scholarly journals Mechanism of interaction of Dictyostelium severin with actin filaments.

1982 ◽  
Vol 95 (3) ◽  
pp. 711-719 ◽  
Author(s):  
K Yamamoto ◽  
J D Pardee ◽  
J Reidler ◽  
L Stryer ◽  
J A Spudich
Keyword(s):  
Energy Transfer ◽  
Actin Filaments ◽  
Exchange Process ◽  
Molar Ratio ◽  
Fluorescence Energy Transfer ◽  
Dependent Manner ◽  
Emission Anisotropy ◽  
Subunit Exchange ◽  
Fluorescence Energy

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.

scholarly journals Ca2+-dependent binding of severin to actin: a one-to-one complex is formed.

1984 ◽  
Vol 98 (5) ◽  
pp. 1796-1803 ◽  
Author(s):  
R G Giffard ◽  
A G Weeds ◽  
J A Spudich
Keyword(s):  
Energy Transfer ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Dnase I ◽  
Fluorescence Energy Transfer ◽  
Dependent Manner ◽  
High Affinity ◽  
Cell Biol ◽  
Fluorescence Energy

Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.

scholarly journals Actin filaments undergo limited subunit exchange in physiological salt conditions.

1982 ◽  
Vol 94 (2) ◽  
pp. 316-324 ◽  
Author(s):  
J D Pardee ◽  
P A Simpson ◽  
L Stryer ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Energy Transfer ◽  
Actin Filament ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Fluorescence Energy Transfer ◽  
Subunit Exchange ◽  
Filament Assembly ◽  
Fluorescence Energy ◽  
Energy Acceptor

The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (IAENS) served as the fluorescent energy donor, and actin labeled with either fluorescein-5-isothiocyanate (FITC) or fluorescein-5-maleimide (FM) served as the energy acceptor. Fluorescent-labeled actins from Dictyostelium amoebae and rabbit skeletal muscle were very similar to their unlabeled counterparts with respect to critical actin concentration for filament assembly, assembly rate, ATP hydrolysis upon assembly, and steady-state ATPase. As evidenced by two different types of fluorescence energy transfer experiments, less than 5% of the actin filament subunits exchanged under a variety of buffer conditions at actin concentrations greater than 0.5 mg/ml. At all actin concentrations limited exchange to a plateau level occurred with a half-time of about 20 min. Nearly identical results were obtained when exchange was quantitated by incorporation of 35S-labeled Dictyostelium actin monomers into unlabeled muscle actin or Dictyostelium actin filaments. Furthermore, the proportion of filament-bound ADP which exchanged with [14C]-ATP was nearly the same as actin subunit exchange measured by fluorescence energy transfer and 35S-labeled actin incorporation. These experiments demonstrate that under approximately physiologic ionic conditions only a small percentage of subunits in highly purified skeletal muscle or Dictyostelium F-actin participate in exchange.

scholarly journals Fusion of phospholipid vesicles produced by the anti-tumour protein α-sarcin

1990 ◽  
Vol 265 (3) ◽  
pp. 815-822 ◽  
Author(s):  
M Gasset ◽  
M Oñaderra ◽  
P G Thomas ◽  
J G Gavilanes
Keyword(s):  
Energy Transfer ◽  
Ionic Strength ◽  
Freeze Fracture ◽  
Molar Ratio ◽  
Phospholipid Vesicles ◽  
Fluorescence Energy Transfer ◽  
Molar Ratios ◽  
Binding Stoichiometry ◽  
Fracture Electron ◽  
Fluorescence Energy

The anti-tumour protein alpha-sarcin causes fusion of bilayers of phospholipid vesicles at neutral pH. This is demonstrated by measuring the decrease in the efficiency of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidylethano lamine (NDB-PE) (donor) and N-(lissamine rhodamine B sulphonyl)-diacylphosphatidylethanolamine (Rh-PE) (acceptor) incorporated in dimyristoylphosphatidylcholine (DMPG) vesicles. The effect of alpha-sarcin is a maximum at 0.15 M ionic strength and is abolished at basic pH. alpha-Sarcin promotes fusion between 1,6-diphenylhexa-1,3,5-triene (DPH)-labelled DMPG and dipalmitoyl-PG (DPPG) vesicles, resulting in a single thermotropic transition for the population of fused phospholipid vesicles. Bilayers composed of DMPC and DMPG, at different molar ratios in the range 1:1 to 1:10 PC/PG, are also fused by alpha-sarcin. Freeze-fracture electron micrographs corroborate the occurrence of fusion induced by the protein. alpha-Sarcin also modifies the permeability of the bilayers, causing the leakage of calcein in dye-trapped PG vesicles. All of the observed effects reach saturation at a 50:1 phospholipid/protein molar ratio, which is coincident with the binding stoichiometry previously described.

scholarly journals Detection of actin assembly by fluorescence energy transfer.

1981 ◽  
Vol 89 (2) ◽  
pp. 362-367 ◽  
Author(s):  
D L Taylor ◽  
J Reidler ◽  
J A Spudich ◽  
L Stryer
Keyword(s):  
Energy Transfer ◽  
Actin Filaments ◽  
Radial Coordinate ◽  
Fluorescence Energy Transfer ◽  
Actin Assembly ◽  
Intact Cells ◽  
Donor And Acceptor ◽  
Fluorescence Energy ◽  
Energy Acceptor

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.

Actin and myosin: control of filament assembly

1982 ◽  
Vol 299 (1095) ◽  
pp. 247-261 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thick Filament ◽  
Light Chains ◽  
Fluorescence Energy Transfer ◽  
Dependent Manner ◽  
Filament Formation ◽  
Tail Region ◽  
Filament Assembly ◽  
Fluorescence Energy

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35 S-labelled actin monomers into unlabelled actin filaments, and exchange of [ 14 C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca 2+ -dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.

Red Laser-Induced Fluorescence Energy Transfer in an Immunosystem

2000 ◽  
Vol 280 (2) ◽  
pp. 272-277 ◽  
Author(s):  
Bernhard Oswald ◽  
Frank Lehmann ◽  
Lydia Simon ◽  
Ewald Terpetschnig ◽  
Otto S. Wolfbeis
Keyword(s):  
Energy Transfer ◽  
Laser Induced Fluorescence ◽  
Fluorescence Energy Transfer ◽  
Fluorescence Energy
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