Abstract
Glucagon-(1-21) was prepared fully synthetically as well as by carboxypeptidase A digestion of natural porcine glucagon. Neither of the two preparations had glucagon agonistic effects with regard to receptor binding or adenylate cyclase activation in purified rat liver plasma membranes. Nor did these preparations contain lipolytic activity in isolated free fat cells. A preliminary batch of glucagon-(1-21) prepared by carboxypeptidase A digestion did, however, contain 1-2% glucagon bioactivity. This activity was separated from glucagon-(1-21) by high-performance liquid chromatography and quantitatively recovered in four minor hind peaks which eluted close to but not in a position identical to the elution position of native glucagon.
Highlights
Glucagon-(1-21) was prepared fully syntheticallyas mmol) waspurchased from Amersham, United Kingdom, and human well as bycarboxypeptidase A digestionofnatural serum albumin wasfrom Boehringer Mannheim
Microfuge tubes containing 200 p1 of 2.5% human serum albumin in HEPES buffer (50 mmol/liter, pH 7.6).Free and membrane-bound l5I-glucagon wereseparated by centrifugation, and supernatantws ere aspirated with a needle which was attached to a vacuum line
The membrane pellets were onist with regard to receptor binding and adenylate cyclase washed once with 200 p1 of cold albumin containing HEPES buffer, activation in ratliver plasma membranes (1)and with regard to glycogenolysis in isolated rat hepatocytes (2)
Summary
From epididymal fat pads of Wistar rats aspreviously described (13). Monoiodinated 'Z51-glucagon(1.1Ci/pmol) (4)and natural porcine a pBondapak C-18 column (3.9 X 300 mm) obtained from Waters glucagon were from Novo Research Institute. [cx-~*P]AT(P13 Ci/ (catalog no. 27324). Azineethanesulfonic acid; HPLC, high-performance liquid chromatogf T o whom correspondence should be addressed. Peptides were isolated from theHPLC effluent by vacuum centrifugation (Savant vacuum centrifuge) in order to remove the acetonitrile, followed by lyophilization in order to remove the ammonium hydrogen carbonate. Natural glucagon-(1-21) was prepared by carboxypeptidase A glucagon-(l-21) on '"I-glucagon binding to purified rat liver digestion of natural glucagon (1, 14), and the peptide was purified plasma membranesT. The amino acid composition of glucagon-(l-21) was determined glucagon-(1-21); A, naturalglucagon-(l-Zl); 0, natural glucagonafter 24 h of hydrolysis with Clz-free N HCI at 110 "C.The compo- (1-21), preliminary batch. Sition of the preparations of glucagon-(l-21) was close to the theoretical (Table I)
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