8. Physiological Active Substances

Puromycin-Sensitive Aminopeptidase (PSA) Inhibitor
PAQ-22, [3-(2,6-diethylphenyl)-2,4(1H,3H)-quinazolinedione]
Wako Cat. No. 165-23581 (10 mg) <for Cellbiology>
Keep at RT

Fluorescent Bioprobe for Visualization of PSA in Living Cells
Wako Cat. No. 049-30761 (2 mg) <for Cellbiology>
Keep at RT

Puromycin-sensitive aminopeptidase (PSA), which is a neutral aminopeptidase with a substrate specifi city similar to that of aminopeptidase N (APN), is distributed mainly in the brain and neurons. The physical roles/functions of PSA remain unclear.
Wako has launched non-peptide, small-molecular, non-competitive PSA Inhibitor, PAQ-22 and the structurally modifi ed fl uorescent bioprobes, DAMPAQ-22. The cellular localization of PSA could be specifi cally visualized by the use of DAMPAQ-221).
These are long-awaited tools for PSA research.
[ Figure 1]
Lineweaver-Burk plot analysis of PSA inhibition by PAQ-22 and Puromycin. Using living human monocytic cell MOLT-4, which is known to express PSA, PSA inhibitory activity of PAQ-22 and Puromycin was determined with an indicator, which is fl uorescence generated from a fl uorescent substrate Ala-MCA broken down by PSA. Lineweaver-Burk plot indicated that PAQ-22 acts as specifi c non-competitive inhibitor. On the other hand, Puromycin acts a competitive inhibitor. In addition, PAQ-22 and DAMPAQ-22 are easily incorporated into MOLT-4 under the general cell culture conditions.
[ Table 1. ]
Aminopeptidase-Inhibitory Activity of PAQ-22 and Puromycin PSA- and APN-inhibitory activities were assayed by the use of L-Ala-MCA with MOLT-4. PAQ-22 is inactive toward APN, indicating that PAQ-22 is specifi c to PSA.
( These data were provided by Dr. Yuichi Hashimoto, Institute of Molecular and Cellular Biosciences, The University of Tokyo)

[ Figure 2. ]
Fluorescent microscopic images of B16F10 mouse melanoma cells treated with 10µmol/L DAMPAQ-22 for 10 min.
(These data were provided by Dr. Yuichi Hashimoto, Institute of Molecular and Cellular Biosciences, The University of Tokyo)
PSA IC50 (µmol/L)APN IC50 (µmol/L)

1) Kakuta, H., Koiso, Y., Nagasawa, K., Hashimoto, Y.: "Fluorescent Bioprobes for Visualization of Puromycin-Sensitive Aminopeptidase in Living Cells" Bioorg. Med. Chem. Lett., 13, 83-6 (2003)
2) Bukowska A, Tadje J, Arndt M, Wolke C, Ka¨hne T, Bartsch J, Faust J, Neubert K, Hashimoto Y, Lendeckel U.: "Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets", Biol. Chem., 384, 657-65 (2003).
3) Kakuta H, Tanatani A, Nagasawa K, Hashimoto Y.: "(1H,3H)-quinazolinedione skeleton", Chem. Pharm. Bull., 51, 1273-82 (2003).
4) Sánchez-Mora´n E, Jones GH, Franklin FC, Santos JL.: "A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana", Plant Cell., 16, 2895-909 (2004).
5) Thielitz A, Bukowska A, Wolke C, Vetter R, Lendeckel U, Wrenger S, Hashimoto Y, Ansorge S, Gollnick H, Reinhold D.: "Identifi cation of extra- and intracellular alanyl aminopeptidases as new targets to modulate keratinocyte growth and diff erentiation", Biochem. Biophys. Res. Commun., 321, 795-801 (2004).

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Wako Product Update Bio-No.1 [ page. 32 ]