Half-maximal Displacement Research Articles

Insulin Action in Isolated Rat Thymocytes: I. BINDING OF 125I-INSULIN AND STIMULATION OF α-AMINOISOBUTYRIC ACID TRANSPORT

Abstract Rat thymocytes, isolated from 13- to 19-day-old animals, responded to insulin with an increased influx of α-aminoisobutyric acid (AIB). Insulin was effective at 1 nm; the half-maximal response was 40 nm, and a maximal response was seen at 1 µm. Biologically inactive insulin derivatives as well as adrenocorticotropic hormone and glucagon were without effect. 125I-Insulin bound specifically to rat thymocyte receptors and was displaced by unlabeled insulin at 1 nm. The insulin concentration that produced half-maximal displacement of 125I-insulin was 20 nm, and complete displacement was seen at 1 µm. Other hormones or inactive insulin derivatives had trivial effects. Insulin increased AIB influx by both raising the maximal influx capacity (Jmax) and lowering the concentration of AIB at which influx was half maximal (Km); insulin did not affect AIB fractional efflux. The full effect of insulin on influx was not immediate; rather, stimulation of influx increased linearly with time. Cycloheximide both inhibited basal AIB influx and reduced the response to insulin. Insulin had only a small effect on [14C]leucine incorporation into cellular proteins and had no effect on the uptake of 3-O-methylglucose. The data suggest that (a) insulin stimulation of AIB transport on rat thymocytes correlates in sensitivity and specificity with 125I-insulin binding to receptors, and (b) insulin stimulation of AIB influx may be mediated by changes in synthesis or degradation of the AIB transport apparatus.

Glucagon Receptors in β-Cells: BINDING OF 125I-GLUCAGON AND ACTIVATION OF ADENYLATE CYCLASE

Abstract Glucagon-sensitive, insulin-secreting tumors of the Syrian (golden) hamster were homogenized in 1 mm NaHCO3 and subjected to differential centrifugation. The 10,000 x g particles were used for both binding of 125I-glucagon and activation of adenylate cyclase. Glucagon was the only hormone that activated adenylate cyclase. 125I-Glucagon bound to receptors rapidly and was competitively displaced by 1 µg per ml or less of unlabeled hormone; other polypeptide hormone were without effect. The glucagon concentration that produced half-maximal activation of adenylate cyclase was 10 ng per ml and half-maximal displacement of 125I-glucagon was 5 ng per ml; 2–29 glucagon, missing only the NH2-terminal histidine, bound to receptors but did not activate adenylate cyclase. When 2–29 glucagon was added to native glucagon, it blocked activation of adenylate cyclase. Extracts of porcine that contained glucagon immunoreactivity also activated adenylate cyclase but were only one-tenth as potent as pancreatic glucagon; 2–29 glucagon inhibited the effect of gut glucagon.