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).     

Isolation and characterization of actin from Entamoeba histolytica

Actin has been identified and purified partially from trophozoites of Entamoeba histolytica HMI-IMSS by a procedure that minimizes proteolysis. In cellular extracts, Entamoeba actin would copolymerize with muscle actin, but would not bind to DNase I or form microfilaments. Fractionation of the extracts by DEAE-cellulose and Sephadex G-150 chromatography yielded a purified actin that would copolymerize with rabbit skeletal muscle actin or polymerize alone into long filaments at 24 degrees C upon addition of 100 mM KC1 and 2 mM MgCl2. These filaments are not cold-stable and will depolymerize at 4 degrees C in 1 or 2 h. Entamoeba actin filaments bind phallotoxin with the same affinity as muscle actin and decorate with rabbit skeletal muscle heavy meromyosin. Entamoeba actin filaments activate the Mg2+ ATPase of heavy meromyosin to the same Vmax as muscle actin, but the Kapp is 2.8 times higher. Entamoeba actin is a single species with a slightly higher molecular weight than muscle actin (45,000) and a more acidic pI (5.4). The purified actin does not bind to DNase I, produce inhibition of the enzymatic activity, or block the binding of muscle actin. Comparison of the peptides obtained by limit digest with protease V8 from Staphylococcus aureus shows sequences with common mobility between alpha-actin and Entamoeba actin, but additional peptides are present which may account for the different properties of the Entamoeba actin. Finally, in vitro translation of mRNA from trophozoites produces a single polypeptide equivalent to the molecule purified from Entamoeba extracts.     

Carp hepatopancreatic DNase I: biochemical,molecular, and immunological properties

A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.     

Cross-linked long-pitch actin dimer forms stoichiometric complexes with gelsolin segment 1 and/or deoxyribonuclease I that nonproductively interact with myosin subfragment 1

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.     

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.     

Limited proteolysis of actin by a specific bacterial protease

A 36 kDa fragment of rabbit skeletal muscle actin resistant to further proteolytic breakdown was obtained with a new bacterial protease. This fragment was the only cleavage product obtained from native actin whereas proteolysis of heat-inactivated actin was unlimited. The 36 kDa fragment failed to polymerize and to inhibit DNase I activity. Binding to DNase I protects actin against proteolysis by protease. The results on actin proteolysis by different proteases are compared.     

A DNase I binding/immunoprecipitation assay for actin

An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.     

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.

     

Production of human skeletal alpha-actin proteins by the baculovirus expression system

Mutations within the human skeletal muscle alpha-actin gene cause three different skeletal muscle diseases. Functional studies of the mutant proteins are necessary to better understand the pathogenesis of these diseases, however, no satisfactory system for the expression of mutant muscle actin proteins has been available. We investigated the baculovirus expression vector system (BEVS) for the abundant production of both normal and mutant skeletal muscle alpha-actin. We show that non-mutated actin produced in the BEVS behaves similarly to native actin, as shown by DNase I affinity purification, Western blotting, and consecutive cycles of polymerisation and depolymerisation. Additionally, we demonstrate the production of mutant actin proteins in the BEVS, without detriment to the insect cells in which they are expressed. The BEVS therefore is the method of choice for studying mutant actin proteins causing human diseases.     

Studies on Trypanosomatid Actin I. Immunochemical and Biochemical Identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai , actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Studies on trypanosomatid actin. I. Immunochemical and biochemical identification

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.     

Isolation and characteristics of actin-like proteins in liver mitochondria

Using affinity chromatography on DNAase I-Sepharose, an actin-like protein was isolated from rat liver mitochondria and purified 60-fold. SDS electrophoresis in polyacrylamide gel revealed that the protein migrated with muscle actin and thus had the molecular weight of 42 000 Da. Evidence for the actin-like nature of the mitochondrial protein could be obtained from the fact that the protein inhibited the activity of pancreatic DNAase I which, similar to the smooth muscle protein, was less conspicuous than that of its muscle counterpart. Unlike striated muscle actin but similar to the smooth muscle protein, the mitochondrial actin weakly stimulated the Mg-ATPase activity of rabbit skeletal muscle myosin. After manyfold washing of the mitochondria with isotonic isolation media, the content of the actin-like protein remained unchanged, which indirectly points to the presence of insignificant cytoplasmic actin contaminations. During isoelectrofocusing, the mitochondrial actin-like protein yielded two forms, i. e., beta- and gamma-isoactins, whose ratio was 8:1. The pI values for the beta- and gamma-isoforms were 5.52 and 5.59, respectively. The identical position of the absorption spectra (260 nm) and fluorescence excitation spectra (around 280 nm) maxima of the actin-like protein and smooth and skeletal muscle actins testify to their homology.     

An actin-like protein is a component of axonemes from Chlamydomonas flagella.

Several lines of evidence indicate that a component of axonemes from Chlamydomonas flagella is similar to actin from rabbit skeletal muscle. The polypeptide has an apparent molecular weight of 42,000 and in a pH gradient has the electrophoretic behavior of beta-actin. It was co-polymerized with rabbit actin and purified by affinity chromatography on DNase I-Sepharose. Incomplete proteolysis of mixed 35S-labeled axonemal protein and cold rabbit actin formed similar sets of peptides as analyzed by one-dimensional gel electrophoresis. The actin-like protein and the tubulin appear to be present in the axoneme in the molar ratio 1:60.     

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.     

Identification of actin from hepatoma Morris 5123 cells

The hepatoma Morris 5123 tumor growth is accompanied by changes in actin content and polymerization (Malicka-B?aszkiewicz et al. (1995) Mat. Med. Pol., 27, 115-118; Nowak et al. (1995) J. Exp. Cancer Res. 14, 37-40). Presently actin isoforms from cytosol and cytoskeleton fractions were separated by SDS/PAGE and identified with antibodies directed against different actin isoforms. Actin isolated from the cytosol by affinity chromatography on DNase I bound to agarose shows the presence of only one protein spot on 2D gel electrophoresis corresponding to the mobility of the rabbit a skeletal muscle actin (Mr 43,000) and isoelectric point equal to 5.3. It interacts only with monoclonal anti beta actin isoform antibodies, posing the question of differential affinity of actin isoforms to DNase I.     

Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin

Two poly(L-proline)-binding proteins (PBP-1 and PBP-2) were purified from chick embryos by using a poly(L-proline)-agarose column. PBP-1 was composed of two different polypeptides (molecular masses of 42 kDa and 15 kDa). The molar ratio of the two proteins in the complex was 1:1. The other poly(L-proline)-binding protein, PBP-2, was the 15-kDa protein itself. The 42-kDa protein was confirmed to be an actin from the amino acid composition, by immunochemical evidence and by its ability to self-polymerize. In addition, the 42 + 15-kDa protein complex (PBP-1) inhibited DNase I, just as a monomeric actin did. The amino acid composition of the 15-kDa protein was similar to that of mammalian profilin and it inhibited the salt-induced polymerization of rabbit skeletal muscle actin. Therefore, we conclude that the two poly(L-proline)-binding proteins from the chick embryo are a profilactin and a profilin in chick embryo. The ability of profilactin to bind poly(L-proline) must be due to profilin itself, because the profilin has a greater affinity for poly(L-proline) than does profilactin. Additionally, both the monomeric and filamentous actin from rabbit skeletal muscle have no affinity for poly(L-proline).     

A cofilin-like protein is involved in the regulation of actin assembly in developing skeletal muscle

An actin-binding protein of 20 kDa (called 20K protein) was purified from the sarcoplasmic fraction of embryonic chicken skeletal muscle. The properties of this protein were very similar to cofilin, which was discovered in porcine brain (Nishida et al. (1984) Biochemistry, 23, 5307-5313): it bound to both G- and F-actin, inhibited actin polymerization in a pH-dependent manner, inhibited binding of tropomyosin to F-actin, and had almost the same molecular size and pI as cofilin. A specific monoclonal antibody to 20K protein (MAB-22) was prepared to examine the expression and location of 20K protein during skeletal muscle development. When the whole protein lysates of embryonic and post-hatched chicken skeletal muscles were examined by means of immunoblotting combined with SDS-PAGE, 20K protein was detected in skeletal muscle through the developmental stages. Location of 20K protein in the cells differed between the embryonic and adult tissues; immunofluorescence staining of the cryosections of embryonic muscle with MAB-22 visualized irregular dot-like structures, but adult muscle sections were stained faintly and uniformly. 20K protein was present as a complex with actin in embryonic muscle, as judged by the ability to bind to a DNase I affinity column, while the same protein was free from actin in the cytoplasm of adult muscle. From these results, it is suggested that 20K protein regulates actin assembly transiently in developing skeletal muscle.     

A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex

A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.     

A simple procedure to produce monospecific polyclonal antibodies of high affinity against actin from muscular sources

A simple and effective technique to produce monospecific polyclonal antibodies of high affinity against actin is described. In this procedure, rabbit skeletal muscle actin in the 1:1 complex with bovine pancreatic deoxyribonuclease I is used as antigen to immunize rabbits. The antisera obtained are shown to contain antibodies against both actin and deoxyribonuclease I. By affinity chromatography the two antibody preparations were separated and characterized. The affinity-purified anti-deoxyribonuclease I and anti-actin do not show cross-reactivity. Thus, anti-deoxyribonuclease I inhibits the enzymic activity of deoxyribonuclease I and stains the enzyme after Western blotting. Affinity-purified anti-actin does not inhibit deoxyribonuclease I activity and stains only actin after Western blotting. The affinity-purified anti-actin can be used in a number of different actin-detecting techniques such as in immunohistochemistry and in immunoblotting techniques. This antibody recognizes only actins from muscular tissues with high affinity. Immunoblots of polyacrylamide gels in the presence of ampholytes (IEF) indicate that this antibody only recognizes the alpha-variants of actin. Thus, the skeletal and cardiac alpha-actins are recognized but not the smooth muscle gamma-isoform and the cytoplasmic actins. Vascular smooth muscle alpha-actin is not recognized when using immunoblotting or enzyme-linked immunosorbent techniques. On frozen sections, however, the anti-actin antibody clearly stained vascular smooth muscle cells. Epitope analysis using actin fragments generated by limited proteolysis and selective cleavage using hydroxylamine indicate that this antibody is directed against a rather limited region within the N-terminus of actin.     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.