Biochemical and immunological characterisation of proteins of the chromaffin granule membrane

• Main Author: Tugal, H. Bülent
• Format: Dissertation
• Language: English
• Published: University of Edinburgh 1991

The aim of the project was to characterise the membrane proteins of the bovine adrenal chromaffin granule by use of mouse monoclonal antibodies raised against them. Such an approach was chosen to overcome the need to purify each antigen from the complex mixture of proteins that are found in preparations of chromaffin granule membranes. The production of material suitable for immunisation involved making fractions of proteins derived from chromaffin granule membranes which were essentially free of the most abundant and immunogenic proteins such as dopamine β-hydroxylase, cytochrome b_561 and the luminal secretory protein chromagranin A. This was done firstly by using the phase separation property of the non-ionic detergent Triton X-114 to obtain a fraction that was enriched with membrane glycoproteins, and secondly by the liberation of tryptic peptides from chromaffin granule membranes that had been washed free of cytosolic and luminal contaminants. Another approach was to obtain a fraction of proteins that bind to chromaffin granule membranes in the presence of elevated concentrations of free calcium ions. Identification of the antibodies that were produced in mice required the development of rapid and sensitive methods of screening the hybridoma supernatants. Procedures for immuno-dotting and enzyme linked immunosorbent assays were developed and these were followed by Western blotting to initially characterise the antibodies. Using the approaches mentioned above several monoclonal antibodies were obtained, in particular a mouse IgM termed cgm67 was selected for further characterisation. Using this antibody and dbh1 (an IgG directed against the chromaffin granule membrane marker protein dopamine β-hydroxylase) the cgm67-reactive antigen was characterised. This antigen was found to have a molecular weight of 67 kDa and pl of 5.4-6.2, and was shown to be an integral chromaffin granule membrane protein. Its susceptibility to the enzyme endoglycosidase F and neuraminidase showed that it was N-glycosylated. Limited trypsin treatment of chromaffin granules and their membranes liberated a 39 kDa soluble fragment of the antigen which corresponded to the whole of its cytoplasmic domain. This domain was shown to contain both the epitope for the monoclonal antibody cgm67 and a calmodulin binding site. This 39 kDa fragment was purified, its N-terminal 20 amino acids were sequenced and found to be identical to a sequence within the rat synaptic vesicle protein p65 which has also been identified as a homologue of a calmodulin-binding protein in chromaffin granule membranes.