Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma.
Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of 125I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, Ke. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for Kd. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D003208:Concanavalin A / Q000378:metabolism*; D005260:Female; D005947:Glucose / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D011972:Receptor, Insulin / Q000378:metabolism*; D011952:Receptors, Concanavalin A / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology*
D007328:Insulin; D011952:Receptors, Concanavalin A; D003208:Concanavalin A; D011392:Proline; D011972:Receptor, Insulin; D014357:Trypsin; D005947:Glucose