High-performance liquid chromatographic-fluorimetric assay for cathepsin A (lysosomal protective protein) activity.
A rapid and sensitive assay for the determination of cathepsin A activity is reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe-L-Leu, after separation by high-performance liquid chromatography using a C18 reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 min per sample for separation and quantitation. The optimum pH for cathepsin A activity was 4.5-5.0. The Km and Vmax values were respectively 14.9 microM and 27.91 pmol/microg/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag+, Hg2+, diisopropylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.
Chikuma
T
T
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Ogura
Y
Y
Kasamatsu
M
M
Taguchi
K
K
Mitsui
K
K
Kato
T
T
Tanaka
A
A
D000818:Animals; D001921:Brain / Q000201:enzymology; D002268:Carboxypeptidases / Q000378:metabolism*; D043402:Cathepsin A; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000201:enzymology; D013737:Testis / Q000201:enzymology
D002268:Carboxypeptidases; D043402:Cathepsin A