Alternate Procedure for the Preparation of the Coenzyme A-Synthesizing Protein Complex of Bakers' Yeast

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Abstract
The CoA-synthesizing protein complex (CoA-SPC) of Baker''s yeast synthesizes CoA in an in vitro system from the precursors ATP, D-pantothenic acid and L-cysteine. CoA-SPC was produced on a small scale by freezing Bakers'' yeast cells in a mixture of diethyl ether and solid CO2, followed by a thawing period, and subsequent removal of the diethyl ether by vacuum. The resulting yeast lysate was then stirred for 18 h in the presence of t-factor to solubilize CoA-SPC. When a greater quantity of CoA-SPC was needed, the danger associated with the use of a large volume of diethyl ether was apparent. Therefore, the freezing step involving diethyl either and solid CO2 was replaced by a process of slowly drying fresh Bakers'' yeast until approximately 34% of the initial weight of the yeast remained as dry solids. The yeast solids were ground to further disrupt the cell wall and membrane structure. The grinding step was followed by rehydration of the dry yeast solids with deionized H2O and stirring the rehydrated yeast for 18 h to solubilize CoA-SPC.