Purification and Radioreceptor Assay of Insulin-Like Growth Factor in Human Serum

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Insulin-like growth factor (IGF) is the genoric designation for serum polypeptides such as somatomedins, multiplication stimulating activity (MSA), and non suppressible insulin-like activity (NSILA), which possess both insulin-like activity and cell growth promoting activity. We have purified one of IGF's from human plasma and established radioreceptor assay (RRA) for this IGF. RRA for insulin, using human placental microsomal membranes, was used to detect insulin-like activity throughout the isolation procedure. The soluble fraction from an acid ethanol extract of Cohn fraction IV was chromatographed on G-75 sephadex in 1% formic acid. The insulin-like activity which binds to insulin receptor (ILA) migrated as a small molecule (Kav=0.56-0.70). The active fraction was subjected to isoelectric focusing. Three peaks of ILA were identified at the position of pH 5.2, 7.0 and 8.5. The major fraction (pI 8.5) designated as ILA-C was further purified by G-50 sephadex gel filtration in 1M acetic acid. The apparent molecular weight of purified ILA-C is around 6,000. ILA-C stimulated 14C-glucose oxidation in rat epididymal fat cells. It also potently augmented 3H-Thymidine incorporation into DNA of 3T3 cells. Thus ILA-C satisfies the characteristics of IGF, but the identification of ILA-C among known IGFs' is not clear at present. Subsequently RRA for ILA-C, utilizing human placental membrane as a receptor, was developped. Placental membrane fraction (100-200μg protein), 125I-ILA-C (50,000cpm) and ILA-C standard or unknown serum in a total volume of 0.5ml were incubated for 16 hrs at 4°. The membrane-bound and the free labelled ILA-C were separated by centrifugation and the pellet was counted in a gamma scintilation counter. An arbitrary unit of ILA-C was expressed as μU/ml insulin equivalent as determined by RRA for insulin. The bound 125I-ILA-C was displaced dosedependently by ILA-C between 0.5μU and 100μU per tube. The binding of labelled ILA-C to the receptor was inhibited partially by somatomedin A and MSA, but only minimally by insulin. Somatomedin B, epidermal growth factor, nerve growth factor, fibroblast growth factor or proinsulin were without effect. Addition 1 to 10μl of sera gave the displacement curve parallel to that obtained by the standard. Comparison of elution patters of ILA-C in human serum on G-50 sephadex at acidic and neutral pH suggests that ILA-C is associated with serum protein and the complex is dissociated by acid. The presence of binding protein in serum might disturb the assay. The dilution curve of native serum between 1μl and 10μl was parallel to that of acid-dissociated serum, and thus ILA-C can be quantitatively measured in native serum at neutral pH in the range between 1μl and 10μl. Therefore all the following assays were done at neutral pH using serum less than 1μl. The intra and inter-assay variences were 6.7% and 20.4%, respectedly. The RRA can measure the concentration of ILA-C in the sera of a variety of mammals. Serum concentrations of ILA-C in normal males and females were 575±46 (mean±SEM), 528±27, respectedly. ILA-C levels were high in acromegalics (3, 133±245) and low in hypopituitary patients (286±47). Administration of hGH to patients with GH deficiency elevated ILA-C levels significantly. In cases of anorexia nervosa, ILA-C levels were generally low but increased as the body weight returned to normal. Very low levels were also found in cord bloods and in patients with liver cirrhosis, whereas uremic patients had high levels. In patients with diabetes mellitus and hypoglycemia due to extrapancreatic tumor ILA-C levels were normal or low. In summary, ILA-C was purified from human plasma using RRA for insulin and sensitive RRA for ILA-C was established. The levels of ILA-C in the serum are under the regulation of GH, as well as nutritional and other factors.
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Purification and Radioreceptor Assay of Insulin-Like Growth Factor in Human Serum

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