A fluorometric assay for measuring deamidase (lysosomal protective protein) using high-performance liquid chromatography.
A rapid and sensitive assay method for the determination of deamidase activity is reported. This method is based on fluorometric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val (N-Dns-D-Tyr-Val), enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val-NH2 (N-Dns-D-Tyr-Val-NH2), after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure N-Dns-D-Tyr-Val at concentrations as low as 100 fmol, yields highly reproducible results and requires less than 8.5 min per sample for separation and quantitation. The optimum pH for deamidase activity was 4.0-4.5. Greater than 5 mM of reduced glutathione was needed for maximal enzyme activity. The Km and Vmax values were respectively 125 microM and 14.12 pmol/micrograms/h with the use of enzyme extract obtained from mouse spleen. Deamidase activity was strongly inhibited by Ag+, Cu2+, diisopropylfluorophosphate, and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in spleen. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this enzyme.
Chikuma
T
T
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Matsumoto
K
K
Furukawa
A
A
Nakayama
N
N
Yajima
R
R
Kato
T
T
Ishii
Y
Y
Tanaka
A
A
D000818:Animals; D002268:Carboxypeptidases / Q000032:analysis* / Q000378:metabolism; D043402:Cathepsin A; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D004151:Dipeptides; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity
D004151:Dipeptides; D002268:Carboxypeptidases; D043402:Cathepsin A