The transsulfuration pathway, through which homocysteine from the methionine cycle provides sulfur for cystathionine formation, which may subsequently be used for glutathione synthesis, has not heretofore been identified as active in mammary cells. Primary human mammary epithelial cells (HMEC’s) were labeled with -methionine for 24 hours following pretreatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthionine sulfoximine. Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compound monochlorobimane to form a fluorescent glutathione-bimane conjugate. Comparison of fluorographic and autoradiographic images indicated that glutathione had incorporated -methionine demonstrating that functional transsulfuration occurs in mammary cells. Pathway inhibitors reduced incorporation by roughly 80%. Measurement of glutathione production in HMEC’s treated with and without hydrogen peroxide and/or pathway inhibitors indicates that the transsulfuration pathway plays a significant role in providing cysteine for glutathione production both normally and under conditions of oxidant stress. 1. Introduction In mammals, cystathionine beta-synthase (CBS) catalyzes the first step in the transsulfuration pathway (see Figure 1) [1], a pyridoxal-5′-phosphate- (PLP-) dependent condensation of serine and homocysteine to cystathionine [2, 3]. The second step of the transsulfuration pathway is the hydrolysis of cystathionine to cysteine, ammonia, and α-ketobutyrate catalyzed by the enzyme γ-cystathionase. These reactions form a metabolic bridge between the methionine cycle and cysteine, a necessary precursor for glutathione biosynthesis. Figure 1: The transsulfuration pathway connects methionine and glutathione biosynthesis. In the methionine cycle, methionine forms S-adenosylmethionine which serves as a methyl donor, generating S-adenosyl homocysteine. This is converted to homocysteine, which is subsequently converted back into methionine. Homocysteine has an alternative fate, however. It can be used to produce cystathionine, which is further converted to cysteine. This latter conversion is catalyzed by gamma-cystathionase and inhibited by propargylglycine. Cysteine can then feed glutathione biosynthesis through production of gamma-glutamylcysteine. This step is catalyzed by gamma-glutamylcysteine synthase and inhibited by buthionine sulfoximine. Homocystinuria is the principal disorder resulting from impairment of transsulfuration (TS) which leads to
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