title: Inhibition of the glucose-6-phosphatase system by N-bromoacetylethanolamine phosphate, a potential affinity label for auxiliary proteins.
abstract: N-Bromoacetylethanolamine phosphate (BAEP) has been used previously as an affinity label to study the hexose phosphate binding sites of fructose-6-P, 2-kinase:fructose-2, 6-bisphosphatase (Sakakibara et al. (1984) J. Biol. Chem. 259, 14023-14028). We have employed this compound to probe components of the glucose-6-phosphatase system using a combination of time-dependent and immediate inhibition kinetic techniques. Inhibition of D-glucose-6-phosphate (G6P) phosphohydrolase activity of native microsomes was irreversible and time- and inhibitor-concentration-dependent. Only a partial time-dependent, irreversible inhibition of the PPi phosphohydrolase activity of native microsomes was observed. BAEP inhibited PPi:glucose phosphotransferase activity of native microsomes in a concentration-dependent, irreversible manner which was more extensive than that seen with PPi phosphohydrolase, but less extensive than was observed with G6P phosphohydrolase. Disruption of microsomal integrity by detergent-treatment either prior to incubation with BAEP or subsequent to preliminary incubation with BAEP but prior to assay for activity abolished the time-dependent inhibition. These irreversible, time- and concentration-dependent inhibitory actions of BAEP thus are manifest at a site or sites where the intact membrane-bound enzyme first makes contact with substrates G6P and PPi. An additional site of inhibition by BAEP, through relatively weak, reversible competitive inhibition at the active catalytic site, is indicated by classical steady-state kinetic analysis. The irreversible, time- and concentration-dependent inhibitions by BAEP seen with G6P and PPi as substrates strongly suggest the potential utility of radio-labeled BAEP as an affinity label for the identification and ultimate isolation and study of uncharacterized auxiliary components of the glucose-6-phosphatase system.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks 58202, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Foster
authors.forename: B A
authors.initials: BA
authors.lastname: Pederson
authors.forename: R C
authors.initials: RC
authors.lastname: Nordlie
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D003902:Detergents / Q000494:pharmacology; D011756:Diphosphates / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004983:Ethanolamines / Q000378:metabolism / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008954:Models, Biological; D017830:Octoxynol / Q000494:pharmacology; D011755:Pyrophosphatases / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D003902:Detergents; D011756:Diphosphates; D004791:Enzyme Inhibitors; D004983:Ethanolamines; D008565:Membrane Proteins; C020161:N-bromoacetylethanolamine phosphate; D019298:Glucose-6-Phosphate; D017830:Octoxynol; D005952:Glucose-6-Phosphatase; D011755:Pyrophosphatases; D005947:Glucose