Radioreceptor assay for alpha-MSH using mouse B16 melanoma cells+.
A radioreceptor assay for alpha-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle4]-alpha-MSH tracer. The assay was used (1) to study the binding characteristics of alpha-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of alpha-MSH to B16 cells reached a stable plateau after 3 h at 15 degrees C. At 25 degrees or 37 degrees C, the binding was transient and at 0-1 degree C, the association was very slow. The hormone-receptor complex was relatively stable between 0 degrees and 15 degrees C whereas a 50% dissociation was reached after 90 min at 25 degrees C and after 35 min at 37 degrees C. The mean KD for alpha-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably lower bioassay values than expected from the binding data. This shows that binding and bioactivity can be dissociated in some of the MSH peptides. The biological activity of MSH from the neurointermediate lobe of the rat pituitary as measured by its binding to B16 cells corresponds fairly well with RIA results; in the anterior lobe, alpha-MSH values are overestimated because of the large amount of ACTH present.
Siegrist
W
W
Department of Research, University Hospital, Basel, Switzerland.
Oestreicher
M
M
Stutz
S
S
Girard
J
J
Eberle
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AN
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D005189:False Positive Reactions; D008546:Melanoma, Experimental / Q000032:analysis*; D051379:Mice; D010618:Phenanthrolines / Q000378:metabolism; D010902:Pituitary Gland / Q000032:analysis*; D011485:Protein Binding; D011869:Radioligand Assay; D011979:Receptors, Pituitary Hormone / Q000032:analysis* / Q000378:metabolism; D013329:Structure-Activity Relationship; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000378:metabolism
D010618:Phenanthrolines; D011979:Receptors, Pituitary Hormone; D000521:alpha-MSH; C024146:MSH, 4-Nle-alpha-; C029446:MSH receptor; C025205:1,10-phenanthroline