Abstract
Abstract Crystalline pig heart fumarase was previously shown to comprise a large number of active components separable by ion exchange chromatography. We have explored the nature of this heterogeneity by dissociating the tetrameric molecule in urea and analyzing the subunits in an electrofocusing column. The results indicate that fumarase contains several subunit types, i.e. at least three major and three minor components, which differ in primary structure. Each subunit type can reassociate to form active enzyme species upon removal of the urea, indicating that the enzyme need not be a heterooligomer to function. Also, hybrid formation from a mixture of subunit types is shown, a result which readily explains the high degree of heterogeneity observed for this enzyme. Fumarase prepared from single pig hearts under conditions designed to minimize proteolysis displays the same isozyme peaks as does the commercial enzyme. This shows that the heterogeneity is not due to pooling of genetically different hearts, and also suggests that the major isozyme components are not the result of enzymatic alteration during isolation. The results suggest that either there are several fumarase genes, or that modification of the enzyme occurs in vivo after synthesis.
Highlights
Crystalline pig heart fumarase was previously shown to comprise a large number of active components separable by ion exchange chromatography
The high degree of heterogeneity is in accord with our earlier chromatographic and gel electrophoretic studies [1]
The results obtained in the present study show that crystalline fumarase preparations contain six or more subunit types which can be separated from each other, and that active hybrids can be formed from a mixture of subunit types, a result which explains the high degree of heterogeneity of this enzyme
Summary
Crystalline pig heart fumarase was previously shown to comprise a large number of active components separable by ion exchange chromatography. We have explored the nature of this heterogeneity by dissociating the tetrameric molecule in urea and analyzing the subunits in an electrofocusing column. The results indicate that fumarase contains several subunit types, i.e. at least three major and three minor components, which differ in primary structure. Each subunit type can reassociate to form active enzyme species upon removal of the urea, indicating that the enzyme need not be a heterooligomer to function. Hybrid formation from a mixture of subunit types is shown, a result which readily explains the high degree of heterogeneity observed for this enzyme
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