Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa.
Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002850:Chromatography, Gel; D005753:Gastric Mucosa / Q000378:metabolism*; D005755:Gastrins / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011949:Receptors, Cholecystokinin; D013696:Temperature; D014238:Trichloroacetic Acid / Q000494:pharmacology
D005755:Gastrins; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011949:Receptors, Cholecystokinin; D014238:Trichloroacetic Acid; C013550:gastrin 17; D010447:Peptide Hydrolases