Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes.
Intracellular enzymes in erythrocytes can be made accessible for in situ kinetic studies by treating the cells with bifunctional reagents to crosslink proteins, thus creating a network that allows subsequent permeabilization by delipidation without escape of intracellular proteins. Dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate, and toluene-2,4-diisocyanate have been used successfully as crosslinking reagents, and digitonin has been used for delipidation. In a systematic study of the in situ behavior of the 11 glycolytic enzymes of rat erythrocytes, it was observed that Km and Vmax values for the majority of the enzymes are essentially the same in situ as in vitro. Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) is inhibited by excess of pyruvate as much in situ as in vitro. Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was allosterically inhibited by glucose 6-phosphate nearly as much in situ as in vitro but was not affected by 2,3-biphosphoglycerate. The allosteric properties of 6-phosphofructokinase (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], and pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) in situ were qualitatively similar to those observed in vitro, but some important quantitative differences were noticed. Particularly striking was the much greater activity of phosphofructokinase in situ compared to that in vitro at physiological concentrations of effector metabolites.
D000494:Allosteric Regulation; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D003432:Cross-Linking Reagents; D004072:Digitonin / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D006019:Glycolysis; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000097:blood; D010732:Phosphofructokinase-1 / Q000097:blood; D011770:Pyruvate Kinase / Q000097:blood; D051381:Rats
D003432:Cross-Linking Reagents; D007770:L-Lactate Dehydrogenase; D010732:Phosphofructokinase-1; D011770:Pyruvate Kinase; D004072:Digitonin