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

Journal Article · Sun Dec 31 23:00:00 EST 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(90)90005-T· OSTI ID:6828461

Fricker, L D; Devi, L ^[1]

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1. Albert Einstein College of Medicine, Bronx, NY (USA)

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 Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). 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 x 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.

• OSTI ID: 6828461
• Journal Information: Analytical Biochemistry; (USA), Journal Name: Analytical Biochemistry; (USA) Vol. 184:1; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CARBOXYPEPTIDASES
CATTLE
CELL CULTURES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
ENDOCRINE GLANDS
ENZYME ACTIVITY
ENZYMES
EXTRACTION
GLANDS
HORSES
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
NUCLEI
ODD-EVEN NUCLEI
ORGANS
PEPTIDE HYDROLASES
PITUITARY GLAND
RADIOASSAY
RADIOISOTOPES
RUMINANTS
SEPARATION PROCESSES
SOLVENT EXTRACTION
SPECTROPHOTOMETRY
SUBSTRATES
SWINE
TRACER TECHNIQUES
VERTEBRATES