INHIBITION OF FRUCTOSE-6-PHOSPHATE,2-KINASE BY N-BROMOACETYLETHANOLAMINE PHOSPHATE IN-VITRO AND IN-VIVO

e-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2-kinase/F ru-2,6-BPase), a bifunctional enzyme, catalyzes the synthesis and degr adation of a potent activator, fructose-2,6-bisphosphate (Fru-2,6-P-2) , of phosphofructokinase, and has been postulated to be an important e nzyme in the regulation of glycolysis in mammalian tissues. The purpos e of this study was to determine whether or not N-bromoacetylethanolam ine phosphate (BrAcNHEtOP), a specific active site-directed inactivato r 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 co ncentrations of 2 and 15 mM, respectively, on 30 min incubation at 30 degrees C. The increases in Fru-6-P,2-kinase activity and the Fru-2,6- P-2 concentration in livers, prepared from fasted rats, induced by hig h glucose (50 mM) perfusion were suppressed in parallel after pre-perf usion with 1 to 10 mM BrAcNHEtOP, dose-dependently. Five hours after i ntraperitoneal injection of BrAcNHEtOP (50 to 150 mg/kg) into mice, th e Fru-6-P,2-kinase activity and Fru-2,6-P-2 concentration in livers ha d decreased in parallel, dose-dependently. These effects continued for 24 h and were accompanied by decreases in the fructose-1,6-bisphospha te, triose phosphates, and lactate contents, although the contents of glucose-6-phosphate and fructose-6-phosphate did not change. These res ults suggested that BrAcNHEtOP inactivates Fru-6-P,2-kinase, resulting in a decrease in the Fru-2,6-P-2 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 BrAcNH EtOP for 4 days, and body weight gain was also suppressed during the a dministration of BrAcNHEtOP. These results suggested that BrAcNHEtOP w ill be a useful reagent for studying the role of Fru-6-P,2-kinase in v ivo.