The role of transglutaminase in the pancreatic b-cell

Lindsay, M.A., 1991. The role of transglutaminase in the pancreatic b-cell. PhD, Nottingham Trent University.

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Electropenneabilised rat islets were employed to investigate a role for transglutaminase as a Ca2+ target during stimulus-secretion coupling. Incubation of electropermeabilised islets in the presence of the primary amine inhibitors of transglutaminase, methylamine, cystamine, glycine methyl ester and monodansylcadaverine inhibited Ca2+-stimulated insulin release. In all cases except for monodansylcadaverine, which appeared to have caused maximum inhibition at the lowest concentration employed, the inhibition was dose- dependent. Incubation with the control compounds of monodansylcadaverine and glycine methyl ester, dimethyl-monodansylcadaverine and sarcosine methyl ester respectively, which lack the primary amine group necessary for inhibition, had no effect on Ca2+-stimulated insulin release. The primary ainines also failed to inhibit basal insulin release from electropermeabilised islets.

These studies suggests a role for transglutaminase during Ca2+-stimulated insulin release in electropermeabilised islets. Incubation of electropermeabilised islets with monodansylcadaverine and glycine methyl ester failed to inhibit insulin release stimulated by either the protein kinase C activator, phorbol myristate acetate or cyclic AMP in the presence of the phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine or arginine. These result suggest that transglutaminase acts at stage prior to these stimulators or that different insulin secretory mechanisms exist.

Using [14C]-methylamine incorporation into, islet proteins by transglutaminase as a probe, attempts were made to identify transglutaminase substrates using both homogenised and electropermeabilised islets. In homogenised islets the major substrate was confirmed as being a high molecular weight polymer which was unable to traverse a 3%(w/v) acrylamide gel following SDS-polyacrylamide gel electrophoresis. The polymer was predominantly associated with 71,000g av. particulate fraction upon subcellular fraction. Studies using electropermeabilised islets showed that three proteins of approximate molecular weights 78kDa, 34kDa and 32kDa were labelled at Ca2+ concentrations required for insulin release whilst the high molecular weight polymer was labelled at supra-physiological concentrations of Ca (100μM and 1mM) which correlated with the loss of insulin release.

Item Type: Thesis
Creators: Lindsay, M.A.
Date: 1991
ISBN: 9781369324204
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 11 Nov 2020 12:15
Last Modified: 11 Oct 2023 09:19

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