Inhibition of fructose-6-phosphate,2-kinase by N-bromoacetylethanolamine phosphate in vitro and in vivo.
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 purpose of 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 degrees 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 BrAcNHEtOP (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 24 h 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.
Harada
Y
Y
Department of Biochemistry, School of Pharmaceutical Sciences, Nagasaki University.
Tominaga
N
N
Watanabe
M
M
Shimokawa
R
R
Ishiguro
M
M
Sakakibara
R
R
D000818:Animals; D001786:Blood Glucose / Q000187:drug effects; D001835:Body Weight / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D004983:Ethanolamines / Q000494:pharmacology*; D019344:Lactic Acid / Q000097:blood; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D010477:Perfusion; D025481:Phosphofructokinase-2; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013737:Testis / Q000201:enzymology; D014280:Triglycerides / Q000097:blood
D001786:Blood Glucose; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D011994:Recombinant Proteins; D014280:Triglycerides; D019344:Lactic Acid; C020161:N-bromoacetylethanolamine phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2