was not a substrate of the cyclic-AMP-dependent protein kinase. Unlike the liver enzyme, which is bifunctional and phosphorylated by fructose 2,6-[2-32P]bisphosphate, the HTC cell enzyme contained no detectable fructose-2,6-bisphosphatase activity and phosphorylation by fructose 2,6-[2-32P]-bisphosphate could not be detected. HTC cell fructose-2,6-bisphosphatase could be separated from 6-phosphofructo-2-kinase activity by purification. Antibodies raised against liver 6-phosphofructo-2-kinase did not precipitate HTC cell fructose-2,6-bisphosphatase whose kinetic properties were completely different from those of the liver enzyme.Fructose 2,6-bisphosphate, Fru(2,6)P2, is an intracellular regulatory molecule that controls glycolysis in mammalian tissues by integrating hormonal and metabolic signals. These signals act through 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2), which catalyze the synthesis and degradation of Fru(2,6)P2, respectively. Both activities are catalysed by a single protein composed of two identical subunits, each of which bears the two catalytic sites. The liver bifunctional enzyme can be phosphorylated on a serine residue by the cyclic-AMP-dependent protein kinase which inactivates PFK-2 and activates FBPase-2. Experimental evidence supports the existence of several PFK-2/FBPase-2 isozymes, which differ from each other in kinetic properties, FBPase-2 content and phosphorylation by protein kinases. (For recent reviews, see [I, 21.) Compared to normal cells, many tumour cells display a high glycolytic rate which is maintained even under aerobic conditions. Recent work has shown that Fru(2,6)P2 might be involved in this phenomenon [3 -61. In addition, several agents or conditions such as cyclic AMP, adenosine, ethanol, and anoxia, which are known to decrease Fru(2,6)P2 concentration in hepatocytes [l, 21, are without effect on this regulator in hepatoma (HTC) cells [6]. This might result from abnormal properties of HTC cell PFK-2. The purpose of this work was to compare PFK-2 from liver and HTC cells. Our results show the two enzymes differ in many respects, but, most of all, in their FBPase-2 content. In HTC cells, FBPase-

Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

“…2). The PFK-2 activity in lysates from clone HTC-25 was immunoprecipitable by BCL-2 antiserum (not shown), as is the case for the liver (Crepin et al, 1988) and HTC cell (Loiseau et al, 1988) Table 1 for the fully coding clones is reminiscent of that described for the muscle bifunctional enzyme, which is 5-10, as compared to 0.3 for liver (Table 1 and . We therefore examined whether the FBPase-2 of the recombinant protein was, like muscle FBPase-2, 5-fold less sensitive to inhibition by fructose 6-phosphate than liver FBPase-2 (see (Fig.…”

“…Comparison of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expressed in E. coli with the HTC cell enzyme HTC cell PFK-2 is not inhibited by sn-glycerol 3-phosphate, in contrast to the liver enzyme, and it is more sensitive than liver PFK-2 to inhibition by citrate, like the muscle enzyme (Loiseau et al, 1988). We therefore determined whether these peculiar kinetic properties of HTC cell PFK-2 were retained following expression in E.…”

“…After shaking the cultures for I h, cells were harvested by centrifugation at 25 "C (8000 g, 10 min) and quickly suspended in a volume of cold lysis buffer (50 mM-Hepes-KOH, pH 7.5, 100 mM-KCl, 1 mmpotassium phosphate and 0.1 mm phenylmethanesulphonyl fluoride) equal to 1/50 of the culture volume. The suspension was frozen in 1 ml aliquots on solid CO2 and allowed to thaw on ice for 3 h. Insoluble material was removed by centrifugation at 4 "C (10000 g, 10 min) and the supernatant was stored at -80 "C. PFK-2 was partially purified by poly(ethylene glycol) (PEG) precipitation followed by Blue-Sepharose chromatography as described (Loiseau et al, 1988). Peptide synthesis A decapeptide GELTQTRLQK corresponding to the N-terminus of liver PFK-2 (to raise a L-antibody) and a nonapeptide EEKASKRTA corresponding to the putative N-terminus of HTC PFK-2 (to raise a M-antibody) were prepared by solid phase on a Beckman 990B automatic peptide synthesizer.…”

“…The reaction was stopped by addition of 100 ,1 of 1 M-NaOH, and [32P]P, was separated from the substrate as described (Loiseau et al, 1988). Thermolysin digestion of PFK-2/FBPase-2 Thermolysin (Boehringer) solutions (0.01-1 mg/ml) were freshly prepared in buffer A (20 mM-Hepes, pH 7.5, 50 mM-KCl, 5 mM-MgCl2, 2 mM-EDTA, 15 mM-2-mercaptoethanol and I mM-potassium phosphate) and clarified by centrifugation.…”

“…As one approach to this problem, we have compared the PFK-2/FBPase-2 purified from HTC cells with that from rat liver (Loiseau et al, 1988). HTC cell PFK-2 has kinetic and antigenic properties different from those in liver and it is not a substrate of cyclic AMP-dependent protein kinase.…”