Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

• Published in: Analytical biochemistry Vol. 184; no. 1; pp. 21 - 27
• Main Authors: Fricker, Lloyd D., Devi, Lakshmi
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 1990; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; ANIMALS; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; Biological and medical sciences; BODY; Carboxypeptidase B; Carboxypeptidase H; CARBOXYPEPTIDASES; Carboxypeptidases - analysis; CATTLE; CELL CULTURES; Cells, Cultured; DAYS LIVING RADIOISOTOPES; DOMESTIC ANIMALS; ELECTRON CAPTURE RADIOISOTOPES; ENDOCRINE GLANDS; ENZYME ACTIVITY; ENZYMES; Enzymes and enzyme inhibitors; EXTRACTION; Fluorometry; Fundamental and applied biological sciences. Psychology; GLANDS; HORSES; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE 125; IODINE ISOTOPES; Iodine Radioisotopes; ISOTOPE APPLICATIONS; ISOTOPES; Kinetics; MAMMALS; Microchemistry - methods; NUCLEI; ODD-EVEN NUCLEI; ORGANS; PEPTIDE HYDROLASES; PITUITARY GLAND; Pituitary Gland - enzymology; RADIOASSAY; RADIOISOTOPES; Radiometry; RUMINANTS; SEPARATION PROCESSES; SOLVENT EXTRACTION; SPECTROPHOTOMETRY; Spectrophotometry, Ultraviolet; Subcellular Fractions - enzymology; Substrate Specificity; SUBSTRATES; SWINE; TRACER TECHNIQUES; VERTEBRATES 550201 > Biochemistry > Tracer Techniques; Substrate; Enzymatic activity; Carboxypeptidase B; Radiolabelling; Peptides; Analog; Reaction product; Radiometry; Spectrophotometry; Comparative study; Fluorescence spectrometry; Quantitative analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(90)90005-T

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg ( 125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity ( 125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with K cat values of 16 s −1 for AcYAR, 13 s −1 for dans-FAR, and 8.5 s −1 for hipp-R. The K m values for CPE with AcYAR (28 μ m) and dans-FAR (34 μ m) are similar, and are much lower than the K m with hipp-R (400 μ m). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10 −15 mol), compared to the fluorescent product (5 × 10 −11 mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described.