Biomedical subjects

S Y Khaitlina

Publications and source records attributed to S Y Khaitlina.

9 recordsLinked to original sources

PubMed2001

Functional specificity of actin isoforms.

Actin, one of the main proteins of muscle and cytoskeleton, exists as a variety of highly conserved isoforms whose distribution in vertebrates is tissue-specific. Synthesis of specific actin isoforms is accompanied by their subcellular compartmentalization, with both processes being regulated by factors of cell proliferation and differentiation. Actin isoforms cannot substitute for each other, and the high-level synthesis of exogenous actins leads to alterations in cell organization and morphology. This indicates that the highly conserved actins are functionally specialized for the tissues in which they predominate. The first goal of this review is to analyze the data on the polymerizability of actin isoforms to show that cytoskeleton isoactins form less stable polymers than skeletal muscle actin. This difference correlates with the dynamics of actin microfilaments versus the stability of myofibrillar systems. The three-dimensional actin structure as well as progress in the analysis of conformational changes in both the actin monomer and the filament allows us to view the data on the structure and polymerization of isoactins in terms of structure-function relationships within the actin molecule. Most of the amino acid substitutions that distinguish actin isoforms are located apart from actin-actin contact sites in the polymer. We suggest that these substitutions can modulate the ability of actin monomers to form more or less stable polymers by long-range (allosteric) regulation of the contact sites.

Actins↗

PubMed2001

Bacterial protease ECP32 specifically hydrolyzing actin and its effect on cytoskeleton in vivo.

A procedure for isolation of bacterial protease ECP32 yielding 100 microg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.

Actins↗

PubMed2000

Sodium channel activity in leukemia cells is directly controlled by actin polymerization.

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable 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 microm [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.

Actins↗

PubMed1999

The structure and dynamics of partially folded actin.

Steady-state and time-resolved intrinsic fluorescence, fluorescence quenching by acrylamide, and surface testing by hydrophobic label ANS were used to study the structure of inactivated alpha-actin. The results are discussed together with that of earlier experiments on sedimentation, anisotropy of fluorescence, and CD spectrum in the near- and far-UV regions. A dramatic increase in ANS binding to inactivated actin in comparison with native and unfolded protein indicates that the inactivated actin has solvent-exposed hydrophobic clusters on the surface. It results in specific association of actin macromolecules (sedimentation constants for native and inactivated actin are 3 and 20 S, respectively) and, consequently, in irreversibility of native-inactivated actin transition. It was found that, though the fluorescence spectrum of inactivated actin is red-shifted, the efficiency of the acrylamide collision quenching is even lower than that of the intact protein. It suggests that tryptophan residues of inactivated actin are located in the inner region of protein formed by polar groups, which are highly packed. It correlates with the pronounced near-UV CD spectrum of inactivated actin. The experimentally found tryptophan fluorescence lifetimes allowed evaluation rotational correlation times on the basis of Perrin plots. It is found that oscillations of tryptophan residues in inactivated actin are restricted in comparison with native one. The inactivated actin properties were invariant with experimental conditions (ionic strength, the presence of reducing agents), the way of inactivation (Ca2+ and/or ATP removal, heating, 3-5 M urea or 1.5 M GdmCl treatment), and protein concentration (within the limits 0.005-1.0 mg/mL). The same state of actin appears on the refolding from the completely unfolded state. Thermodynamic stability, pronounced secondary structure, and the existing hydrophobic clusters, tested by ANS fluorescence and reversibility of transition inactivated-unfolded forms, allowed us to suggest that inactivated actin can be intermediate in the folding-unfolding pathway.

Acrylamide↗

PubMed1999

Effect of self-association on the structural organization of partially folded proteins: inactivated actin.

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.

Acrylamide↗

PubMed1997

Ca-dependent regulation of Na+-selective channels via actin cytoskeleton modification in leukemia cells.

With the use of the patch-clamp technique, physiological mechanisms of Na+ channel regulation involving submembranous actin rearrangements were examined in human myeloid leukemia K562 cells. We found that the actin-severing protein gelsolin applied to cytoplasmic surface of membrane fragments at a high level of [Ca2+]i (1 microM) increased drastically the activity of Na-selective channels of 12 pS unitary conductance. In the experiments on intact cells, the elevation of [Ca2+]i using the ionophore 4Br-A23187 also resulted in Na+ channel activation. Addition of actin to the cytoplasmic surface of membrane patches reduced this activity to background level, likely due to actin polymerization. Our data imply that Ca-dependent modulations of the actin cytoskeleton may represent one of the general mechanisms of channel regulation and cell signalling.

Actins↗

PubMed1996

Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain.

The proteinase previously described as an unidentified component of E. coli A2 extracts which hydrolyses actin at a new cleavage site (Khaitlina et al. (1991) FEBS Lett. 279, 49) was isolated and further characterized. A chromatographic method of proteinase purification was developed by which a purity of more than 80% was attained. The enzyme was identified as a single, 32 kDa polypeptide (ECP 32) by SDS-PAGE and non-denaturing electrophoresis as well as by ion-exchange chromatography and gel filtration. The N-terminal sequence of ECP 32 was determined to be: AKTSSAGVVIRDIFL. The activity of ECP 32 is inhibited by o-phenanthroline, EDTA, EGTA and zincone. The EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions. Based on these properties ECP 32 was classified as a metalloproteinase (EC 3.4.24). Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 was observed at enzyme substrate mass ratios of 1:25 to 1:3000. Two more sites between Ala-29 and Val-30, and between Ser-33 and Ile-34 were cleaved by ECP 32 in heat- or EDTA-inactivated actin. Besides actin, only histones and DNA-binding protein HU were found to be substrates of the proteinase, confirming its high substrate specificity. Its molecular mass, N-terminal sequence and enzymatic properties distinguish ECP 32 from any known metalloproteinases of E. coli, and we therefore conclude that it is a new enzyme.

Actins↗

PubMed1993

The actin/actin interactions involving the N-terminus of the DNase-I-binding loop are crucial for stabilization of the actin filament.

Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I.M., Pershina, V.P., Synakevich, I.G., Turoverov, K.K. & Usmanova, A.M. (1991) FEBS Lett. 279, 49-51]. The resulting C-terminal and N-terminal fragments remained associated to one another in the presence of either Ca2+ or Mg2+. The protease-treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+. Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease-treated actin to inhibit the DNase I activity. The critical concentration for KCl-induced polymerization of ECP-treated ATP-Mg-G-actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisin, and 36-fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP-treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl-labelled actins and chemical cross-linking with N,N'-1,2-phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP-treated actin and those of intact as well as subtilisin-treated actin. Moreover, the filaments of ECP-treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F-actin [Holmes, K.C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44-49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.

Actins↗

PubMed1993

Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion.

Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment. Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39-51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.

Actins↗