Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I).

The aim of this study was to isolate and to characterize actin from the carp liver cytosol and to examine its ability to polymerize and interact with bovine pancreatic DNase I. Carp liver actin was isolated by ion-exchange chromatography, followed by gel filtration and a polymerization/depolymerization cycle or by affinity chromatography using DNase I immobilized to agarose. The purified carp liver actin was a cytoplasmic beta-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle alpha-actin. Polymerization of purified carp liver actin by 2 mM MgCl(2) or CaCl(2) was only obtained after addition of phalloidin or in the presence of 1 M potassium phosphate. Carp liver actin interacted with DNase I leading to the formation of a stable complex with concomitant inhibition of the DNA degrading activity of DNase I and its ability to polymerize. The estimated binding constant (K(b)) of carp liver actin to DNase I was calculated to be 1.85x10(8) M(-1) which is about 5-fold lower than the affinity of rabbit skeletal muscle alpha-actin to DNase I.     

Polarized distribution of actin isoforms in gastric parietal cells.

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.     

Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase.

The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49].     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

Interaction of myosin subfragment 1 with forms of monomeric actin

The ability of myosin subfragment 1 to interact with monomeric actin complexed to sequestering proteins was tested by a number of different techniques such as affinity absorption, chemical cross-linking, fluorescence titration, and competition procedures. For affinity absorption, actin was attached to agarose immobilized DNase I. Both chymotryptic subfragment 1 isoforms (S1A1 and S1A2) were retained by this affinity matrix. Fluorescence titration employing pyrenyl-actin in complex with deoxyribonuclease I (DNase I) or thymosin beta4 demonstrated S1 binding to these actin complexes. A K(D) of 5 x 10(-8) M for S1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicate binding of S1 to actin in complex with gelsolin segment 1 (G1) or vitamin D-binding protein (DBP). However, fluorescence competition experiments and analysis of tryptic cleavage patterns of S1 indicated its interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complex was directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitive to ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable to significantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant of Dictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within an insertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase I complex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higher affinity than wild-type S1 and was less sensitive to mono- and divalent cations.     

Purification and biochemical characteristics of actin from the rat malignancy sarcoma-45]

Actin was purified from rat sarcoma-45 by using affinity chromatography on DNase I agarose. Actin was detected in the soluble and cytoskeletal fractions. The molecular mass of the protein was found to be equal to 45 kDa. The tumour actin specifically reacted with the antibody against skeletal muscle actin, inhibited the DNAase I activity and activated in the fibrillar state Mg(2+)-ATPases of sarcoma-45 and skeletal muscle myosins. The activating effect of the tumour protein was lower than that of its skeletal muscle counterpart. V8-protease peptide mapping revealed a similarity between tumour and brain actins. Sarcoma-45 actin was found to contain beta- and gamma-actin isoforms and an unusual isoform which appeared to be more acidic than the alpha-actin isoform.     

Characterization of heart cytosolic proteins capable of modulating calcium uptake by the sarcoplasmic reticulum. 2. Identification of actin isoforms with inhibitory activity

Ca uptake by isolated SR membranes is inhibited by a cytosolic factor derived from heart cells. The inhibitory activity resides in the fraction of soluble proteins which precipitates in 30% saturated (NH4)2SO4 [Narayanan et al. (1982) Biochem. Biophys. Res. Commun. 108, 1158-1164]. In the present study, the mechanism of inhibition and the properties of the inhibitor have been analysed. The cytosolic inhibitor activates a Ca-release pathway, thereby uncoupling Ca loading and Ca-dependent ATPase activity of SR vesicles. Analysis of some general physiochemical characteristics of the endogenous inhibitor (e.g. thermolability, protein profile, solubility properties, interaction with ion-exchange resins) showed it to be distinct from free fatty acids which might contaminate the cytosolic fraction. Rather, it indicated that the inhibitor is related to myofibrillar or cytoskeletal structures. By means of an affinity-chromatography procedure using muscle albumin coupled to Sepharose 4B, a protein component was obtained from the inhibitor fraction. The characteristics of this protein closely resembled those of the endogenous inhibitor. A protein with similar characteristics was also obtained using a DNase-I-affinity chromatography column. The isolated protein was identified as actin. Inhibition of Ca uptake by the isolated inhibitor protein was reversed by muscle albumin and by stoichiometric amounts of DNase I. The potency of inhibition of various actin preparations was found to be highly variable and dependent on the tissue source. Our results indicate that particular minor actin isoforms present in heart cytosol display the greatest inhibitory activity (IC50 15-20 micrograms/ml).     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Carp liver DNase--isolation, further characterization and interaction with endogenous actin

Deoxyribonuclease I (DNase I)-like enzyme from the liver of the carp (Cyprinus carpio) was purified to homogeneity and further characterized. Ion exchange chromatography on DEAE-cellulose, molecular filtration on Sephacryl S-300 and Con A-Sepharose affinity chromatography were applied for enzyme isolation. Carp liver DNase, similarly to DNase I from bovine pancreas, was found to be an endonuclease that hydrolyses linear DNA from salmon sperm as well as circular DNA forms--plasmid and cosmid. The purified enzyme is a glycoprotein and shows microheterogeneity, as observed in DNase zymograms prepared after native and two-dimensional electrophoresis (2D-PAGE). The composition of sugar component of the enzyme was characterized. Special attention was focused on the ability of carp liver DNase to interact with carp liver actin. The carp liver enzyme was inhibited by endogenous actin. The estimated binding constant of carp liver DNase to carp liver actin was calculated to be 1.1 x 10(6) M(-1).     

Myosin subfragment 1 and structural elements of G-actin: effects of S-1(A2) on sequences 39-52 and 61-69 in subdomain 2 of G-actin.

The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH2-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the experimental conditions of this work, no polymerization of actin was induced by S-1(A2) [Chen & Reisler (1991) Biochemistry 30, 4546-4552]. S-1(A2) did not change the rates of subtilisin and chymotryptic digestion of G-actin at loop 39-52. In contrast to this, the second protease-sensitive region on G-actin, segment 61-69, was protected strongly by S-1(A2) from tryptic cleavage. The minor if any involvement of loop 39-52 in S-1 binding was confirmed by determining the binding constants of S-1(A2) for pyrene-labeled G-actin (1.2 x 10(6) M-1), subtilisin-cleaved pyrenyl G-actin (0.3 x 10(6) M-1), and DNase I-pyrenyl G-actin complexes (0.3 x 10(6) M-1). Consistent with this, the activity of DNase I, which binds to actin loop 39-52 [Kabsch et al. (1990) Nature 347, 37-44], was inhibited almost equally well by actin in the presence and absence of S-1(A2). These results confirm the observation that DNase I and S-1(A2) bind to distinct sites on actin [Bettache et al. (1990) Biochemistry 29, 9085-9091] and demonstrate myosin-induced changes in segment 61-69 of G-actin.     

Evidence for the presence of DNase—actin complex in L1210 leukemia cells

L1210 leukemia cell cytosol was analysed for the presence of DNase I activity. No free activity was determined in crude cytosol. DNase I enzyme was found to occur in a latent form bound to cytoplasmic actin. DNase-actin complex was partially isolated by Sephadex filtration and DNase I-like activity was demonstrated after SDS gel electrophoresis of the complex and enzyme renaturation. The results were compared with those for synthetic complex of pancreatic bovine DNase I and chicken muscle actin.     

Rat pancreas actin: purification and characterization

Isolation of rat pancreas actin was performed with three different technics: polymerization-depolymerization method, affinity chromatography on DNase I-Sepharose 4B or ion exchange chromatography on DEAE-cellulose. Inhibition of DNase I activity, localization by SDS polyacrylamide slab gel electrophoresis and presence of microfilaments allowed its identification. Affinity process led us to obtain actin which kept inhibitory activity (30,000 U per mg) on DNase I when using vacuum dialysis. Actin eluted from DEAE-cellulose associated reversibly in 50-70 A microfilaments in the presence of phalloidin, was pure at 95% and had a satisfactory inhibitor activity (77,000 U per mg).     

The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography

Native actin can be isolated from pea (Pisum sativum L.) roots by DNase I affinity chromatography, but the resulting yields and quality of actin are variable. By use of two assays for actin, a DNase I inhibition assay and a gel scanning assay, we identified several factors that increased actin yield. ATP is required for the actin in crude pea root extracts to bind to immobilized DNase I. Low amounts of ATP are hydrolyzed rapidly by an endogenous ATPase in the extract, and the actin then irreversibly loses the ability to bind to DNase I. High ATP concentrations (5-10 mm) or inhibition of the ATPase (with 10 mm pyrophosphate) are required for pea actin to retain DNase I binding ability. When adequate amounts of ATP are present, actin binding from the extract is further enhanced by basic pH, formamide, and soluble polyvinyl-pyrrolidone. Once actin is bound to the DNase I-agarose and washed free of extract, high ATP concentrations are not required to keep actin bound. Actin eluted from the DNase I-agarose with formamide retained its ability to polymerize into filaments with the addition of KCl and Mg²⁺. The advantages and disadvantages of this procedure and its application to other plant materials are discussed.     

DNase I-like activity and actin content in the liver of some vertebrates

Total actin content and F:G actin ratio were determined in the liver cytosol of fish, frogs and mouse by measurements of inhibition of exogenous crystalline bovine pancreatic DNase I. Endogenous DNase I-like activity, was found in all examined livers after electrophoresis in the presence of sodium dodecyl sulfate and subsequent enzyme renaturation. It is concluded that DNase I-like enzymes occur in the liver cytosol in a latent form, probably bound to cytoplasmic actin.     

Tissue-specific tropomyosin isoform composition.

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells (Gunning et al. 1998b). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.     

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and on the DNase I-G actin complex

Effects of isoflurane on the DNase I activity in an isolated enzyme preparation and in the DNase I-globular (G) actin complex were investigated. DNase I, DNase I-G actin complex, and G actin were exposed to various (0.2-4.0 vol%) isoflurane concentrations for 180 min. Thereafter, DNase I activity was determined. DNase I activity was inhibited in relation to time and concentration of isoflurane exposure. At concentrations ranging from 0.2 to 1.0 vol% of isoflurane inactive DNase I was activated in the DNase I-G actin complex. The DNase I inhibitor G actin showed a reduced capability to inhibit DNase I following isoflurane exposure. Albumin can inhibit the DNase I inactivation possibly by competition in the reactions between DNase I/albumin and isoflurane. After exposure to isoflurane the absorption maximum of DNase I was identical with the absorption maximum of heat-denatured DNase I. The results suggest a mechanism by which isoflurane may affect DNA in an indirect way at concentrations to which the patient is exposed during clinical anesthesia.     

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.     

The binding of mutant actins to profilin, ATP and DNase I.

Twenty-five mutations were created in the Drosophila melanogaster Act88F actin gene by in vitro mutagenesis and the mutant actins expressed in vitro. The affinity of the mutant actins for ATP, profilin and DNase I was determined. They were also tested for conformational changes by non-denaturing gel electrophoresis. Mutations at positions 364 (highly conserved) and 366 (invariant) caused changes in conformation, reduced ATP binding and increased profilin binding. At position 362 (invariant) only the conservative change from tyrosine to phenylalanine had no effect; other changes at this position affected conformation, ATP and profilin binding. Although only glycine or serine occur naturally at position 368, changes to threonine or glutamine had no effect on the actin. The mutant in which Asp363 was replaced by His and that in which Glu364 was replaced by Lys decreased DNase I binding, yet neither amino acid occurs in the DNase I binding site. Likewise several mutations affect ATP and profilin binding but are distant from the binding sites. We conclude that, although actin has a highly conserved amino acid sequence, individual amino acids can have variable tolerance for substitutions. Also amino acid changes can exert significant effects on the binding of ligands to distant parts of the actin structure.