The role of transglutaminase in stimulus-secretion coupling in the pancreatic b-cell

Owen, RA, 1988. The role of transglutaminase in stimulus-secretion coupling in the pancreatic b-cell. PhD, Nottingham Trent University.

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Abstract

The sensitivity of rat pancreatic islet transglutaminase to activation by Ca2+ was investigated using a modified assay incorporating dephosphorylated N,N'-dimethylcasein as substrate protein. The Km for Ca2+ obtained (3μM approx.) was an order of magnitude lower than previously reported for the islet enzyme (Bungay et al., 1984). Magnesium (2mM) was found to have little effect on the sensitivity of the enzyme to Ca2+.

A number of primary amine inhibitors were tested for the specificity of their action as inhibitors of transglutaminase activity. Monodansylcadaverine (IC50 = 43μM ; Ki = 9.8μM ) was found to be a specific inhibitor of transglutaminase activity. Monodansylcadaverine (30-100 ?M) significantly inhibited glucose-stimulated insulin release, but had no effect on basal insulin release. Monodansylcadaverine (75μM) inhibited the first phase of glucose-stimulated insulin release and greatly diminished the second phase. The control compound, dimethyl-monodansyl-cadaverine, inhibited neither transglutaminase activity or glucose-stimulated insulin release. These studies strongly implicate transglutaminase in the process of glucose-stimulated insulin release. The test compound N-(8-amino-octyl)-5-iodo-naphthalene -1-sulphonamide was found to be a specific antagonist of calmodulin.

Incubation of intact islets in the presence of [32P]Pi and stimulatory levels of glucose followed by separation of phosphorylated islet proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of a high molecular weight phosphopolymer which did not traverse a 3% (w/v) acrylamide gel. This polymer was found to be predominantly composed of protein. The majority of this phosphopolymer (70% approx.) was present in the 600g av. sedimented fraction of islet homogenates. Incubation of islet homogenates, obtained from intact islets previously incubated with [32P]Pi and stimulatory levels of glucose, under conditions that activated the islet transglutaminase resulted in an increase in the amount of phosphopolymer present in the 600g av. sedimented fraction. Inhibitors of transglutaminase activity which are known to inhibit glucose-stimulated insulin release led to a significant reduction in the fraction of phosphopolymer present in the glucose-stimulated intact islet. These findings suggest that protein cross-linking and phosphorylation reactions may be closely linked in the pancreatic β-cell.

Item Type:	Thesis
Creators:	Owen, R.A.
Date:	1988
ISBN:	9781369324310
Identifiers:	Number Type PQ10290182 Other
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	11 Nov 2020 15:28
Last Modified:	11 Oct 2023 10:14
URI:	https://irep.ntu.ac.uk/id/eprint/41620

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