This suggests that transport of D4TCA is a limiting factor at bot

This suggests that transport of D4TCA is a limiting factor at both the peroxisomal membrane and the plasma membrane.

Only small amounts of D4TCA were detected in peroxisomes. This may be expected, as D4TCA only transiently resides in peroxisomes. Moreover, D4TCA may disappear from the peroxisomal fraction during their isolation BAY 80-6946 due to (1) mechanical rupture of the peroxisomal membrane, and (2) maintained export of D4TCA without new production in peroxisomes. However, at present we are not able to discriminate between tauro/glyco-CA formed in the peroxisomes followed by transport to the cytosol and tauro/glycol-CA that is formed in the cytosol directly. This requires manipulation of the peroxisomal bile salt shuttle, either by inhibiting the to-be-identified-peroxisomal bile salt transporters or manipulating peroxisome biogenesis. It is relevant to note

that this is the first report that demonstrates the presence of a specific product of peroxisomal metabolism in the peroxisome-enriched fractions after a full cell fractionation procedure. Mechanical breakage of peroxisomes was kept to a minimum by using optimized protocols that stabilize these organelles,13 which also further reconfirmed the predominant peroxisomal location of BAAT because it remained (almost) undetectable in the cytosol-enriched fractions after Nycodenz gradient centrifugation. Remarkably, significant amounts of BAAT, catalase, and PMP70 were also detected in low density gradient fractions cofractionating, in part, with mitochondria. In these fractions also D4TCA this website was detected. It remains to be determined whether these fractions contain a subpopulation of peroxisomes that may be involved in the bile salt conjugation as well. To obtain independent evidence for the peroxisomal shuttle we also analyzed the subcellular distribution of several variants of 4-nitrobenzo-2-oxa-1,3-diazole

(NBD)-labeled cholic acid (with the NBD group at the 3alpha, 3beta, 7alpha, and 7beta position, respectively)27 by fluorescence microscopy. Only 3alpha-NBD-cholate was taurine-conjugated and exported to the medium by cultured rat hepatocytes. However, the aminophylline efficiency of conjugation is much lower (>90%) compared to D4CA. Interestingly, a clear accumulation of 3alpha-NBD-CA in subcellular structures was detected at early timepoints (see Supporting Fig. S2). Unfortunately, due to technical limitations we were unable so far to identify these subcellular structures (see Supporting data for details). Still, the detection of a clear punctuate staining pattern for 3alpha NBD-cholate in hepatocytes supports our data that bile salts (transiently) accumulate in membrane enclosed organelles. Remarkably, we did not detect D4GCA in the peroxisomal fractions. Still, BAAT is believed to be responsible for both taurine and glycine-conjugation of bile salts.3 This may indicate that the peroxisomal bile salt exporter in rat hepatocytes has a higher affinity for GCA compared to TCA.

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