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. 1993 Mar 1;290(Pt 2):389–394. doi: 10.1042/bj2900389

Kinetic characterization of guanine-nucleotide-induced exocytosis from permeabilized rat mast cells.

T H Lillie 1, B D Gomperts 1
PMCID: PMC1132285  PMID: 8452525

Abstract

We have measured the time course of secretion of hexosaminidase from rat mast cells permeabilized (in simple buffered NaCl solutions) in response to guanine nucleotides [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])] and Ca2+. In these experiments, ATP was excluded from the system (and the cells were pretreated with metabolic inhibitors). For cells permeabilized in the absence of Mg2+ but in the presence of Ca2+, secretion commences promptly in response to addition of GTP; when Mg2+ (2 mM) is provided, secretion commences after an extended delay, much higher concentrations of GTP are required, and the final extent of secretion is decreased. Ongoing secretion due to GTP and Ca2+ is abruptly terminated by addition of Mg2+ to cells initially stimulated in its absence. In contrast, although Mg2+ has no effect on the sensitivity to the non-hydrolysable analogue GTP[S], its absence does nevertheless cause delays in the onset of secretion triggered by the addition of GTP[S] to cells initially permeabilized in the presence of Ca2+ (micromolar range, again in the absence of ATP). However, exocytosis from cells triggered with Ca2+ after permeabilization in the presence of high concentrations of GTP[S] is instantaneous. The delays due to triggering by GTP[S] have GTP[S]-concentration-dependent and -independent components. The guanine-nucleotide-concentration-dependent component is expressed as an extended duration of delay as the concentration of GTP[S] is decreased, and may reflect the binding of GTP[S] to GE. The concentration-independent component is manifested as a limiting delay which cannot be further diminished by increasing the guanine nucleotide concentration. The duration of the limiting delay is sensitive to the identity of the stimulating nucleotide (GTP < GTP[S] < p[NH]ppG) and may reflect the time taken for an activating conformational change to occur after binding. Since both components of the delays are abolished by the presence of Mg2+, both the binding of guanine nucleotide and the activation of GE appear to be Mg(2+)-dependent. We therefore conclude that nucleotide binding, activation and the GTPase activity of GE are strongly dependent on Mg2+, in common with the same three processes in Gs and Gi.

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Selected References

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