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JHC exPRESS: First Published June 16, 2006. doi:10.1369/jhc.5A6900.2006
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A more recent version of this article appeared on September 1, 2006.
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Articles

Detection of Tripeptidyl Peptidase I Activity in Living Cells by Fluorogenic Substrates

Robert Steinfeld 1*, Jens C. Fuhrmann 1 and Jutta Gärtner 1

1 Department of Pediatrics and Pediatric Neurology, University of Göttingen, Göttingen, Germany (RS,JG), and Centre of Molecular Neurobiology (ZMNH), University of Hamburg, Hamburg, Germany (JCF)

* To whom correspondence should be addressed. E-mail: rsteinfeld{at}med.uni-goettingen.de.

Submitted on December 11, 2005
Accepted on 31 May 2006


  Abstract
Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase 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 colocalize 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 to determine TPP-I activity in living cells and also its intracellular localization. Further, these substrates could be a valuable tool to study the neuronal pathology underlying classical late infantile NCL. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Key Words: neuronal ceroid lipofuscinosis, NCL, tripeptidyl peptidase I, TPP-I, neurodegeneration, living cell cytochemistry, protease


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