Transcarboxylase

Abstract

Abstract Transcarboxylase labeled with 60Co or 65Zn was isolated and purified to near homogeneity from Propionibacterium shermanii. The enzyme which is a metallo-biotin enzyme, was found to contain 7 to 8 g atoms of zinc plus cobalt per mole of transcarboxylase (molecular weight ∼790,000). The 60Co- or 65Zn-labeled enzymes were dissociated under controlled conditions to yield subunits with s20,w ≃ 12 and 6 S, which were separated by density gradient centrifugation. The 6 S subunit contains the metals and the 12 S subunit little or none. The 6 S subunit dissociates further to a subunit containing the metals and to the biotin containing carboxyl carrier protein. The metals in transcarboxylase as well as in the subunits are tightly bound. Only small amounts of the bound cobalt or zinc are removed when transcarboxylase labeled with these metals is passed over Sephadex equilibrated with 10 mm o-phenanthroline, 8-hydroxyquinoline 5-sulfonate, or EDTA. Dialysis at alkaline or neutral pH does not remove the metals but some is removed when the enzyme is dialyzed at acidic pH values (pH 5, 4.5, or 4). Dialysis against 0.1% sodium dodecyl sulfate or 4 to 6 m urea causes release of the metals but these conditions cause irreversible dissociation of the enzyme to its constituent peptides.