Ultra‐violet fluorescence of actin. Determination of native actin content in actin preparations

Turoverov, Konstantin K.; Haitlina, S.Yu.; Пинаев, Г. П.

doi:10.1016/0014-5793(76)80003-8

Cited by 85 publications

(58 citation statements)

References 6 publications

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“…3 shows that no polymerization of 12 M ECP32-cleaved Mg-actin was observed during at least 10 min after addition of 0.1 M KCl. In addition, heat-inactivated actin, which cannot polymerize under any conditions (19), was used in the following patch The results of inside-out experiments with application of different ionic conditions and different actin species are summarized in Fig. 4.…”

Section: Resultsmentioning

confidence: 99%

“…Inactivated actin was obtained from intact actin by heating for 5 min at 70°C. Inactivation of the sample was checked using the parameter A ϭ I 320 /I 365 of the intrinsic fluorescence spectrum, where I 320 and I 365 are the fluorescence intensities at 320 and 365 nm, respectively (19). The values of the parameter A were 2.5 and 1.3 for intact and inactivated actin, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

Negulyaev¹,

Khaitlina²,

Hinssen³

et al. 2000

Journal of Biological Chemistry

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The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in nonexcitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 M [Ca 2؉ ] i . The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mM MgCl 2 to induce actin polymerization. In the absence of MgCl 2 , addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.Functional coupling between channel proteins and the cortical cytoskeleton may play a key role in membrane ion transport and cellular signaling. Involvement of F-actin in ion channel functioning has been established and studied extensively in polarized epithelial cells (1-7). Specifically, several lines of evidence revealed an association between the amiloride-sensitive sodium channels and the actin-based cytoskeleton in renal epithelia. Indirect immunofluorescence and confocal microscopy demonstrated that sodium channels in the apical membrane colocalize with actin, spectrin (fodrin), and ankyrin (5, 6). Electrophysiological studies on epithelial A6 cells showed that disruption of actin microfilament networks by cytochalasin D induced sodium channel activity both in cell-attached and excised patches (1). Similar effects were observed in the presence of actin or actin-gelsolin complexes added to the cytoplasmic side of excised inside-out patches, whereas the actin-DNase I complexes did not activate sodium channels. These results were explained by a model suggesting that the channels are activated by short actin filaments produced either by severing of endogenous long filaments with cytochalasin or by assembly from monomeric actin during spontaneous or gelsolin-mediated polymerization (1). Similar observations were made on planar lipid bilayers containing cloned epithelial sodium channels (4, 7). In addition, interaction of actin with epithelial channels was reported to modulate considerably the intrinsic...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

Negulyaev¹,

Khaitlina²,

Hinssen³

et al. 2000

Journal of Biological Chemistry

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“…The analysis of the intrinsic Trp fluorescence by Parameter A and fitting were performed according to the published procedures [He et al, 2005;Su et al, 2007]. Parameter A, which reflects the shape and position of the Trp fluorescence [Turoverov et al, 1976], is obtained by dividing the emission intensity at 320 nm (I 320 ) by that at 365 nm (I 365 ). The fitting of the Trp fluorescence spectra was performed using the discrete states model of Trp fluorophores .…”

Section: Spectroscopic Experimentsmentioning

confidence: 99%

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens

Cai

Zhou

et al. 2011

Hum. Mutat.

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“…1). The native-like structure of ECP and subtilisincleaved actins was routinely controlled by intrinsic fuorescence using the parameter A = 132o]1365 as a measure of actin nativity [1,9]. Upon cleavage, the parameter A remained practically unchanged (2.56, 2.52 and 2.54 for intact, subtilisin-and ECP-cleaved actins, respectively).…”

Section: Proteolytic Digestionmentioning

confidence: 99%

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

et al. 1996

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Alteration of the actin polypeptide chain within the DNnse I-binding loop by cleavage with E. coli A2 protease or suhtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys-374. Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can he energy transfer donors. It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin. These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer. We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling.

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Ultra‐violet fluorescence of actin. Determination of native actin content in actin preparations

Cited by 85 publications

References 6 publications

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

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