The preparation of bioactive 125I-gastrin, using Iodo-gen as oxidizing agent, and the use of this tracer in receptor studies.
The preparation of 125I-labelled gastrin 1-17, using Iodo-gen as oxidizing agent, is described. Mono-125I-gastrin was purified from the iodination mixture by gel chromatography, followed by ion-exchange chromatography. The Iodo-gen-labelled 125I-gastrin tracer showed a slightly higher immunoreactivity than a similarly purified tracer produced by a gentle chloramine-T method (50% binding of 0.45 fmol tracer at an antibody dilution of 945,000 versus 780,000, respectively). The Iodo-gen-labelled 125I-gastrin, which had the same biological activity as native gastrin when tested in conscious gastric fistula rats, was tested for specific binding at 30 degrees C in pronase-isolated rat gastric fundic cells and plasma membranes from the same area. A specific binding with a Kd of 4.5 X 10(-9) M was found to isolated fundic cells. This binding was rapid and reached equilibrium within 40 min. The dissociation induced by a 10-fold dilution of the incubation medium was biphasic, with a rapid initial phase and a slow late phase, indicating two different binding sites. On the other hand, no reproducible specific binding of the tracer to plasma membranes was obtained. This study shows that Iodo-gen is suitable as an oxidizing agent in iodination of gastrin without loss of biological activity. The tracer produced may be used in receptor studies for isolated cells, whereas the use of plasma membranes for studying gastrin receptors needs reevaluation.
Kleveland
P M
PM
Haugen
S E
SE
Waldum
H L
HL
D000818:Animals; D005748:Gastric Fundus / Q000166:cytology / Q000378:metabolism; D005753:Gastric Mucosa / Q000166:cytology / Q000378:metabolism*; D005755:Gastrins; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008297:Male; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000502:physiology*; D011949:Receptors, Cholecystokinin; D014508:Urea / Q000031:analogs & derivatives*
D005755:Gastrins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011949:Receptors, Cholecystokinin; C012385:1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril; D014508:Urea