Chemical Characteristics of Glucagon

Abstract

The object of this chapter is to describe the covalent chemistry of pancreatic glucagon to provide a background for subsequent chapters. Modifications of the primary structure are considered with respect to biologic activity, but not immunologic activity which is discussed in Chap. 9. Conformation and function are considered in Chap. 3. B. Isolation and PurificationGlucagon is effectively extracted from animal pancreas by any of the acidic ethanol methods commonly employed to extract insulin (e.g., SUTHERLAND and DEDuVE 1948;KENNY 1955). Such methods have been successfully used with mammalian, avian, and piscine pancreas (for details see Chap. 8). The glucagon content of freshly collected bovine or porcine pancreas is about 5-10 J.tg/g wet tissue. RHOTEN (1976) found as much as 5 mg/g glucagon in splenic pancreas from lizards. In a typical insulin purification scheme, glucagon and insulin are purified together to the point of insulin crystallization, where most of the glucagon is found in the mother liquor (SUNDBY and MARKUSSEN 1971). Even at this stage the glucagon fraction is less than 2% pure. The first successful purification of glucagon by STAUB et al. (1955) employed repeated precipitations culminating in crystallization, which was in itself an excellent purification step. In more recent studies, purification was often achieved by separations based more directly on molecular size and on charge. For example, in the purification of chicken glucagon, POLLOCK and KIMMEL (1975) used ion exchange chromatography on columns of CM-Sephadex and QAE-Sephadex. A particulary elegant procedure applicable to small-scale purification was developed by SUNDBY and MARKUSSEN (1971) for rat glucagon and was applied subsequently to glucagon from several other species: (a) the mother liquor fraction from the citrate crystallization of insulin was brought to about half-saturation with NaCl; (b) the resulting precipitate was dissolved in acidic urea solution and gel-filtered in dilute acetic acid; ( c) glucagon fractions (4 % pure) were lyophilized and fractionated by preparative isoelectric focusing in polyacrylamide gels; (d) the glucagon fraction was eluted and crystallized. If desirable, the isoelectric focusing step may most likely be replaced by ion exchange chromatography using both anion and cation exchange media.The first glucagon crystals contained about 10% of a minor acidic component which was separable by electrophoresis (STAUB et al. 1955); reelectrophoresis of the P. J. Lefèbvre (ed.), Glucagon I

Mechanisms of glucagon degradation at alkaline pH

“…In terms of cAMP generation in cultured hepatocytes, oxidized glucagon was found to be slightly less potent than glucagon by one group [12] and much less potent by another [23]. Another group found that the potency of oxidized glucagon in causing glycogenolysis was normal [3]. In our study, rather than oxidizing native glucagon oxidation, we synthesized Met sulfoxide de novo and found that its potency and power to be essentially normal.…”

Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line.

“…Earlier studies of the structure-function relationships ofglucagon indicate that the amino terminus is involved in the signal transduction and biological potency of the peptide, whereas the carboxyl terminus is primarily responsible for recognition and binding to receptors (25). Hence, GLP-I-(1-37) may be an inactive precursor, which undergoes further processing to expose a new amino-terminal region that confers bioactivity.…”

Amyloid Fibrils of Glucagon Characterized by High-Resolution Atomic Force Microscopy

“…Glucagon, a 29 amino acid peptide hormone playing an important endogenous role in maintaining normal circulating glucose levels, is commercially available as a pharmaceutical preparation for the treatment of hypoglycemia and other clinical applications (11). Due to limitations in both chemical and physical stability of glucagon, the pharmaceutical product is supplied as a lyophilized powder that must be used immediately after reconstitution with its supplied aqueous diluent (12). Of particular concern is the propensity of glucagon to aggregate resulting in the formation of fibrils and gels; a process that can be stimulated by acidic pH, increased ionic strength, peptide concentration, agitation, and/or elevated temperatures (12,13).…”