Activation of Lipoic Acid and Its Transfer from the Free Pool to the Protein-bound State*

That the adenyl lipoate (AMP-lipoate) is the product of activation of lipoate was demonstrated by using the apopyruvate dehydrogenase system of Streptococcus jaecalis as well as Escherichiacoli. Pyruvate dismutation assay was used to demonstrate the activation of apoenzyme with either a mixture of ATP and lipoate or adenyl lipoate alone. That an active intermediate was formed was demonstrated by the hydroxamate reaction. In an attempt to use ATP-32P32Pi exchange assay for activation of lipoate, difficulties were experienced due to considerable endogenous exchange catalyzed by the microbial and plant extracts. Subsequnetly, it was possible to demonstrate Reaction 1 in the backward direction with the use of synthetically prepared adenyl lipoate and “P3’Pi (3). In the present experiments, the formation of adenyl lipoate as the product of activation of lipoate will be directly demonstrated with the use of AT32P as well as 35S-labeled lipoate which was prepared by a new biosynthetic method (4). Transfer of the %-labeled lipoate from the free to the protein-bound state will also be demonstrated. Similar demonstrations have recently been made in case of activation of biotin and its transfer from the free pool to the protein-bound state (5, 6).