J. Biochem, 1997, Vol. 121, No. 4 724-730
© 1997 Japanese Biochemical Society
research-article |
Inhibition of Fructose-6-Phosphate,2-Kinase by N-Bromoacetylethanolamine Phosphate In Vitro and In Vivo1
*Department of Biochemistry, School of Pharmaceutical Sciences
Center of Instrumental Analysis, Nagasaki University 1–14 Bunkyo-machi, Nagasaki, Nagasaki 852
3To whom correspondence should be addressed.
Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-BPase), a bifunctional enzyme, catalyzes the synthesis and degradation of a potent activator, fructose-2,6-bisphosphate (Fru-2,6-P2), of phosphofructokinase, and has been postulated to be an important enzyme in the regulation of glycolysis in mammalian tissues. The purposeof this study was to determine whether or not N-bromoacetylethanolamine phosphate (BrAcNHEtOP), a specific active site-directed inactivator of Fru-6-P,2-kinase, is useful for studies on the role of Fru-6-P,2-kinase in the regulation of glycolysis in vivo. BrAcNHEtOP inactivated purified recombinant rat testis-type Fru-6-P,2-kinase as well as Fru-6-P,2-kinase in a rat liver extract, with half maximum inactivation concentrations of 2 and 15 mM, respectively, on 30 min incubation at 30°C. The increases in Fru-6-P,2-kinase activity and the Fru-2,6-P2 concentration in livers, prepared from fasted rats, induced by high glucose (50 mM) perfusion were suppressed in parallel after pre-perfusion with 1 to 10 mM BrAcNHEtOP, dose-dependently. Five hours after intraperitoneal injection of BrAc-NHEtOP (50 to 150 mg/kg) into mice, the Fru-6-P,2-kinase activity and Fru-2,6-P2 concentration in livers had decreased in parallel, dose-dependently. These effects continued for 24h and were accompanied by decreases in the fructose-1,6-bisphosphate, triose phosphates, and lactate contents, although the contents of glucose-6-phosphate and fructose-6-phosphate did not change. These results suggested that BrAcNHEtOP inactivates Fru-6-P, 2-kinase, resulting in a decrease in the Fru-2,6-P2 level, which causes inactivation of phosphofructokinase and consequently inhibition of glycolysis in liver. Furthermore, the suppressed levels of Fru-6-P,2-kinase activity and metabolites in mice livers were sustained by daily injection of BrAcNHEtOP for 4 days, and body weight gain was also suppressed during the administration of BrAcNHEtOP. These results suggested that BrAcNHEtOP will be a useful reagent for studying the role of Fru-6-P,2-kinase in vivo.
1This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
2Present address: Faculty of Protein Chemistry and Engineering, Graduate School of Genetic Resources & Technology, Kyushu University, 6–10–1 Hakozaki, Higashi-ku, Fukuoka 812.