Studies on the mode of action of cyclic AMP in regulating the release of insulin from islets of Langerhans.
thesisposted on 19.11.2015, 09:07 by Yvonne. Soars
The subcellular distribution of protein kinase activity in isolated islets of Langerhans was determined. The majority (70%) of cyclic AMP-dependent protein kinase activity was located in the S-100 (soluble) fraction, while the majority (42%) of cyclic AMP-independent activity was located in the solublised P-100 (containing mitochondria, secretory granules and microsomes) fraction. Partial characterisation of the islet cyclic AMP-dependent protein kinase activity revealed the presence of two isozymes designated Type I and Type II. Type II kinase was the predominant isozyme of the S-100 fraction and Type I was the predominant isozyme found in the solublised P-100 fraction. Nonidet-P40 (a non-ionic detergent) was found to 'activate' the S-100 cyclic AMP-dependent protein kinase activity, although no significant increase in protein kinase activity was observed when the P-0.6 (containing nuclei and cellular debris) fraction and P-100 fraction were solublised with Nonidet-P40. This 'activation' was not directly due to the observed Nonidet-P40 inhibition of (?-32P)-ATP hydrolysis and is likely to be related to changes in the charged state of the holoenzyme induced by Nonidet-P40. The phosphorylation of islet cell proteins by islet cell protein kinases has been studied, using a cell free system. The incorporation of 32P from (?-32P)-ATP into islet non-nuclear proteins was found to vary with pH. In the presence of Walsh protein (a specific inhibitor of cyclic AMP-dependent protein kinases) cyclic AMP mediated phosphorylation of four polypeptides was demonstrated at pH 6.0. At pH 7.0, cyclic AMP inhibition of 32P incorporation into a S-100 54,000 dalton polypeptide was observed. At pH 7.0, Nonidet-P40 inhibited 32P incorporation into a 88,000 dalton polypeptide. This protein was tentatively identified as an ATPase whose activity was modulated by its phosphorylation state. The results of these studies are discussed in relation to the role of cyclic AMP in insulin secretion.