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PubMed2000

Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

Actins↗
PubMed2002

Differential response of fast and slow myosin ATPase from skeletal muscle to F-actin and to phalloidin F-actin.

Fast muscle myosin responds in similar way to F-actin and to phalloidin F-actin. It is activated 7.5 fold at infinite F-actin concentration and 6.8 fold at infinite phalloidin F-actin. The actomyosin dissociation constants are 0.89 +/- 0.34 microM with F-actin and 0.90 +/- 0.71 microM with phalloidin F-actin. Slow muscle myosin responds differently to F-actin and to phalloidin F-actin. It is activated 3.76 fold at infinite F-actin concentration and only 2.27 fold at infinite phalloidin F-actin concentration. The actomyosin dissociation constants are 1.95 +/- 1.27 microM with F-actin and 0.27 +/- 0.16 microM with phalloidin F-actin. At first glance this means that substitution of F-actin with phalloidin F-actin magnifies the difference between fast muscle and slow muscle myosins. Furthermore the change of the dissociation constants may affect the contractile force of the attached crossbridge.

Actins↗
PubMed1992

Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets.

Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.

Actins↗
PubMed2001

Beneficial role of amino acids in mitigating cytoskeletal actin glycation and improving F-actin content: in vitro.

AIMS: The actin filaments present in circulating leukocytes facilitate their passage through microvenules and capillaries by helping in their deformability. Decreased deformability of granulocytes is now known to cause occlusion of the retinal microcapillaries leading to hypoxia and the subsequent development of diabetic retinopathy. Structural and functional loss of proteins, due to non-enzymatic glycation and glycoxidation, has been reported to cause diabetic pathogenesis. As amino acids have been earlier reported to have antidiabetic properties, the present study involves the investigation of the susceptibility of the cytoskeletal actin to glycation and its mitigation by free amino acids. This study also involves quantifying F-actin in cultured mononuclear cells obtained from diabetic and normal healthy volunteers and on the effect of glucose and free amino acids on F-actin content. METHODS: Commercial non-muscle actin and actin immuno-pre-cipitated from granulocytes obtained from (a) normal healthy human volunteers and (b) patients with type 2 diabetes mellitus were subjected to glycation studies using [U] (14)C glucose. The effect of free amino acids, as antiglycating agents, was determined using various concentrations of lysine, arginine, alanine, aspartic acid and glutamic acid. F-actin content in cultured mononuclear cells was estimated by flow cytometry using fluorescein isothiocynate (FITC)-Phalloidin. RESULTS: Commercial actin at physiological conditions of pH and temperature was found to undergo non-enzymatic glycation. The extent of in vitro glycation was significantly low (P<0.001) in actin isolated from patients with type2 diabetes when compared to the non-diabetic group, suggesting an increased in vitro structural modification of actin in patients with diabetes. All the free amino acids tested were found to have varying degrees of antiglycating effect. The F-actin content in the intact mononuclear cells obtained from diabetic patients was found to be low when compared with normal healthy volunteers (P<0.001). Similarly the F-actin content was significantly low when the normal mononuclear cells were incubated with glucose. This effect was reversed upon the addition of free amino acids to the incubation mixture. CONCLUSIONS: Free amino acids can play a positive role in improving leukocyte deformability by mitigating cytoskeletal actin glycation and improving F-actin content.

Actins↗
PubMed2000

Thermal unfolding of G-actin monitored with the DNase I-inhibition assay stabilities of actin isoforms.

Actin is one of the proteins that rely on chaperonins for proper folding. This paper shows that the thermal unfolding of G-actin, as studied by CD and ultraviolet difference spectrometry, coincides with a loss in DNase I-inhibiting activity of the protein. Thus, the DNase I inhibition assay should be useful for systematic studies of actin unfolding and refolding. Using this assay, we have investigated how the thermal stability of actin is affected by either Ca2 + or Mg2 + at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in different states of phosphorylation (ATP, ADP.Pi, ADP. Vi, ADP.AlF4, ADP.BeFx, and ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclude that the thermal unfolding of G-actin is a three-state process, in which an equilibrium exists between native actin with bound nucleotide and an intermediate free of nucleotide. Actins in the Mg-form were less stable than the Ca-forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle alpha-actin = bovine cytoplasmic gamma-actin > yeast actin > cytoplasmic beta-actin. The activation energies for the thermal unfolding reactions were in the range 200-290 kJ.mol- 1, depending on the bound ligands. Generally, the stability of the actin depended on the degree with which the nucleotide contributed to the connectivity between the two domains of the protein.

Actins↗
PubMed2001

The Abl-related gene (Arg) nonreceptor tyrosine kinase uses two F-actin-binding domains to bundle F-actin.

Abl family nonreceptor tyrosine kinases regulate cellular morphogenesis and motility through functional interactions with the actin cytoskeleton. Although Abl family kinases are known to contain filamentous (F)-actin-binding domains at their C termini, it is unclear how Abl family kinases regulate the structure and/or function of the actin cytoskeleton. We show here that the Abl-related kinase Arg binds with positive cooperativity to F-actin in vitro with binding saturating at a ratio of one Arg/two actin molecules. Measurements of the F-actin-binding properties of Arg deletion mutants led to the identification of a second, previously uncharacterized internal F-actin-binding domain in Arg. Purified Arg can bundle F-actin in vitro, and this bundling activity requires both F-actin-binding domains. An Arg-yellow fluorescent protein fusion protein can induce the formation of actin-rich structures at the lamellipodia of Swiss 3T3 fibroblasts. Both of Arg's F-actin-binding domains are necessary and sufficient for the formation of these actin-rich structures. Together, our data suggest that Arg can use its F-actin-bundling activity to directly regulate actin cytoskeletal structure in vivo.

3T3 Cells↗
PubMed1997

Actin-binding verprolin is a polarity development protein required for the morphogenesis and function of the yeast actin cytoskeleton.

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70- amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.

Actins↗
PubMed1980

Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

Actins↗
PubMed1984

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.

Actins↗
PubMed2002

Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor.

Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics. SRF activity can be inhibited by ectopic expression of beta-actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations. To distinguish between these possibilities, we studied actin mutants. Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies. These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio. Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor. SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B. Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals. These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding. Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF.

3T3 Cells↗
PubMed1986

Rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex.

The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1.

Actins↗
PubMed1993

The actin monomers in the ternary gelsolin: 2 actin complex are in an antiparallel orientation.

Gelsolin forms ternary complexes with two actin monomers in the presence of Ca2+, which nucleate actin polymerization and cap the barbed ends of filaments. It has therefore been assumed that the two actins are oriented in a similar manner to the terminal subunits in the genetic helix of F-actin. We have tested this using chemical cross-linking with N,N'-1,4-phenylenedimaleimide. For all conditions tested, we identified as the only cross-linked dimeric species an actin dimer indistinguishable from the lower actin dimer of 86 kDa. This lower dimer was previously identified in the initial phase of actin polymerization and also when actin paracrystals are chemically cross-linked [Millonig, R., Salvo, H. & Aebi, U. (1988) J. Cell Biol. 106, 785-796]. It probably defines a contact between adjacent monomers oriented in an antiparallel orientation. In contrast, when F-actin is cross-linked by the same reagent, an upper dimer of apparent molecular mass 115 kDa is formed, which corresponds to adjacent monomers in the genetic helix. The formation of this upper dimer was specifically inhibited by addition of gelsolin to F-actin. Evidence is presented for a Cys374-Cys374 cross-link in the lower dimer. Isolated lower dimer binds to gelsolin in a 1:1 stoichiometry, but it inhibits nucleation of polymerization by gelsolin. Other gelsolin constructs that bind two actin subunits (e.g. the N-terminal half of the molecule, which has severing and capping but no nucleating activity) also form only lower dimer when cross-linked with N,N'-1,4-phenylenedimaleimide. Only segment 2-6 (gelsolin fragment devoid of the N-terminal segment 1) induces an upper dimer orientation of the two actins under nucleating conditions. Our evidence suggests that the two actins associated with gelsolin are not fixed in the orientation of adjacent subunits in F-actin; instead they have a flexible orientation with respect to each other, which permits cross-linking into a stable antiparallel form that does not correspond to the presumed nucleating conformation.

Actins↗
PubMed1983

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

Actins↗
PubMed1988

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.

Actins↗
PubMed1993

TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction.

Tumor necrosis factor-alpha (TNF-alpha) influences pulmonary vascular endothelial barrier function in vitro. We studied whether recombinant TNF-alpha (rTNF-alpha) regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to human rTNF-alpha (1,000 U/ml) and evaluated for 1) transendothelial [14C]albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the deoxyribonuclease I inhibition assay. rTNF-alpha induced increments in [14C]albumin flux (P < 0.04) and intercellular gap formation at > or = 2-6 h. During this same time, the endothelial F-actin pool decreased (P = 0.0064), with reciprocal increases in the G-actin pool (P < 0.0001). Prior F-actin stabilization with phallicidin protected against the rTNF-alpha-induced increments in G-actin (P < 0.002) as well as changes in barrier function (P < 0.01). Prior protein synthesis inhibition enhanced the rTNF-alpha-induced decrement in F-actin (P < 0.0001), blunted the G-actin increment (P < 0.002), and increased rTNF-alpha-induced changes in endothelial barrier function (P < 0.003). Therefore, rTNF-alpha induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. rTNF-alpha also increased total actin (P < 0.02) and new actin synthesis (P < 0.002), which may be a compensatory endothelial cell response to rTNF-alpha-induced F-actin depolymerization.

Actins↗
PubMed2005

Contribution of sequence variation in Drosophila actins to their incorporation into actin-based structures in vivo.

Actin is a highly conserved protein important for many cellular functions including motility, contraction in muscles and intracellular transport. Many eukaryotic genomes encode multiple actin protein isoforms that differ from each other by only a few residues. We addressed whether the sequence differences between actin paralogues in one species affect their ability to integrate into the large variety of structures generated by filamentous actin. We thus ectopically expressed all six Drosophila actins as fusion proteins with green fluorescent protein (GFP) in a variety of embryonic, larval and adult fly tissues. We found that each actin was able to integrate into most actin structures analysed. For example, in contrast to studies in mammalian cells, the two Drosophila cytoplasmic actins were incorporated into muscle sarcomeres. However, there were differences in the efficiency with which each actin was incorporated into specific actin structures. The most striking difference was observed within the Z-lines of the sarcomeres: one actin was specifically excluded and we mapped this feature to one or both of two residues within the C-terminal half of the protein. Thus, in Drosophila, the primary sequence of different actins does affect their ability to incorporate into actin structures, and so specific GFPactins may be used to label certain actin structures particularly well.

Actins↗
PubMed2005

Role of actin DNase-I-binding loop in myosin subfragment 1-induced polymerization of G-actin: implications for the mechanism of polymerization.

Proteolytic cleavage of actin between Gly(42) and Val(43) within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln(41) indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the changes in filament-length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization.

Actins↗
PubMed1986

Phosphorylation of Amoeba G-actin and its effect on actin polymerization.

Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.

Actins↗