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Characterization of Cholecystokinin Binding Sites in Rat Cerebral Cortex Using a 125 I‐CCK‐8 Probe Resistant to Degradation
Author(s) -
Praissman M.,
Martinez P. A.,
Saladino C. F.,
Berkowitz J. M.,
Steggles A. W.,
Finkelstein J. A.
Publication year - 1983
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1983.tb13583.x
Subject(s) - radioligand , cholecystokinin , chemistry , membrane , binding site , cholecystokinin receptor , radioligand assay , secretin , gastrin , cerebral cortex , receptor , biochemistry , endocrinology , biology , secretion
Specific binding sites for cholecystokinin (CCK) have been characterized in a particulate membrane fraction of rat cerebral cortex using a biologically active 125 I‐labeled derivative of the C‐terminal octapeptide of CCK (CCK‐8) prepared by reaction with the iodinated form of the imidoester ( 125 IIE), methyl‐ p ‐hydroxybenzimidate. The time course of binding to cortical membranes was rapid, temperature dependent, and saturable. Half‐maximal binding at 24°C was reached in 30 min and full binding at 120 min. At 37°C there was only a slight increase in 125 IIE‐CCK‐8 bound after 15 min. The addition of a large excess of CCK‐8 after 30 min of binding at 24°C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of 125 IIE‐CCK‐8 bound to membranes with increasing concentrations of CCK‐8 and other structurally related peptides. CCK‐8 displaced 50% of the radioligand at 4 n M , CCK‐33 at 10 n M , and gastrin (desulfated CCK‐8) at 60 n M . Secretin, a structurally unrelated peptide, was unable to displace the radioligand from cortical membranes at 1.0 μM . Finally, 125 IIE‐CCK‐8 exposed to cortical membranes or to buffers that had previously contained such membranes for 60 min at 24°C bound equally as well to fresh cortical membranes as control radioligand that had not been exposed to the same conditions. Thus the 125 I‐CCK‐8 radioligand used in this study was highly resistant to degradative processes in rat brain tissue.