Abstract
Preincubation
of rat islets of Langerhans with the potent inhibitors of islet
transglutaminase activity, monodansylcadaverine (30-100 microM) and
N-(5-aminopentyl)-2-naphthalenesulphonamide (100-200 microM), led to
significant inhibition of glucose-stimulated insulin release from
islets.
In contrast, the respective N'-dimethylated derivatives of these
two compounds, which did not inhibit islet transglutaminase activity,
were much less effective as inhibitors of glucose-stimulated insulin
release. None of the compounds inhibited rat spleen protein kinase C
activity at concentrations which gave rise to inhibition of
glucose-stimulated insulin release.
When tested for their effects on
calmodulin-stimulated bovine heart phosphodiesterase activity, of the
compounds that inhibited insulin release, only monodansylcadaverine did
not act as an effective antagonist of calmodulin at concentrations (up
to 50 microM) that gave rise to significant inhibition of
glucose-stimulated insulin release
. Furthermore, at 50 microM,
monodansylcadaverine did not inhibit methylation of islet lipids.
The
inhibition of glucose-stimulated insulin release by monodansylcadaverine
is therefore likely to be attributable to its interference with islet
transglutaminase activity.
The sensitivity of islet transglutaminase to
activation by Ca2+ was investigated by using a modified assay
incorporating dephosphorylated NN'-dimethylcasein as a substrate
protein. The Km for Ca2+ obtained (approx. 3 microM) was an order of
magnitude lower than previously reported for the islet enzyme [Bungay,
Potter & Griffin (1984) Biochem. J. 219, 819-827]. Mg2+ (2 mM) was
found to have little effect on the sensitivity of the enzyme to Ca2+.
Investigation of the endogenous substrate proteins of islet
transglutaminase by using the Ca2+-dependent incorporation of
[14C]methylamine into proteins of islet homogenates demonstrated that
most of the incorporated radiolabel was present in cross-linked
polymeric aggregates which did not traverse 3% (w/v) acrylamide gels.
The radiolabelled polymeric aggregates were present in 71 000
g-sedimented material of homogenates, and their formation was
transglutaminase-mediated.
These findings provide new evidence for the
involvement of islet transglutaminase in the membrane-mediated events
necessary for glucose-stimulated insulin release.
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