Abstract
To study (pro)insulin biosynthesis, pancreases from calf fetuses 27-30 cm long were incubated with 3H-leucine (100 µC/ml) and glucose (3.0 mg/ml) for periods from six to twelve hours. Sephadex G-100 chromatography of the acid-ethanol extracts demonstrated that a large proportion of the biosynthetic activity was directed towards the production of substances of very high molecular weight. Two major areas of radioactivity and immunoreactive insulin (IRI) activity were found by Sephadex G-50 chromatography: higher molecular weight IRI (HMWIRI) and IRI. The movement of radioactivity from the HMWIRI into the IRI peak was shown in a pulse-chase experiment. The seemingly homogenous central regions of Sephadex G-50 IRI peaks were further separated into three major fractions by Polyacrylamide disc-gel electrophoresis. The first fraction behaved electrophoretically as insulin. The two more basic fractions accounted for over 50% of both radioactivity and IRI and exhibited decreased biological activities. Mild tryptic hydrolysis of these two fractions yielded material with the electrophoretic mobility of insulin. The molecular size, charge and susceptibility to tryptic hydrolysis suggest that these two fractions represent mono- and di-arginine insulins.
Key words
Proinsulin - Fetal Calf Pancreas -
3H-Leucine - Immunoreactive Insulin
1 Supported in part by the Fonds national Suisse de la recherche (Grant no. 3.384.70 SR), Berne, Switzerland, and by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany
