The enzyme splitting pantethine into pantothenic acid and cystamine has been purified about 3000 times starting from horse kidney. The purification procedure is reported. The enzyme, whose optimal activity lies in the pH range 4.0-5.5, requires the presence of a reduced thiol for the full activity : mercaptoethanol and dithiothreitol give the higher effect. This activation seems not to be specific. The enzyme has a K , value for the substrate of the order of 5 mM and shows inhibition by excess substrate and by products. Substrate specificity studies show that the enzyme splits only the intact substrate molecule, which is not hydrolyzed by several hydrolytic enzymes, even with a broad speci6city (as bacterial pronase and subtilisin). The enzyme could be responsible for the production in vivo of cysteamine, which is known to be oxydized by a specific oxygenase, isolated recently in this laboratory.Identification of an enzyme able of hydrolyzing pantethine t o pantothenic acid and cystaminel has been reported in previous communications from this laboratory [1,2]. The purification of this enzyme is described in this paper. Some characteristic features of the protein and the catalyzed reaction are also presented. The enzyme is named throughout this paper, for our convenience, pantethinase.
EXPERIMENTAL PROCEDURE
Determination of ProteinThe protein concentration of the starting homogenate was determined by dry weight of samples kept at 110" to constant weight. I n the other steps of purification proteins were determined by the biuret reaction according t o the method of Goa [3].
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