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At least 19 recordsLinked to original sources
PubMed1990

Destrin, a mammalian actin-depolymerizing protein, is closely related to cofilin. Cloning and expression of porcine brain destrin cDNA.

Destrin is a mammalian 19-kDa protein that rapidly depolymerizes F-actin in a stoichiometric manner. In this study, we isolated cDNA clones coding for destrin from a porcine brain cDNA library. The deduced amino acid sequence of destrin is 165 residues long and is very similar (71% identical) to that of cofilin, a widely distributed, pH-sensitive actin-modulating protein. Destrin contains a sequence nearly identical with the putative nuclear transport signal sequence of cofilin and a hexapeptide sequence identical with the amino-terminal sequence (residues 2-7) of tropomyosin, which is shown to be involved in cofilin binding to actin. Destrin, like cofilin, also has in its carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. We have expressed destrin as well as cofilin in Escherichia coli, purified them, and examined their function in vitro. The two proteins were found to differ in their interaction with actin, like destrin and cofilin isolated from porcine brain. This suggests that the difference in the function of the two proteins results from the subtle difference in their amino acid sequence rather than possible differences in post-translational modifications. Northern blot analyses indicated that both destrin mRNA and cofilin mRNA are widely distributed in various tissues, but both mRNAs differ in their relative abundance among tissues.

Acanthamoeba↗
PubMed1985

An actin-depolymerizing protein (destrin) from porcine kidney. Its action on F-actin containing or lacking tropomyosin.

An Mr 19 000 protein (destrin) that has the ability to rapidly depolymerize F-actin in a stoichiometric manner was purified from porcine kidney by sequential chromatography on DNase I-agarose, hydroxyapatite, and Sephadex G-75. Its actin-depolymerizing activity is reversibly controlled by changes in KCl concentration but is insensitive to Ca2+ concentration. The rate of depolymerization of F-actin by destrin is much faster than that of spontaneous depolymerization induced by dilution and is not markedly decreased by the addition of end-blocking reagents such as cytochalasin B. These results suggest that destrin depolymerizes F-actin by interacting directly with F-actin protomers. Binding of muscle tropomyosin to F-actin slows down the rate of destrin-induced depolymerization of F-actin by about 30-fold. The data suggest that the destrin-induced depolymerization occurs from the ends of F-actin when F-actin is complexed with tropomyosin, but it takes place from the entire length of F-actin in the absence of tropomyosin.

Actin Depolymerizing Factors↗
PubMed1992

Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin.

Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.

Actin Depolymerizing Factors↗
PubMed1990

Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides.

Cofilin is a widely distributed actin-modulating protein that has the ability to bind along the side of F-actin and to depolymerize F-actin in a pH-dependent manner. We found that phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibited both actions of cofilin in a dose-dependent manner, while inositol 1,4,5-triphosphate (IP3), 1-oleoyl-2-acetylglycerol (OAG), phosphatidylserine (PS), or phosphatidylcholine (PC) had little or no effect on them. Gel filtration analyses showed that PIP2 bound to cofilin and thereby inhibited the binding of cofilin to G-actin. Destrin is a mammalian, pH-independent actin-depolymerizing protein. The actin-depolymerizing activity of destrin was also inhibited by PI, PIP, and PIP2, but not by IP3, OAG, PS, or PC. In addition, we found further that an actin-depolymerizing activity of bovine pancreas deoxyribonuclease I, a G-actin-sequestering protein, was inhibited by PIP and PIP2, but not by PI, IP3, OAG, PS, or PC. These results together with previous findings (Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474; Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364) suggest that the sensitivity to polyphosphoinositides may be a common feature in vitro among actin-binding proteins that can bind to G-actin and regulate the state of actin polymerization.

Actin Depolymerizing Factors↗
PubMed1991

Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin.

Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.

Actin Depolymerizing Factors↗
PubMed1991

Variant cDNAs encoding proteins similar to the alpha subunit of chicken CapZ.

Chicken adult muscle and liver cDNA libraries were screened with a cDNA, alpha 1, previously isolated from a chicken embryo library by screening with antibodies against the alpha subunit of chicken CapZ. cDNAs with a new coding region, called alpha 2, were found in addition to ones with the alpha 1 coding region. alpha 2 predicts a protein sequence that matches exactly the N-terminal sequence of 5 peptides prepared from CapZ alpha purified from chicken muscle, while the protein sequence predicted by alpha 1 matches the peptides well, but not exactly. The predicted protein sequences of alpha 1 and alpha 2 are very similar to each other, and they are similar to those of the alpha subunit of capping protein from Dictyostelium [Hartmann et al., J. Biol. Chem. 163:5254-5254, 1989] and an actin-binding protein from Xenopus [Ankenbauer et al., Nature 342:822-824, 1989]. Other conserved features of the predicted primary and secondary structures are noted. Chicken alpha 1 and alpha 2 are transcribed in all of 7 adult chicken muscle and non-muscle tissues in comparable amounts by Northern analysis. alpha 2 has four poly(A)+RNA transcripts, one of which is rare in liver. alpha 1 has two transcripts. alpha 1 and alpha 2 are encoded by different single-copy genes by Southern analysis of chicken genomic DNA.

Actin Depolymerizing Factors↗
PubMed1987

The F-actin capping proteins of Physarum polycephalum: cap42(a) is very similar, if not identical, to fragmin and is structurally and functionally very homologous to gelsolin; cap42(b) is Physarum actin.

We have carried out a primary structure analysis of the F-actin capping proteins of Physarum polycephalum. Cap42(b) was completely sequenced and was found to be identical with Physarum actin. Approximately 88% of the sequence of cap42(a) was determined. Cap42(a) and fragmin were found to be identical by amino acid composition, isoelectric point, mol. wt, elution time on reversed-phase chromatography and amino acid sequence of their tryptic peptides. The available sequence of cap42(a) is greater than 36% homologous with the NH2-terminal 42-kd domain of human gelsolin. A highly homologous region of 16 amino acids is also shared between cap42(a), gelsolin and the Acanthamoeba profilins. Cap42(a) binds two actin molecules in a similar way to gelsolin suggesting a mechanism of F-actin modulation that has been conserved during evolution.

Actin Depolymerizing Factors↗
PubMed1991

Actin-depolymerizing factor (ADF) in the cerebellum of the developing rat: a quantitative and immunocytochemical study.

A specific antiserum against actin-depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton-soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization.

Actin Depolymerizing Factors↗
PubMed1988

Identification of two species of actin depolymerizing factor in cultures of BHK cells.

High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.

Actin Depolymerizing Factors↗
PubMed1985

A Ca2+-sensitive actin regulatory protein from smooth muscle.

Using a procedure involving DNase I affinity chromatography and Sephadex G-200 gel filtration, we partially purified a Ca2+-sensitive actin regulatory 90-kDa protein from bovine aorta. The 90-kDa protein existed in the form of a complex with actin on a DNase I column even in the presence of 5 mM EGTA, indicating that the 90-kDa protein binds tightly to actin in a Ca2+-insensitive manner. The isolation procedure described above indicates that the 90-kDa protein is present in smooth muscles including aorta, uterus and bladder, but not in skeletal and heart muscles. When added to G-actin before polymerization, the 90-kDa protein increases the initial rate of actin polymerization and lowers the final viscosity at Ca2+ concentrations greater than 10(-7) M. This decrease in viscosity is due to the generation of filaments which cannot be readily sedimented.

Actin Depolymerizing Factors↗
PubMed1989

Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments.

The effect of nonmuscle actin ADP-ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP-ribosylation. ADP-ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends. ADP-ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin. The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments. The results suggest that nonmuscle actin ADP-ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.

Actin Depolymerizing Factors↗
PubMed1990

Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization.

In striated muscle the pointed ends of polar actin filaments are directed toward the center of the sarcomere. Formed filaments keep a constant length of about 1 micron. As polymerization and depolymerization at free pointed ends are not sufficiently slow to account for the constant length of the filaments, we searched for proteins which occur in sarcomeres and can stabilize the pointed ends of actin filaments. We observed that tropomyosin-troponin complex reduces the rate of association and dissociation of actin molecules at the pointed ends more than 30-fold. On the average, every 600 s one association or dissociation reaction has been found to occur at the pointed ends near the critical actin monomer concentration.

Actin Depolymerizing Factors↗
PubMed1988

Inhibition of deoxyribonuclease I activity by actin covalently cross-linked to chick brain actin depolymerizing factor through exposed sulfhydryls.

All but one of the six free sulfhydryl groups of chick brain actin depolymerizing factor (ADF) are protected from modification when ADF forms a 1:1 complex with actin. This exposed sulfhydryl can be cross-linked to cys 374 of actin with N,N'-phenylenedimaleimide. The cross-linked complex inhibits the hydrolytic activity of pancreatic deoxyribonuclease (DNase I) to an identical extent as both the untreated complex and an equivalent amount of free actin. These data indicate that ADF binds to actin at a site which does not overlap with the DNase I binding site.

Actin Depolymerizing Factors↗
PubMed1991

Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids.

Chicken muscle CapZ, a member of the capping protein family of actin-binding proteins, binds to the barbed end of actin filaments and nucleates actin polymerization. No regulation of the capping protein family has been described. We report that micelles of phosphatidylinositol 4,5-bisphosphate (PIP2) bind to CapZ and completely inhibit its ability to affect actin polymerization as measured by several independent assays. Higher concentrations of other anionic phospholipids also completely inhibit the activity of CapZ. Neutral phospholipids have no effect. Mixed vesicles of PIP2 with phosphatidylcholine or phosphatidylethanolamine also inhibit CapZ, but addition of Triton X-100 both prevents and reverses PIP2's inhibition of CapZ.

Actin Capping Proteins↗
PubMed1985

Effect of capping protein on the kinetics of actin polymerization.

Acanthamoeba capping protein increased the rate of actin polymerization from monomers with and without calcium. In the absence of calcium, capping protein also increased the critical concentration for polymerization. Various models were evaluated for their ability to predict the effect of capping protein on kinetic curves for actin polymerization under conditions where the critical concentration was not changed. Several models, which might explain the increased rate of polymerization from monomers, were tested. Two models which predicted the experimental data poorly were (1) capping protein was similar to an actin filament, bypassing nucleation, and (2) capping protein fragmented filaments. Three models in which capping protein accelerated, but did not bypass, nucleation predicted the data well. In the best one, capping protein resembled a nondissociable actin dimer. Several lines of evidence have supported the idea that capping protein blocks the barbed end of actin filaments, preventing the addition and loss of monomers [Cooper, J. A., Blum, J. D., & Pollard, T. D. (1984) J. Cell Biol. 99, 217-225; Isenberg, G. A., Aebi, U., & Pollard, T. D. (1980) Nature (London) 288, 455-459]. This mechanism was also supported here by the effect of capping protein on the kinetics of actin polymerization which was nucleated by preformed actin filaments. Low capping protein concentrations slowed nucleated polymerization, presumably because capping protein blocked elongation at barbed ends of filaments. High capping protein concentrations accelerated nucleated polymerization because of capping protein's ability to interact with monomers and accelerate nucleation.

Actin Depolymerizing Factors↗
PubMed1985

Equilibrium constant for binding of an actin filament capping protein to the barbed end of actin filaments.

Depolymerization of treadmilling actin filaments by a capping protein isolated from bovine brain was used for determination of the equilibrium constant for binding of the capping protein to the barbed ends of actin filaments. When the capping protein blocks monomer consumption at the lengthening barbed ends, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. In this way the ratio of capped to uncapped filaments could be determined as a function of the capping protein concentration. Under the experimental conditions (100 mM KCl and 2 mM MgCl2, pH 7.5, 37 degrees C) the binding constant was found to be about 2 X 10(9) M-1. Capping proteins effect the actin monomer concentration only at capping protein concentrations far above the reciprocal of their binding constant. Half-maximal increase of the monomer concentration requires capping of about 99% of the actin filaments. A low proportion of uncapped filaments has a great weight in determining the monomer concentration because association and dissociation reactions occur at the dynamic barbed ends with higher frequencies than at the pointed ends.

Actin Depolymerizing Factors↗
PubMed1986

Actin-fragmin interactions as revealed by chemical cross-linking.

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.

Actin Depolymerizing Factors↗