| Summary: | Gelsolin, a protein involved in the regulation of actin filament length,
was isolated from horse plasma and modified with the amine-selective reagent
fluorescein-5-isothiocyanate (FITC). The extent of FITC incorporation per
gelsolin molecule was 4.8 ± 0.6, based upon 4 labelling trials. FITC-gelsolin
analysed by SDS-PAGE revealed a single fluorescent band under UV light
prior to staining, and then a single Coomassie Blue stained band. Viscosity
studies showed that gelsolin’s F-actin severing activity was not impaired upon
reaction with FITC, but incorporation of the fluorophores did increase the
the temperature at which the onset of irreversible precipitation occurred.
Under physiological ionic conditions, fluorescence studies failed to
detect a C2a effect on FITC-gelsolin’s spectroscopic characteristics, but in
low ionic strength solutions, C2a increased the fluorescence intensity by
approximately 25%. A calcium effect was also evident in bimolecular
fluorescence quenching studies with KI, which showed that C2a rendered the
fluorescence more susceptible to quenching. Subsequent protein denaturation
with guanidine-HC1 resulted in further exposure of the fluorescein
chromophores to iodide in solvent.
An investigation of reactivity with the thiol selective reagents acrylodan
(ACR) and pyrene iodoacetamide (HA) revealed that native gelsolin exposes
1 to 2 cysteine groups for reaction. Under denaturing conditions, the extent of reaction increased to approximately 3 with ACR and 4 with PTA. In
comparison, the average number of thiol groups available for reaction with
5,5’-dithiobis(2-nitrobenzoic acid) was approximately 1, and 3 to 4 in native
and denaturing conditions, respectively.
Actin-gelsolin complexation was revealed by fluorescence titrations and
fluorescence polarization measurements. Addition of actin to FITC-labelled
gelsolin increased the fluorescence intensity of the sample in a Ca2 specific
manner, while binding of actin to ACR-gelsolin resulted in a fluorescence
decrease. Fluorescence polarization values increased on addition of actin to
both FITC- and ACR-labelled gelsolin solutions only in the presence of Ca2,
establishing the calcium requirement for actin binding.
Tropomyosin binding to gelsolin was evident from an increase in the
fluorescence intensity of FITC-labelled gelsolin, and from retention of
labelled gelsolins on a tropomyosin-agarose affinity column. Quantitative
assessment of a direct titration of FI’l’C-labelled gelsolin with tropomyosin
yielded two independent tropomyosin binding sites on gelsolin, with a
dissociation constant of approximately 0.6 IIM. Both of these effects required
the presence of micromolar Ca2. Photoactivation of solutions of
benzophenone-4-isothiocyanate labelled gelsolin and tropomyosin resulted in
the appearance of high molecular weight bands on SDS-PAGE, indicative of
intermolecular crosslinking.
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