Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate.
The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004120:Dimethyl Suberimidate / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism; D007096:Imidoesters / Q000494:pharmacology*; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000097:blood*; D051381:Rats; D011919:Rats, Inbred Strains; D012156:Reticulocytes / Q000201:enzymology*
D003432:Cross-Linking Reagents; D005636:Fructosephosphates; D007096:Imidoesters; C024244:dithiobispropionimidate; D004120:Dimethyl Suberimidate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1