Extraction of glycolytic enzymes: myo-inositol as a marker of membrane porosity.
Detergent extraction of brain slices and mouse fibroblast 3T3 cells was performed to determine rates and relative amounts of extraction of inositol versus the glycolytic enzymes. The two detergents, Triton X-100 and Brij 58, led to similar results for extraction of myo-inositol. The extraction of enzymes from brain slices or cells varied with the detergent. In brain slices, a buffered solution containing 0.2% of the detergent Brij 58 led to the extraction of 85% of the inositol before 3% of the aldolase or before 37% of either lactate dehydrogenase or triose phosphate isomerase was extracted. In contrast, with 0.1% Triton X-100 in isotonic phosphate-buffered saline, when 70% of the inositol was extracted, 33% of the aldolase and 48% of the triose phosphate isomerase were extracted. Lesser amounts of aldolase and glyceraldehyde phosphate dehydrogenase were extracted than most of the other glycolytic enzymes under all conditions, implying that these enzymes may be interacting with non-extractable subcellular components. In 3T3 cells, both detergents were of similar effectiveness for inositol extraction. Triton X-100 caused 89% of the inositol to be released and Brij 58 caused 84% to be released. With the enzymes, Brij 58 caused between 15 and 38% extraction and Triton X-100 caused between 61 and 85% extraction of the different glycolytic enzymes. Thus Brij 58 was as effective as Triton X-100 in inositol extraction but not nearly as effective in glycolytic enzyme extraction. The results demonstrate that inositol leakage from tissues or cells is a better indicator of detergent-mediated alterations in membrane porosity than glycolytic enzyme leakage.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000201:enzymology* / Q000648:ultrastructure; D003902:Detergents; D005634:Fructose-Bisphosphate Aldolase / Q000032:analysis; D005956:Glucose-6-Phosphate Isomerase / Q000032:analysis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D006019:Glycolysis; D066298:In Vitro Techniques; D007294:Inositol / Q000032:analysis*; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D051379:Mice; D008854:Microscopy, Electron; D010735:Phosphoglycerate Kinase / Q000032:analysis; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D011770:Pyruvate Kinase / Q000032:analysis; D051381:Rats; D014305:Triose-Phosphate Isomerase / Q000032:analysis
D002021:Buffers; D003902:Detergents; D007294:Inositol; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011770:Pyruvate Kinase; D010735:Phosphoglycerate Kinase; D005634:Fructose-Bisphosphate Aldolase; D010751:Phosphopyruvate Hydratase; D014305:Triose-Phosphate Isomerase; D005956:Glucose-6-Phosphate Isomerase