This is a proposal requesting support for research and student training to study the biochemistry and biology of glycosylated human growth hormone (G- hGH) which was recently discovered in our laboratory. It is hypothesized that because of its altered structure G-hGH has altered biological activity. To test the hypothesis the hypothesis the research plan will focus on ascertaining the structure and biological activities of G-hGH. The specific objectives are; 1. Structural analysis of G-hGH by : a) micropreparative purification of G-hGH, b) comparative HPLC tryptic peptide mapping of hGH and G-hGH to identify peptide peak shifts that point ot location(s) within hGH that are altered to form G-hGH. c) determination of the amino acid compositions (s) and amino acid sequence(s) of the shifted tryptic peptides, d) identification of hGH glycosylation site(s) by glycopeptide dot blot analysis of shifted tryptic peptide peaks, and e) carbohydrate composition. oligosaccharide mapping and carbohydrate sequence analysis of glycosylated tryptic peptides. 2. Generation of a procedure for preparative isolation of native non-denatured G-hGH using a combination of chromatographic and electrophoretic techniques to provide material for biological testing. 3. Assessment of the potency of G-hGH in a standard hGH radioimmunoassay, a human liver GH radioreceptorassay and in a radioreceptorassay for lactogenic hormones as well as determination of its isoelectric point-molecular weight coordinates in a two-dimensional polyacrylamide gel for its unequivocal identification in the presence of other hGH to promote growth, test its effects on carbohydrate metabolism, measure its influence on fat metabolism,a and assess its ability to stimulate lymphoid cell division. We will also determine the metabolic clearance rate of G-hGH. 5. Develop a radioimmunoassay which is specific for G-hGH with monoclonal antibodies. 6. Research training of graduate and undergraduate students in biomedical research with special emphasis on assisting under-represented minority students to excel in the scientific and biomedical fields. Results of these studies will elucidate the structural nature and physiological significance of G-hGH. This information is important to our current understanding of the structure-function relationships of hGH. Student training will augment the participation of ethnic minorities and women pursuing bio medical careers.
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