Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three-step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenylSepharose); and third, anion-exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50 "/.Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35 000, determined by exclusion chromatography, and 32 000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidiciamidated residues. The N-terminal amino acid sequence was Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala.

A RIA has been developed for somatomedin A (SM-A) utilizing Sepharose-bound antibodies. This assay, measuring SM-A, the insulin-like growth factors 1 and 2, and somatomedin C, allows determination in serum samples. In comparison with a serum standard, the mean serum levels in patients with acromegaly or GH deficiency and healthy subjects were 8.7 %/- 0.7 (n = 25), 0.24 +/- 0.02 (n = 25), and 1.15 +/- 0.11 U/ml, respectively. The correlation coefficient between immunoreactive SM-A and SM-A by radioreceptor assay was highly significant (r = 0.93), although the potency ratio of SM-A between the two groups of patients was higher in the RIA than in the radioreceptor assay. Gel chromatography revealed that SM-A in acromegalic serum is bound to a carrier protein which is absent in patients with GH deficiency. After gel chromatography at low pH, 90% of applied immunoreactive SM-A was recovered in the low molecular weight fraction and consisted mainly of neutral polypeptides.

Binding of human GH (hGH) and insulin-like growth factors I and II (IGFI and II) to isolated human adipocytes from adult subjects was studied. Binding equilibrium for hGH at 24°C was reached at 120 min and half-maximal specific binding at 6-8 ng/ml. Apparent K, was 2.1 x log M-l and B,,, 7.3 x 10 -ii M/lo6 cells. The human fat cell growth hormone receptor recognized neither bovine, ovine or rat GH nor human prolactin or placental lactogen. No specific receptors for human IGFII could be demonstrated.Thus, human adipocytes do not possess IGF receptors but have specific GH receptors which recognize hGH but not GH from lower species. Growth hormone InsulinInsulin-like growth factor Adipocyte

Abstract. Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and fl-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13-acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to ~70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I.