An aluminum silicate binding assay for quantitation of degradation of cholecystokinin octapeptide and other short peptides.
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 degrees 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.
Janas
R M
RM
Department of Internal Medicine, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905.
Marks
D L
DL
LaRusso
N F
NF
D000538:Aluminum Silicates / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D007457:Iodine Radioisotopes; D017633:Magnesium Silicates; D008722:Methods; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012824:Silicic Acid / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism*
D000538:Aluminum Silicates; D007457:Iodine Radioisotopes; D017633:Magnesium Silicates; D009842:Oligopeptides; C005013:Florisil; D012824:Silicic Acid; D012844:Sincalide