期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1980
卷号：77
期号：2
页码：730-734
DOI：10.1073/pnas.77.2.730
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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:-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.
