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Isolation of the Subunits of Transcarboxylase and Reconstitution of the Active Enzyme from the Subunits

H. G. Wood,F. Ahmad,B. Jacobson,M. Chuang,W. Brattin

Published 1975 in Journal of Biological Chemistry

ABSTRACT

Abstract The separation of the 12 SH central subunit, the 5 SE peripheral metallo subunit, and the 1.3 SE biotinyl carboxyl carrier protein which are formed on the dissociation of transcarboxylase has been accomplished by molecular sieving on Bio-Gel. The 12 SH and 5 SE subunits have been obtained in nearly homogeneous form as judged by the sedimentation velocity profiles and by acrylamide gel electrophoresis. The 1.3 SE carboxyl carrier protein has given less consistent results; sometimes a single band of molecular weight of approximately 12,000 is obtained on gel electrophoresis in sodium dodecyl sulfate but sometimes there is an additional band of lower molecular weight of approximately 11,000. This lower molecular weight component may result from limited proteolytic degradation in spite of efforts to prevent it. Two or more bands are obtained in the absence of dodecyl sulfate. This heterogeneity may result from aggregation. subunits. The most effective reconstitution is accomplished by a two-step process. First, the 1.3 SE carboxyl carrier protein and 5 SE metallo subunit are combined to form the 6 SE complex; then this product is combined with the 12 SH subunit to yield active enzyme. With a limiting amount of the 6 SE complex and an excess of the 12 SH subunit the resulting enzymatic activity is proportional to the concentration of 6 SE complex. Likewise, with an excess of the 6 SF, complex and the 12 SH subunit limiting, the enzymatic activity is proportional to the concentration of the 12 SH subunit. The maximum specific activities of the reconstituted 6 SE complex and the 12 SH subunit were approximately 60% and 50%, respectively of the value estimated for their specific activities in the native 18 S form of the enzyme with three peripheral 6 SE subunits. Because assay of the 6 SE complex is done using an excess of the 12 SH subunit, this may yield enzyme with a single peripheral subunit. The 6 SE subunit may be less effective in this form. In the case of the 12 SH subunit, the activity is most likely low because the reconstituted 6 SE complex does not contain the full complement of 1.3 SE biotinyl carboxyl carrier proteins. The carboxyl carrier protein provides the groups which link together the 5 SE peripheral subunits with the central subunit. Evidence is presented that the 1.3 SE biotinyl carboxyl carrier must first combine with the 5 SE subunit to assume a form which effectively provides the combining groups for association with the 12 SH subunit.

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