A radioimmunoassay based on a monoclonal antibody, Mc-ab 1, which was raised against growth hormone but cross-reacted with human placental lactogen yielded higher GH immunoreactivity levels in serum than one based on a polyclonal antiserum. This discrepancy was noted in subjects with normal GH secretion as well as in patients with GH insufficiency. To characterize this GH immunoreactivity detected by Mc-ab 1, affinity purification and molecular sieve chromatography of serum were performed. High molecular weight proteins with GH immunoreactivity were found with both techniques. These proteins were associated with carbohydrates. Affinity cross-linking showed specific binding of radiolabelled GH to high molecular weight proteins in the serum. After fractionation of serum, the GH immunoreactivity became detectable by the polyclonal antiserum assay as well as by an immunoradiometric assay. GH immunoreactive material with an approximate mass of 80 kD was subjected to isoelectric focusing. When GH immunoreactive fractions at pH 5 were re-chromatographed, GH immunoreactivity was recovered in the elution volume corresponding to monomeric GH. Our results show that sera from normal subjects as well as from patients with deficient GH secretion contain notable amounts of high molecular weight GH which is undetectable by antibodies generally used for GH measurements, but which can be revealed after fractionation of serum.The aim of this work was to elucidate the discre¬ pancies found when growth hormone was mea¬ sured in human serum using a polyclonal antiserum (1) and a monoclonal antibody, Mc-ab 1, raised against GH but cross-reacting with human placental lactogen (2): Patients with GH insufficiency and non-detectable GH levels according to the polyclo¬ nal antiserum showed consistently detectable GH immunoreactivity with Mc-ab 1.Several monoclonal antibodies have been pro¬ duced against the closely related hormones human GH and human placental lactogen. These hor¬ mones consist of a single polypeptide chain, crosslinked by two disulfide bridges and have a mole¬ cular mass of approximately 22 kD (3). Pituitary GH does not, however, occur as only one molecular species, but shows both size and charge variations (4-6). In spite of the heterogeneity of GH, the radioimmunoassays based on monoclonal antibodies have correlated well with RIAs using polyclonal antisera (7-9). Subjects and MethodsSerum samples were obtained from 3 healthy volunteers, 2 patients with panhypopituitarism, 3 with acromegaly, and 2 with prolactinomas. The panhypopituitarism of the 2 was due to a non-secreting pituitary adenoma and a craniopharyngioma, respectively; these 2 patients had non-detectable GH levels in 24-h diurnal profiles, after oral administration of 500 mg L-dopa and during insulininduced hypoglycemia determined with the polyclonal antiserum.From the patients with acromegaly and newly diag¬ nosed disease, pieces of growth hormone producing adenomatous tissue were obtained during pituitary micro-
Polyethylene glycols 4,000 and 6,000 were determined in different plasma protein preparations by the formation of complexes with barium and iodide ions after precipitation of the proteins with perchloric acid. The method, which is fast and reliable, can be used for the determinations of PEG down to 0.005% (w/w) of the total protein content.
GH has acute stimulatory effects on amino acid transport and protein synthesis in a variety of tissues, but it has not been established whether these effects are expressions of the growth-promoting property of GH or of its separate insulin-like action. The 20,000-dalton structural variant of human GH (20K hGH) has been shown to have a high ratio of growth-promoting to insulin-like activity compared to native hGH (22K hGH), suggesting that it could be used as a tool to address the above question. Therefore, experiments were conducted to compare the relative abilities of native 22K hGH and 20K hGH, when added in vitro, to stimulate amino acid transport and protein synthesis in the isolated diaphragm of the female hypophysectomized rat. Paired intact hemidiaphragms were preincubated for 1 h in the absence or presence of various concentrations of 22K or 20K hGH. Then, 3-O-[14C]methylglucose was added to the medium to measure sugar transport as a test of insulin-like activity, and either alpha-[3H]aminoisobutyric acid acid or [3H] phenylalanine was also added to measure amino acid transport or protein synthesis, respectively, during a final hour of incubation. When the responses to the various concentrations of 22K and 20K were compared, 20K hGH was only about 20% as effective as 22K in stimulating 3-O-methylglucose transport, reflecting its markedly attenuated insulin-like activity on the diaphragm. Similarly, 20K hGH was only 20% as effective as 22K hGH in stimulating alpha-aminoisobutyric acid transport and phenylalanine incorporation into protein in the same muscles. Therefore, these findings support the idea that the rapid stimulatory effects of GH on amino acid transport and protein synthesis are expressions of the insulin-like action of GH and are not components of the response of target cells to its growth-promoting action.
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