This chapter discusses the use of enzymes of nucleotide metabolism to illustrate procedures for the purification and characterization or multi-enzyme complexes. A significant result of these procedures is the successful use of ammonium sulfate precipitation to concentrate and enrich each of the three complexes. It might be expected that the complexes would be disrupted at the high ionic strength present during ammonium sulfate precipitation. The most direct evidence for the existence of a multi-enzyme complex is the association of multiple activities through several fractionation steps. In the isolation of a putative complex, several activities must be monitored through the purification steps and their ratios should remain constant. With a biological system amenable to genetic analysis, the power of such approaches has enormously increased. Bifunctional protein cross-linking reagents can be used to identify neighbor relationships among proteins in a purified complex, to demonstrate that such relationships occur in intact cells, or as an adjunct to purification of a complex. If the enzyme–intermediate complex is preferred as a substrate to that same intermediate in free solution, then this constitutes evidence for direct transfer of the intermediate from one enzyme to the next.
ASJC Scopus subject areas
- Molecular Biology