Abstract
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 S35-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 S35-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.
Highlights
In mammals, cystathionine beta-synthase (CBS) catalyzes the first step in the transsulfuration pathway [1], a pyridoxal-5-phosphate- (PLP-) dependent condensation of serine and homocysteine to cystathionine [2, 3]
Based on our results obtained using a radioactive tracer method involving 35S-methionine incorporation into glutathione, we report that the transsulfuration pathway is active in human mammary epithelial cells
To test our hypothesis that transsulfuration occurs in mammary tissue, we incubated primary human mammary epithelial cells (HEMC’s) with a radioactive 35S-methionine tracer (Perkin Elmer, Waltham, MA), isolated cellular material following incubation, and determined the extent to which the 35S-methionine incorporated into glutathione
Summary
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]. Transsulfuration impairment is associated with other disorders such as autism [4,5,6,7,8], cirrhosis [9,10,11,12,13], coronary artery disease [14,15,16,17,18], goiter [19], impaired coagulation [20,21,22], immune function [23], neurodegenerative disease [24,25,26], and pancreatitis [27]. Despite the widespread nature of tissues involved in these disorders, our knowledge of transsulfuration is confined to the liver and a small number of other tissues. These include the brain [28], certain lymphoid cells [29], and the pancreas [27, 30]
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