Influence of dimyristoylphosphatidylcholine vesicles on the proteolysis of bound glucagon

Abstract

The interactions between dimyristoylphosphatidylcholine (DMPC) vesicles and mammalian glucagon have been investigated by intrinsic fluorescence and centrifugal separation of DMPC vesicle-glucagon complexes. The influence of the phospholipid vesicles on the hydrolysis of glucagon by trypsin and chymotrypsion have been studied. The hydrolysis process was monitored by HPLC of glucagon and its digestion products. The topology of bound glucagon was predicted indirectly by sequencing the protected fragments of glucagon from proteolytic enzymes. DMPC vesicles induced a blue-shift in the wavelength of maximum emission of fluorescence of glucagon by about 14 nm and the fluorescence intensity was increased by about 230\%. Glucagon interact with DMPC vesicles electrostatically and hydrophobically. The hydrophobic interaction appears to protect glucagon against proteolysis by trypsin and chymotrypsion. Protection of glucagon by hydrophobic interaction with membrane was caused by physiological barrier of membrane. Prolonged tryps in treatment cleaved only Arg(18) site out of three possible sites. From this abservation possible structure of irreversibly bound glucagon on the vesicle surface is discussed.