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Fluorogenic Substrates for Bacterial Aminopeptidase P and Its Analogs Detected in Human Serum and Calf Lung
- Source :
- European Journal of Biochemistry. 125:609-615
- Publication Year :
- 1982
- Publisher :
- Wiley, 1982.
-
Abstract
- A sensitive fluorimetric assay was developed for bacterial aminopeptidase P, based on intramolecularly quenched fluorogenic substrates. Two substrates were synthesized. Phe(NO2)-Pro-HN-CH2-CH2-NH-ABz (substrate I) and Phe(NO2)-Pro-Pro-HN-CH2-CH2-NH-ABz (substrate II), in which the Phe(NO2) group (rho-nitro-L-phenylalanyl) quenches the fluorescence of the ABz group (omicron-aminobenzoyl). Both substrates were readily cleaved by aminopeptidase P from Escherichia coli, releasing rho-nitro-L-phenylalanine and causing a proportional increase in fluorescence. Complete hydrolysis of the two substrates resulted in a 7.5-fold and 3.4-fold fluorescence increase, respectively. Applying this fluorogenic assay, we were able to detect and measure quantitatively amino-peptidase P-like activity in the human serum and calf-lung extracts. Substrate II was shown to be specifically cleaved by aminopeptidase P in these preparations, while substrate I was apparently cleaved by other enzymes as well. In both preparations, the enzyme activity was independent of Co2+ ions, and Pro-HN-CH2-CH2-NH-ABz (Cbz) was inhibitory. The kinetic constant Km was determined as 0.35 mM and 0.28 mM for the human serum and the calf-lung enzymes respectively. The enzyme activity was only slightly dependent on pH in the range 7.0-8.4.
- Subjects :
- medicine.disease_cause
Aminopeptidases
Biochemistry
Substrate Specificity
Hydrolysis
Escherichia coli
medicine
Animals
Humans
Fluorometry
Kinetic constant
Lung
chemistry.chemical_classification
Chromatography
biology
Aminopeptidase P
Substrate (chemistry)
Hydrogen-Ion Concentration
Fluorescence
Enzyme assay
Kinetics
Enzyme
Chromogenic Compounds
chemistry
biology.protein
Cattle
lipids (amino acids, peptides, and proteins)
Oligopeptides
Subjects
Details
- ISSN :
- 00142956
- Volume :
- 125
- Database :
- OpenAIRE
- Journal :
- European Journal of Biochemistry
- Accession number :
- edsair.doi.dedup.....19066a5e34437d41ee6fd8daa769be19