TY - JOUR
T1 - An aluminum silicate binding assay for quantitationof degradation of cholecystokinin octapeptide and other short peptides
AU - Janas, Roman M.
AU - Marks, David L.
AU - LaRusso, Nicholas F.
N1 - Funding Information:
We thank Diane Roddy and Lou Kost for their superb technical assistance and Maureen Craft for typing the manuscript. We also thank Stephen Powers for helpful discussion. This work was supported by a grant from Merck, Sharp and Dohme, by Grant DK24031 from the National Institutes of Health, and by the Mayo Foundation. A preliminary report of part of this work was published in abstract form (Gastroenterology 98, 501, 1990).
PY - 1992/10
Y1 - 1992/10
N2 - Most available techniques for the quantitation of enzymatic degradation of peptide hormones are time-consuming and require expensive equipment and/or novel reagents. Our aim here was to develop a rapid and sensitive assay for the measurement of degradation of cholecystokinin octapeptide (CCK-8) as well as other short, hydrophobic peptides. The proposed technique is based on our novel observation that intact CCK-8, but not its degradation product(s), binds to Lloyd reagent, a form of aluminum silicate. When radiolabeled CCK-8 was exposed to rat liver cytosol containing endogenous CCK-degrading activity, there was a time-dependent decrease in the binding of radiolabel to aluminum silicate [from 86 to 8% over 60 min at 37°C]. The decrease in binding closely paralleled the extent of CCK-8 degradation over time as assessed by high-performance liquid chromatography and immunoprecipitation with specific polyclonal antibodies to CCK-8. While aluminum silicate did not efficiently bind to C-terminal and N-terminal CCK tetrapeptides, magnesium silicate bound to both tetrapeptides (>82%), but not to their radiolabeled degradation products. Both aluminum and magnesium silicate also extensively bound (>82%) to other peptide hormones including Met-enkephalin, somatostatin, and secretin, but did not bind their degradation products. These binding assays will be useful in studies of peptidases which degrade cholecystokinin or other small, hydrophobic peptides.
AB - Most available techniques for the quantitation of enzymatic degradation of peptide hormones are time-consuming and require expensive equipment and/or novel reagents. Our aim here was to develop a rapid and sensitive assay for the measurement of degradation of cholecystokinin octapeptide (CCK-8) as well as other short, hydrophobic peptides. The proposed technique is based on our novel observation that intact CCK-8, but not its degradation product(s), binds to Lloyd reagent, a form of aluminum silicate. When radiolabeled CCK-8 was exposed to rat liver cytosol containing endogenous CCK-degrading activity, there was a time-dependent decrease in the binding of radiolabel to aluminum silicate [from 86 to 8% over 60 min at 37°C]. The decrease in binding closely paralleled the extent of CCK-8 degradation over time as assessed by high-performance liquid chromatography and immunoprecipitation with specific polyclonal antibodies to CCK-8. While aluminum silicate did not efficiently bind to C-terminal and N-terminal CCK tetrapeptides, magnesium silicate bound to both tetrapeptides (>82%), but not to their radiolabeled degradation products. Both aluminum and magnesium silicate also extensively bound (>82%) to other peptide hormones including Met-enkephalin, somatostatin, and secretin, but did not bind their degradation products. These binding assays will be useful in studies of peptidases which degrade cholecystokinin or other small, hydrophobic peptides.
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U2 - 10.1016/S0003-2697(05)80003-X
DO - 10.1016/S0003-2697(05)80003-X
M3 - Article
C2 - 1456442
AN - SCOPUS:0026793178
SN - 0003-2697
VL - 206
SP - 6
EP - 11
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -