Isolation and properties of noncovalent complex of transketolase with RNA.

A method for isolation of homogenous transketolase from baker's yeast using immunoaffinity chromatography was significantly simplified. It was demonstrated that transketolase could be isolated from fresh yeast in the form of a complex with a high molecular weight RNA. Storage of yeast led to the dissociation of the complex to a low molecular weight complex and then to the free enzyme. Conditions were chosen for complex dissociation and free enzyme isolation. In comparison to the free enzyme, the specific activities of the high and low molecular weight complexes were decreased 20-25- and 3-5.5-fold, respectively. The affinity to the cofactor thiamine diphosphate and to xylulose-5-phosphate (donor substrate) did not change for the low molecular weight complex, while the time of binding to calcium increased. The latter was necessary for the complete manifestation of the enzymatic activity. Changes in the circular dichroism spectrum between 300 and 360 nm after the addition of thiamine diphosphate, which characterize the formation of the catalytically active holoenzyme, were significantly lower for the low molecular weight complex than for the free enzyme.