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Erythropoietin regulates the growth and proliferation of red blood cell progenitors. We demonstrated previously the important structural and functional roles of the amino acid region 99–110 in the biological activity of the hormone. [Chern, Y., Chung, T. & Sytkowski, A. J. (1991) Eur. J. Biochem. 202, 225–229]. We have now performed alanine scanning mutagenesis to identify which specific residues in this region are essential for function. Four substitutions reduced the biological activity of erythropoietin. Alanine substitution of Ser104, Leu105 and Leu108 reduced activity to 16, 44, and 37% that of the wild-type protein, respectively. Most significantly, substitution of Arg103 with Ala reduced activity to undetectable levels, indicating a > 230-fold reduction in specific activity. Immunochemical analyses using anti-peptide monoclonal antibodies demonstrated that this substitution does not cause major changes in the conformation of the protein or large, localized distortions within the amino acid region 99–129 of the molecule. Heat-denaturation kinetics for the arginine mutant and wild-type erythropoietin are virtually identical, further indicating the structural similarity between these two molecules. Based upon these finding, we propose that Arg103 plays an essential role in the biological activity of erythropoietin, presumably by interacting directly with the erythropoietin receptor. [ABSTRACT FROM AUTHOR]

Studies the amount of glucose-6-phosphatase activity in the intact beta-cell and to assess its control strength on glycolytic flux. Presence of mM 2-deoxyglucose; Islet preparation and incubation protocol; Ion transport, biosynthesis and secretion; Stimulation of insulin secretion by the production of ATP.