Detection of tripeptidyl peptidase I activity in living cells by fluorogenic substrates
2006 | journal article; research paper. A publication with affiliation to the University of Göttingen.
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- Authors
- Steinfeld, Robert ; Fuhrmann, Jens C.; Gärtner, Jutta
- Abstract
- Tripepticlyl pepticlase I (TPP-1) is a lysosomal pepticlase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe](2)-rhodamine 110 and [Arg-Nle-Nle](2)-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of (Ala-Ala-Phe](2)-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe](2)-rhodamine 110 and [Arg-Nle-Nle](2)-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the I neuronal pathology underlying classical late-infantile NCL.
- Issue Date
- 2006
- Publisher
- Histochemical Soc Inc
- Journal
- Journal of Histochemistry and Cytochemistry
- ISSN
- 0022-1554