Renaturation and localization of enzymes in polyacrylamide gels: studies with UDPglucose pyrophosphorylase of Dictyostelium.
We have developed procedures for renaturing, in polyacrylamide isoelectric focusing gels, homomeric enzymes (i.e., enzymes with identical subunits) that have been denatured with sodium dodecyl sulfate or urea or both. The renatured enzymes can then be localized as discrete species by conventional histochemical staining. One of these enzymes, uridine diphosphoglucose pyrophosphorylase (UTP:alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) of Dictyostelium discoideum, was studied in detail. Conditions have been established for renaturing and localizing this enzyme and for quantitating the amount of activity recovered. Up to 40% of the activity can be recovered after renaturation. This procedure is widely applicable because several enzymes, including alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27), can be localized. It is sensitive enough to resolve isozymes and enzyme variants that differ by a single charged amino acid. It can be used to localize enzymes in crude cell extracts that have been resolved in two-dimensional slab gels by sodium dodecyl sulfate electrophoresis and isoelectric focusing. These methods should allow detailed analysis of genes and their enzyme proteins that, though present in small amounts in eukaryotic cells, perform important metabolic or developmental functions.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification; D004023:Dictyostelium / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007526:Isoelectric Point; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D009994:Osmolar Concentration; D011489:Protein Denaturation; D013194:Staining and Labeling; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification*; D014508:Urea
D014508:Urea; D000429:Alcohol Oxidoreductases; D009713:Nucleotidyltransferases; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase