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Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I)
Biochimica et Biophysica Acta 1451 (1999) 141^152 www.elsevier.com/locate/bba
Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I) Lidia Ciszak 1;a , Agnieszka Krawczenko 1;a , Bernhard Polzar b , Hans Georg Mannherz b , Maria Malicka-Blaszkiewicz a; * a b
Department of Cell Pathology, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland Cytoskeletal Laboratory, Department of Anatomy and Embryology, Ruhr-University, Universita«tsstrasse 150, D-44780 Bochum, Germany Received 12 April 1999; received in revised form 3 June 1999; accepted 11 June 1999
Abstract 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 L-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle K-actin. Polymerization of purified carp liver actin by 2 mM MgCl2 or CaCl2 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 (Kb ) of carp liver actin to DNase I was calculated to be 1.85U108 M31 which is about 5-fold lower than the affinity of rabbit skeletal muscle K-actin to DNase I. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Actin; DNase I; Actin-DNase I interaction; Actin polymerization; Carp liver actin
1. Introduction Actin is one of the most abundant proteins in nature occurring in all eucaryotic cells [1^3]. In striated muscle cells, actin is the main constituent of the thin ¢laments. In non-muscle cells, actin is one of the three major components of the cytoskeleton and is
* Corresponding author. Fax: +48 (71) 3252930; E-mail: [email protected] 1 Present address: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland.
involved in many cellular functions including cell motility, contractile ring formation during cytokinesis and the maintenance of cell shape. Actin plays a very substantial role in organelle movement [4], secretion [5], neurotransmitter release [5,6], the regulation of the epithelial Na -K channel [7,8], and in signal transduction [9]. In hepatocytes actin ¢laments participate in the processes of bile formation like the transport of secretory vesicles, in exocytosis and contraction of the canalicular walls [10]. For all these functions of actin, a dynamic conversion between monomeric (G-actin) and ¢lamentous actin (F-actin) is necessary [1,2]. Intracellularly, actin polymerization is regulated by a large number of
0167-4889 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 9 ) 0 0 0 8 3 - X
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actin-binding proteins [1,2]. Some of these proteins are able to stabilize monomeric actin by direct complex formation and thus sequester it from the polymeric pool. Deoxyribonuclease I from bovine pancreas (DNase I) has been shown to interact both with monomeric and with ¢lamentous actin [11^13]. Both proteins form a 1:1 complex with concomitant inhibition of the DNA degrading activity of DNase I and the ability of actin to form high molecular mass polymers [12^14]. The biological role of this interaction is still unclear. The study of the interaction between various DNase I-like enzymes and di¡erent actin isoforms might help to determine whether one of the many functions of actin is to control the degradative activity of DNase I-like enzymes towards cellular DNA. Due to di¡erences in sequence and isoelectric points, a number of actin isoforms have been distinguished [15^18], which in mammals are distributed in a tissue speci¢c manner: muscle (K-cardiac, K-skeletal, K-vascular and Q-smooth muscle actin) and nonmuscle (L- and Q-cytoplasmic) actins. In spite of the documented very high conservation among actin genes and their encoded proteins, recent results have indicated that there exist functional di¡erences among the actin isoforms [19^22]. Puri¢ed isoforms and actins isolated from di¡erent species di¡er in their thermodynamic stability, polymerization characteristics, binding of Ca2 ions and ATP and their a¤nity for particular actin-binding proteins like for instance DNase I [19,22]. Little information is available on the actins occurring in tissues and organs of the lower vertebrates. Particularly, information on the properties and stability of actin in ¢sh cells is lacking. Several investigators have puri¢ed actin from the back muscle of the milk¢sh [23] and from the liver and back muscle of the electric ¢sh [24]. Electron microscopic and immuno£uorescence studies have allowed to localize actin micro¢laments in microridges of the oral mucosal epithelium of carp [25] and in horizontal cells in carp retina [26]. In contrast, detailed information on the actin content in ¢sh liver, its stability and ability to interact with other actin binding proteins like DNase I is not available. Therefore, the aim of this study was to isolate and characterize actin from the carp liver. Special attention was given to its ability to interact with bovine pancreatic DNase I.
2. Materials and methods 2.1. Isolation procedures for actins Rabbit skeletal muscle K-actin was puri¢ed from acetone powder according to Spudich and Watt [27]. To purify ¢sh liver actin, we used carp (Cyprinus carpio). Fresh livers were homogenized in 3 vols. of freshly made 10 mM Tris-HCl bu¡er, pH 7.4, containing 0.1 mM ATP, 1 mM DTT, 0.1 mM CaCl2 and 0.25 M sucrose (bu¡er A), as described previously by Malicka-Blaszkiewicz [28,29], except that 0.1 mM PMSF, 0.5 mg/1 aprotinin and 1 mg/1 leupeptin were added to the homogenization bu¡er. Homogenates were centrifuged at 105 000Ug for 1 h at 4³C. The supernatant served as the cytosolic fraction and was used immediately or kept frozen at 370³C for further analysis. The cytosol sample was dialyzed for 24 h against bu¡er A without sucrose containing 0.1 mM PMSF before it was loaded on a DEAE-cellulose column (2.5U36 cm) according to Gordon et al. [30]. Proteins adsorbed were eluted from the column with increasing concentration of NaCl (linear gradient from 0.1 to 0.5 M) in bu¡er A without sucrose. Actin containing fractions were identi¢ed by SDS-PAGE, pooled, concentrated by pressure dialysis and loaded on a Sephacryl S-300 column (1.6U92 cm) equilibrated with bu¡er A without sucrose. Proteins were eluted with the same bu¡er. All operations were done at 4³C. Finally a cycle of polymerization and depolymerization was employed to further purify the actin obtained by gel ¢ltration [30,31]. To this aim, the pooled fractions obtained after Sephacryl S-300 were pressure concentrated to 2 ml in the cold (using Amicon with PM 10 membrane) and incubated for 2 h at room temperature precipitated with an equal volume of 2 M potassium phosphate bu¡er, pH 7.6, containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT, and then centrifuged for 30 min at 20 000Ug at 15³C [32]. The supernatant was discarded, the loose precipitate was suspended in 2 ml of polymerizing bu¡er (5 mM Tris-HCl, pH 7.6, containing 0.5 mM ATP, 2 mM MgCl2 , 0.5 mM DTT, 0.2 mM CaCl2 , 10 WM EDTA) and centrifuged for 2 h at 100 000Ug at 15³C. For further experiments, the actin pellet was homogenized in bu¡er A without su-
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crose and the F-actin was depolymerized by dialysis against the same bu¡er for 24 h at 4³C. 2.1.1. A¤nity chromatography on DNase I agarose Alternatively, we performed DNase I a¤nity chromatography essentially as described by Zechel [33]. DNase I was immobilized on vinylsulfone-agarose (Mini Leak, Kem-En-Tec, Copenhagen, Denmark) following the instructions of the supplier. DNase Iagarose was mixed gently overnight at 4³C with the actin samples using a rotary shaker and then centrifuged at 15 000 rpm for 15 min in order to remove unbound protein. DNase I-agarose with adsorbed proteins was poured into a column, which was washed successively with bu¡ers according to the procedure of Zechel [33]. The proteins strongly adsorbed to DNase I-agarose were eluted with bu¡er A containing 40% formamide plus 20% glycerol in order to minimize the denaturation of actin. The fractions collected were immediately diluted 1/2 with bu¡er G (2 mM Tris-HCl, pH 7.9, 0.2 mM CaCl2 , 0.2 mM ATP, 0.2 mM DTE). The actin containing fractions were combined and concentrated by pressure ¢ltration in an Amicon cell using a PM 10 membrane. The formamide was removed by repeated addition of bu¡er G. All operations were done at 4³C. 2.2. Enzymatic tests DNase I activity was determined spectrophotometrically as described earlier by Malicka-Blaszkiewicz and Roth [34]. Highly polymerized calf thymus DNA was used in all experiments as substrate. In some experiments DNase I activity was measured by the hyperchromicity assay of Kunitz [35] at 260 nm and room temperature. 0.2 Wg DNase I was added to the cuvette containing 10 mM Tris-HCl, pH 8.0, 1 mM MgCl2 , 0.1 mM CaCl2 , 1 mM NaN3 and 50 Wg DNA. Activity was expressed in Kunitz units; one Kunitz unit corresponds to an increase in absorbance of 0.001 min31 at 260 nm. The actin content was measured as inhibitor of bovine pancreatic DNase I (Sigma) under standard DNase assay conditions [29,34,36,37] and expressed as units of DNase I inhibitor per mg of protein. One unit of actin was de¢ned as the amount which decreases the activity of 20 ng of DNase I by 10%. In the part of experiments the actin content was
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measured as an inhibitor of DNase I under Kunitz assay conditions [35]. Various actin concentrations were incubated with 0.2 Wg DNase I for 15 min at room temperature and then transferred to ice to stop the reaction. 2.3. Determination of the binding constant of carp liver actin to bovine DNase I The binding constant of carp liver actin to bovine DNase I was determined according to Dixon [38]. DNase I (at a constant concentration) and increasing concentrations of actin were added sequentially to cuvettes containing 40 mM HEPES-OH, pH 7.0, 5 mM MgCl2 , 0.1 mM CaCl2 and incubated for 30 s at room temperature. The reaction was started by addition of substrate (calf thymus DNA, Sigma). The initial velocity of the reaction was measured for 200 s at 260 nm. Both enzyme and substrate concentrations were selected such that the increase in reaction products was directly proportional to the amount of inhibitor (actin). The measurements were performed in the presence of two substrate concentrations: 30 Wg and 70 Wg DNA. The dissociation constant Kd and the binding constant Kb were calculated from double-reciprocal plots of the dependence of the enzymatic activity on the inhibitor concentration (1/v against actin concentration [I]). The measurements were performed on a diode spectrophotometer HP 8452 A (Hewlett-Packard, Palo Alto, CA, USA). 2.4. Fluorescence measurements Polymerization of actin from the carp liver cytosol was determined by the £uorescence increase of pyrene-labeled K-actin. Carp liver actin was mixed with 1.4 WM pyrene-labeled rabbit skeletal muscle actin [39] at room temperature under di¡erent bu¡er conditions. The £uorescence intensity was measured at 407 nm after excitation at 365 nm at room temperature on a spectro£uorometer Labmath Shimadzu RF-5001 PC V2.00. The identical procedure was used to examine the e¡ect of bovine DNase I on the carp liver polymerization. Carp liver actin was mixed with 1.4 WM pyrenyl-actin and incubated for 15 min at room temperature with bovine pancreatic DNase I at various
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molar ratios. Filament elongation was started by addition of 1 M potassium phosphate bu¡er, pH 7.6, containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT. The control sample did not contain DNase I. 2.5. Electrophoretic procedures and immunoblotting SDS-PAGE was performed according to Laemmli [40] in 10% gels, at 30 mA per slab, using 0.1% SDS in electrode bu¡ers. Gels were stained with Coomassie brilliant blue R-250 or the silver method [41]. Isoelectric focusing was performed essentially as described by O'Farrell [42] in cylindrical gels cast into glass tubes (75 mmU1.5 mm) in the presence of 9.5 M urea, 2% Nonidet P40, 3.3% acrylamide and 2% ampholine mixture (pH 3^5/5^7). The gels were prefocused for 30 min at 4³C at 250 V before the proteins samples were layered onto the cathodic end of the gels. At the end of the isoelectric focusing, the gels were extruded from the tubes and washed for 10 min in 0.125 M Tris-HCl bu¡er, pH 6.8, containing 10% glycerol, 4.9 mM DTT and 2% SDS in order to remove the Ampholines. The gels were subsequently used for protein separation by SDS-PAGE or kept frozen at 370³C for further analysis. pI values of actin from the carp liver cytosol was calculated based on the comparison with isoelectric focusing of muscle K-actin. The transfer of proteins to nitrocellulose sheets was achieved according to Towbin et al. [43]. For immunoblotting we used a monoclonal antibody (Sigma) directed against L-cytoplasmic actin. Immunoreactivity was shown by the avidin-biotin peroxidase (Sigma) technique, using 4-chloro-1-naphthol as substrate. The presence of actin was also veri¢ed by using a goat antiserum directed against skeletal muscle K-actin. Immunoreactivity was detected as described for the monoclonal antibodies. 2.6. Determination of protein concentration Protein concentrations were determined either by following the procedure given by Lowry et al. [44] or spectrophotometrically at 280 nm or 290 nm (when ATP was present in the used bu¡er). The concentration of carp liver actin was calculated using A1% 290nm = 6.3 [45].
3. Results 3.1. Isolation of actin from carp liver cytosol The procedure developed by Gordon et al. [30,31] to purify actin from non-muscle cells was also applied to isolate actin from carp liver. Fractions of each puri¢cation step were analyzed by SDS-PAGE and for actin content measured as an inhibitor of DNase I. 3.1.1. Ion exchange chromatography The cytosol of carp liver was loaded on a DEAEcellulose column and eluted as described in Section 2 (Fig. 1a). DNase I inhibitory activity eluted in the two peaks (peak I and peak II) at 0.19 and 0.26 M NaCl probably due to the presence of complexes of actin with actin binding proteins. As shown in Fig. 1a, the fractions of the peak II revealed higher inhibitory activity than the fractions of peak I. Therefore the fractions of peak II were used for further actin puri¢cation. 3.1.2. Gel ¢ltration The combined fractions of peak II were concentrated by dialysis against sucrose and chromatographed on Sephacryl S-300 using the conditions described in Section 2. The elution pro¢le is shown in Fig. 1b. The proteins were separated into two distinct peaks, whereas DNase I inhibitory activity was eluted in one symmetrical peak within the descending arm of the ¢rst protein peak. The fractions with highest inhibitory activity (59 to 68) were collected and subjected to further analysis. SDS-PAGE analysis of the collected fractions showed the presence of only a few protein bands with a predominance of two bands: one with a mobility equal to skeletal muscle K-actin and another one with a mobility corresponding to Mr of 21 kDa (compare Fig. 2, lanes 4, 5 and 7). 3.1.3. Polymerization of actin In order to further purify the actin present in the fractions obtained after Sephacryl S-300 ¢ltration, the pooled material (fractions 59^68, Fig. 1b), was subjected to a polymerization-depolymerization cycle according to Gordon et al. [30,31]. However, no pellet was seen under applied conditions after centrifu-
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Fig. 1. Puri¢cation of carp liver actin. (a) Ion exchange chromatography. DEAE-cellulose column (2.5U36 cm) was equilibrated with bu¡er A without sucrose containing 0.1 M NaCl. Elution was carried out with the same bu¡er and then with linear NaCl concentration gradient (0.1^0.5 M). 4 ml fractions were collected at a £ow rate 60 ml/h. 1 g of protein was applied to the column. +, absorption at 290 nm; a, inhibition capacity of DNase I. (b) Gel ¢ltration. Sephacryl S-300 column (1.6U92 cm) was equilibrated with bu¡er A without sucrose and elution was carried out with the same bu¡er. 65 mg of protein of the pooled fractions of peak II was applied to the column. 2 ml fractions were collected at a £ow rate 10 ml/h. +, absorption at 290 nm ; a, inhibition capacity of DNase I.
gation at 100 000Ug. Therefore we concluded that carp liver actin had not polymerized after addition of 2 mM MgCl2 and 0.1 M KCl to bu¡er A without sucrose. In order to dissociate possible complexes of actin with actin binding proteins (AbPs), which might have inhibited its polymerization, we applied a procedure described by Weber et al. [32], i.e., we incubated the pooled fractions in 2 M potassium bu¡er, pH 7.6, containing 4 mM ATP, 2 mM MgCl2 and 2 mM DTT to induce polymerization of the carp liver actin. After centrifugation only one protein band was seen after SDS-PAGE of equal mobility to K-actin (Fig. 2, lanes 5 and 6). 3.1.4. A¤nity chromatography on DNase I agarose Alternatively, a¤nity chromatography using agarose immobilized DNase I was employed to purify carp liver cytosolic actin. For this purpose we used the pooled fractions collected from peak I and peak
Fig. 2. Electrophoretic analysis of samples obtained during puri¢cation of carp liver actin. Slab SDS-PAGE was performed according to Laemmli [40] on 10% gels. The proteins were stained with Coomassie brilliant blue. Lanes: 1, carp liver cytosol, 55 Wg; 2, pooled fractions of peak I (147^156, Fig. 1a), 80 Wg; 3, pooled fractions of peak II (169^184, Fig. 1a), 75 Wg; 4, pooled fractions (59^68, Fig. 1b), 70 Wg; 5, standard K-rabbit skeletal muscle actin, 10 Wg; 6, actin puri¢ed from the carp liver, 15 Wg; 7, marker proteins, 10 Wg.
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Using DNase I a¤nity chromatography we obtained two actin preparations: preparation A (obtained from peak I) and preparation B (from peak II). SDS-PAGE analysis of the preparation A showed the presence of only one protein band with a mobility identical to skeletal muscle K-actin, whereas in the preparation B a few additional protein bands were seen (Fig. 3a, lanes 1^3). For further analysis we used the actin obtained from preparation A.
Fig. 3. Electrophoretic analysis and immunoblotting of the actin preparations obtained after a¤nity chromatography on DNase I agarose. Slab SDS-PAGE was performed in the same conditions as in Fig. 2. (a) The proteins were stained with Coomassie brilliant blue. (b) Immunoblotting. Murine monoclonal antibodies directed against L-cytoplasmic actin were used. Lanes; 1, 1P, preparation A, 8 Wg; 2, 2P, preparation B, 4.5 Wg; 3, 3P, standard K-rabbit skeletal muscle actin, 7 Wg; 4, 4P, marker proteins, 10 Wg.
II of the DEAE-cellulose column (see Fig. 1a). The elution pro¢le was analyzed for DNase I inhibitor activity measured in the DNase assay conditions according to Kunitz [35]. The actin present in peak I as well as in peak II was strongly adsorbed on DNase Iagarose: DNase I inhibitory activity was only associated with the fractions eluted with 40% formamide.
Fig. 4. Two-dimensional PAGE. Isoelectric focusing according to O'Farrell [42], SDS-PAGE in 10% gels according to Laemmli [40]. (a) Puri¢ed carp liver actin, 10 Wg (stained with silver method, [41]); (b) standard K-rabbit skeletal muscle actin, 7 Wg (stained with Coomassie brilliant blue).
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3.2. Characterization of carp liver actin 3.2.1. Two-dimensional PAGE Two-dimensional PAGE was applied to examine the homogeneity, isoform composition and isoelectric point (pI) of carp liver actin. The results obtained were compared with rabbit skeletal muscle K-actin. No di¡erences were found in the protein pattern after two-dimensional PAGE of the actin obtained after the polymerization-depolymerization cycle and for both preparations puri¢ed by DNase I-agarose a¤nity chromatography. Only one protein spot was seen after two-dimensional PAGE with a pI slightly higher than the pI of skeletal muscle K-actin, i.e., approx. 5.4 (compare Fig. 4a and b). 3.2.2. Immunoblotting using isoform speci¢c antibodies The 2D gel electrophoresis indicated the presence of only one actin variant in carp liver. Therefore the identity of the puri¢ed band with actin was veri¢ed by immunoblotting using polyclonal antibodies directed against K-actin (Fig. 5a) and a monoclonal antibody directed against cytoplasmic L-actin (Fig. 5b). As can be seen, carp liver actin interacted weakly with the polyclonal antibodies directed against K-actin, but strongly with the monoclonal antibody against cytoplasmic L-actin (compare Fig. 5a, lane 2 and Fig. 5b, lane 2P). In contrast, skeletal
Fig. 5. Immunoblotting of carp liver actin; comparison with Kactin. The proteins were separated in SDS-PAGE [40] in 10% gels, transferred to nitrocellulose sheets [43] and incubated with the antibodies. Goat antiserum directed against rabbit skeletal muscle K-actin (a) and murine monoclonal antibodies directed against L-cytoplasmic actin (b) were used. Lanes: 1, 1P, standard K-rabbit skeletal muscle actin, 7 Wg; 2, 2P, actin from the carp liver, 15 Wg.
Fig. 6. E¡ect of bu¡er composition on polymerization of carp liver actin. Actin (3.25 WM, preparation A from a¤nity chromatography on DNase I-agarose) was mixed with 1.4U1036 M of K-actin conjugated with N-(1-pyrenyl)iodoacetamide. Fluorescence intensity was measured at 407 nm after excitation at 365 nm at room temperature. The following bu¡ers were used to start the ¢lament elongation: E, bu¡er G+2 mM MgCl2 + phalloidin (5 WM); O, bu¡er G+2 mM CaCl2 ; a, bu¡er G+2 mM MgCl2 ; +, 1 M potassium phosphate bu¡er pH 7.6 containing 4 mM ATP; 2 mM MgCl2 ; 2 mM DTT.
muscle actin was only recognized by the antibodies directed against K-actin (compare Fig. 5a, lane 1 and Fig. 5b, lane 1P). This result was obtained irrespective of the ¢nal puri¢cation procedure (Fig. 3b, lane 1P^ 3P). Based on this comparison we concluded that the puri¢ed carp liver actin is a cytoplasmic L-actin variant. 3.2.3. Fluorescence measurements of carp liver actin polymerization Optimal conditions for carp liver actin polymerization were examined spectro£uorometrically after addition of 4% of pyrene-labeled K-actin. First, we measured the £uorescence increase obtained at room temperature in the presence of bu¡er G (2 mM TrisHCl, pH 7.9, 0.2 mM CaCl2 , 0.2 mM ATP, 0.2 mM DTE) supplemented by either 2 mM MgCl2 or 2 mM CaCl2 . As can be seen from Fig. 6, carp liver actin did not polymerize under these ionic conditions. Only after addition of phalloidin and 2 mM MgCl2 we observed an increase in £uorescence intensity. However, we found that carp liver actin polymerized in the presence of 1 M potassium phosphate bu¡er,
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Fig. 7. Inhibition of the standard DNase I from bovine pancreas by carp liver actin. (a) Actin was puri¢ed by polymerization-depolymerization cycle. Inhibition was performed in standard assay conditions [34], in the presence of 0.75 nM DNase I. (b) Actin puri¢ed by a¤nity chromatography on DNase I-agarose was used. Inhibition was carried out in the DNase assay conditions of Kunitz [35], in the presence of 6.2 nM DNase I.
pH 7.6, containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT [32]. The formation of regular micro¢laments by puri¢ed carp liver actin under these conditions was observed in electron microscopy (data not shown).
that about 80 nM carp liver actin was required for decrease the DNase I activity by 50% (at a molar ratio of actin to DNase I of about 13, see Fig. 7b). Thus both actin preparations exhibited a similar abil-
3.3. Interaction of carp liver actin with bovine pancreatic DNase I 3.3.1. Inhibition of DNase I by carp liver actin Actins isolated from non-muscle cells di¡er in their a¤nity to DNase I [22]. We examined the inhibitory e¡ect of carp liver actin on puri¢ed bovine pancreatic DNase I using two di¡erent actin preparations: (i) the actin obtained after the polymerization-depolymerization cycle and (ii) the actin obtained by a¤nity chromatography on DNase I-agarose (preparation A). In the case of actin obtained by polymerization and depolymerization cycle DNase I inhibition was measured under standard assay conditions [29,34,36]. As shown in Fig. 7a 12 nM actin was required to decrease the activity of 0.75 nM DNase I by 50% (molar ratio of 16). When using the actin obtained after a¤nity chromatography on DNase I-agarose (preparation A), the inhibitory activity was measured immediately after removal of the formamide by ultra¢ltration (see Section 2). The inhibition was measured using assay conditions according to Kunitz [35]. We found
Fig. 8. E¡ect of DNase I on polymerization of actin from carp liver cytosol. Carp liver actin was mixed with 1.4U1036 M pyrene-labeled K-actin and incubated for 15 min at room temperature with standard DNase I in various molar ratio of actin to DNase I. Filament elongation was started by simultaneous addition of 1 M potassium phosphate bu¡er pH 7.6 containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT. Fluorescence intensity was measured at 407 nm after excitation at 365 nm at room temperature. E, actin without DNase I (3.25U1036 M); O, actin plus DNase I mixed in molar ratio of actin to DNase I 18:1; a, molar ratio of actin to DNase I 8:1; b, molar ratio of actin to DNase I 3:1.
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Fig. 9. Determination of the binding constant of bovine pancreas DNase I to carp liver actin. (a) Inhibition of DNase I by actin. DNase I was added to the test solution (see Section 2) to give a ¢nal concentration of 3.8U1039 M, mixed with increasing concentrations of inhibitor (carp liver actin) and incubated for 30 s. Reaction was started by addition of 30 Wg DNA and carried out at room temperature. Identical measurements were done for 70 Wg DNA (data not shown). a, DNase I without actin; O, DNase I plus 2.1U1039 M actin; b, DNase I plus 3.3U1039 M actin; +, DNase I plus 6.6U1039 M actin; E, DNase I plus 8.7U1039 M actin. (b) Dixon plots analysis of the dependence of DNase I activity on actin concentration. The actin concentration (I0 ) was calculated usa, data obtained in the presence of 70 Wg DNA. ing A1% 290nm = 6.3 [45]. b, data obtained in the presence of 30 Wg DNA;
ity to inhibit DNase I activity. These results con¢rmed previous observations [46] which had demonstrated that actin from carp liver cytosol is able to inhibit bovine DNase I. 3.3.2. E¡ect of DNase I on the polymerization of carp liver actin The e¡ect of DNase I on the ability of carp liver actin to polymerize was analyzed by the changes of the £uorescence intensity in the presence of pyrenelabeled K-actin. Actin (preparation A obtained after a¤nity chromatography on DNase I-agarose) was incubated with bovine DNase I in the molar ratios of actin to DNase I of 18:1; 8:1 and 3:1. A pronounced inhibitory e¡ect of DNase I on carp liver actin polymerization was observed (Fig. 8). Filament elongation was completely inhibited, when the molar ratio of actin to DNase I was 3:1. These results indicated that actin from carp liver cytosol interacts with bovine pancreatic DNase I leading to the formation of a stable complex with concomitant inhibition of DNA degrading activity of DNase I and ability of carp liver actin to polymerize. 3.3.3. Determination of the binding constant of bovine DNase I to actin The actin dependent DNase I inhibition was em-
ployed for computation of the binding constant according to Dixon (see Section 2). Actin puri¢ed by polymerization-depolymerization was used in these experiments. From the data given in Fig. 9a double-reciprocal plots were constructed relating the dependence of the DNase I activity on inhibitor concentration (1/v against actin concentration [I]) in the presence of 30 Wg and 70 Wg DNA, Fig. 9b. It can be seen that straight lines were obtained, which intercepted at a common point above the negative [I] axis. The dissociation constant Kd was estimated to be 5.4U1039 M resulting in a Kb of carp liver L-actin to bovine DNase I of 1.85U108 M31 . 4. Discussion Here we describe the puri¢cation of actin from the carp liver cytosol. The isolated actin was used to examine some of its properties. The puri¢cation procedure given by Gordon et al. [30,31] was particularly useful to isolate actin from these cells. By this procedure actin was obtained in two peaks after ion exchange chromatography most probably being due to the occurrence of actin in complexes with other cytosolic AbPs. After the subsequent gel ¢ltration on Sephacryl S-300 and a cycle of de- and polymeriza-
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tion a highly pure actin preparation was obtained (see also the SDS-PAGE of Fig. 2, lanes 1 and 4). The obtained actin preparation was solely composed of the cytoplasmic L-isoform indicating that carp liver contains this L-variant as only actin isoform. Our results are in agreement with data from Zechel and Weber [24] demonstrating the presence of only L-actin in the liver of the electric ¢sh Torpedo marmorata. The isoelectric points of both ¢sh liver L-actins were slightly higher than of skeletal muscle K-actin. In contrast, mammalian livers were shown to contain both L- and Q-cytoplasmic actins [31,47]. We have con¢rmed the presence of L-actin in the carp hepatocytes also by immunogold labeling at the ultrastructural level. It was localized in the cytoplasm and nuclei (data not shown). Although the exact amino acid composition of carp liver actin has not yet been determined, ¢sh liver will represent a reliable source for future studies of the isotype speci¢c properties of cytoplasmic L-actin. Polymerization of puri¢ed carp liver actin was only induced in the presence of the high ionic strength phosphate bu¡er as described by Weber et al. [32]. We did not obtain its polymerization under standard ionic conditions (like in 10 mM Tris-HCl, pH 7.4, 0.1 mM ATP, 1 mM DTT, 0.1 mM CaCl2 , 2 mM MgCl2 and 100 mM KCl) except when phalloidin was present indicating its ability to bind this drug. It is probable that the occurrence of AbPs might have inhibited its polymerization. The inhibitor may be the observed 21 kDa protein, whose presence was shown by SDS-PAGE (see Fig. 2, lane 4). Miron et al. [48] have reported the presence of an inhibitor of Mr = 25 kDa in many chicken tissues including liver. Rouhnau and coworkers [49] puri¢ed a polymerization inhibiting protein of 21 kDa from chicken gizzard smooth muscle. It is also probable that actin depolymerizing or sequestering proteins were present in the preparation obtained after gel ¢ltration, such as co¢lin (Mr = 19 kDa) or actin depolymerizing factor (Mr = 18.5 kDa) [22,50]. A¤nity chromatography on DNase I-agarose was also applied for further actin puri¢cation using both peaks obtained after ion-exchange chromatography on DEAE-cellulose. Actin present in both peaks strongly adsorbed onto DNase I-agarose from which it was eluted using a bu¡er containing 40% form-
amide and 20% glycerol. We obtained two actin preparations, A and B from the protein peaks I and II, respectively, initially separated by ion-exchange chromatography (see Fig. 1a). SDS-PAGE analysis showed that in preparation B a few additional bands occurred (see Fig. 3a, lane 2). It is interesting that these higher Mr bands (corresponding to Mr from 80 kDa to 130 kDa) interact similarly as the 43 kDa band (actin) with the monoclonal antibodies directed against L-cytoplasmic actin (see Fig. 3b, lane 2P). We have shown that puri¢ed carp liver actin inhibited DNase I to 50% when added in a 13^16-fold molar excess. A molar binding ratio of skeletal muscle K-actin to bovine pancreatic DNase I of 1:1 was reported earlier by Mannherz et al. [51]. The discrepancy may have been due to partial denaturation of carp liver actin during elution from DNase Iagarose or an intrinsic property of this cytoplasmic actin. Indeed, the ability of cytoplasmic actins from di¡erent sources to inhibit DNase I was reported to be very labile and variable [29,34]. The estimated the binding constant of carp liver actin to DNase I (Kb ) was calculated to be 1.85U108 M31 . This is the ¢rst determination of the binding constant of cytoplasmic L-actin to bovine pancreatic DNase I. Previously, the binding constant of K-rabbit skeletal muscle actin to DNase I was shown to range from 5 to 10U108 M31 [51] indicating an approx. 5-fold lower a¤nity of carp liver actin to bovine pancreatic DNase I. We have suggested previously that one of the many functions of non-muscle actin may be to inhibit intracellular DNase I-like activities [28,29,36,37, 46]. Here we demonstrated that carp liver L-actin is indeed able to inhibit bovine pancreatic DNase I. In parallel experiments we have isolated a DNase I-like activity from carp liver cytosol (data not shown). The enzymatic activity of the endogenous carp liver DNase I-like enzyme was inhibited by both K-rabbit skeletal muscle and carp liver actin (to be published). The presence of natural complex of carp liver actin with carp liver DNase was suggested by Krawczenko et al. [52]. Experiments using DNase I-like enzymes and its inhibitor (actin) from the same source may help to understand the role of this interaction in biological systems. In this respect carp liver may be an advantageous system, since it contains high concentrations of both proteins. Such studies may also be of value in understanding some clinical aspects of liver
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pathology like hepatic ¢brosis [53] where actin may serve as functional markers. Acknowledgements The studies were supported by a research grant from the University of Wroclaw (Wroclaw, Poland). The ¢nancial support from the Deutsche Akademischer Austauschdienst (DAAD, Bonn, Germany) and the Deutsche Forschungsgemeinschaft (Bonn, Germany) is gratefully acknowledged.
References [1] E.D. Korn, Actin polymerization and its regulation by proteins from nonmuscle cells, Physiol. Rev. 62 (1982) 672^737. [2] T.D. Pollard, J.A. Cooper, Actin and actin binding proteins. A critical evaluation of mechanisms and functions, Annu. Rev. Biochem. 55 (1986) 987^1035. [3] I. Sauman, S.J. Berry, An actin infrastructure is associated with eucaryotic chromosomes: structural and functional signi¢cance, Eur. J. Cell Biol. 64 (1994) 348^356. [4] F. Grolig, Actin-based organelle movements in interphase Spirogyra, Protoplasma 155 (1990) 29^42. [5] J.M. Trifaro, M.L. Vitale, Cytoskeleton dynamics during neurotransmitter release, Trends Neurol. Sci. 16 (1993) 466^472. [6] P. Greengard, F. Benfenati, F. Valtorta, Synapsin I, an actin-binding protein regulating synaptic vesicle tra¤c in the nerve terminal, in: Molecular and Cellular Mechanism of Neurotransmitter Release, Raven Press, New York, 1994, pp. 31^45. [7] H.F. Cantiello, J.L. Stow, A.G. Prat, D.A. Ausiello, Actin ¢laments regulate epithelial Na channel activity, American Physiological Society (1991) C882^C888. [8] W. Wang, A. Cassola, G. Giebisch, Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct, American Physiological Society (1994) F592^F598. [9] T.P. Stossel, From signal to pseudopod, J. Biol. Chem. 264 (1989) 18261^18264. [10] M. Ishii, H. Washioka, A. Tonosaki, T. Toyota, Regional orientation of actin ¢laments in the pericanalicular cytoplasm of rat hepatocytes, Gastroenterology 101 (1991) 1663^1672. [11] E. Lazarides, U. Lindberg, Actin is the naturally occurring inhibitor of deoxyribonuclease I, Proc. Natl. Acad. Sci. USA 71 (1974) 4742^4746. [12] S.E. Hitchcock, L. Carlsson, U. Lindberg, Depolymerization of F-actin by deoxyribonuclease I, Cell 7 (1976) 531^ 542.
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[13] H.G. Mannherz, J. Barrington Leigh, R. Leberman, H. Pfrang, A speci¢c 1:1 G-actin: DNase I complex formed by the action of DNase I on F-actin, FEBS Lett. 60 (1975) 34^38. [14] H.G. Mannherz, W. Kabsch, R. Leberman, Crystals of skeletal muscle actin: pancreatic DNase I complex, FEBS Lett. 73 (1977) 141^143. [15] J. Vandekerckhove, K. Weber, At least six di¡erent actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide, J. Mol. Biol. 126 (1978) 783^802. [16] J. Vandekerckhove, K. Weber, Mammalian cytoplasmic actins are the products of at least two genes and di¡er in primary structure in at least 25 identi¢ed positions from skeletal muscle actins, Proc. Natl. Acad. Sci. USA 75 (1978) 1106^1110. [17] J. Vandekerckhove, K. Weber, The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle and rabbit slow skeletal muscle, Di¡erentiation 142 (1979) 123^133. [18] P.A. Rubenstein, The functional importance of multiple actin isoforms, Bioessays 12 (1990) 309^315. [19] J. Vandekerckhove, The vertebrate isoactins: their characterization and possible functional di¡erences, in: International Meeting Actin '87, 1987, pp. 89^98. [20] K.M. McHugh, K. Crawford, J.L. Lessard, A comprehensive analysis of the developmental and tissue-speci¢c expression of the isoactin multigene family in the rat, Dev. Biol. 148 (1991) 442^458. [21] I.M. Herman, Actin isoforms, Curr. Opin. Cell Biol. 5 (1993) 48^55. [22] P. Sheterline, J. Clayton, J.C. Sparrow, Actin. Protein Pro¢le 1, Academic Press, London, 1994. [23] S.-T. Jiang, F.-J. Wang, C.-S. Chen, Properties of actin and stability of the actomyosin reconstituted from milk¢sh (Chanos chanos) actin and myosin, J. Agric. Food Chem. 37 (1989) 1232^1235. [24] K. Zechel, K. Weber, Actins from mammals, bird, ¢sh and slime mold characterized by isoelectric focusing in polyacrylamide gels, Eur. J. Biochem. 89 (1978) 105^112. [25] K. Uehara, M. Miyoshi, S. Miyoshi, Actin ¢laments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio, Cell Tissue Res. 261 (1990) 419^422. [26] R. Weiler, U. Janssen-Bienhold, Spinule-type neurite outgrowth from horizontal cells during light adaptation in the carp retina: an actin-dependent process, J. Neurocytol. 22 (1993) 129^139. [27] J.A. Spudich, J. Watt, The regulation of rabbit skeletal muscle contraction, J. Biol. Chem. 246 (1971) 4866^4871. [28] M. Malicka-Blaszkiewicz, DNase I-like activity and actin content in the liver of some vertebrates, Comp. Biochem. Physiol. 84B (1986) 207^209. [29] M. Malicka-Blaszkiewicz, Rat liver DNase I-like activity and its interaction with actin, Z. Naturforsch. 45c (1990) 1165^ 1170. [30] D.J. Gordon, E. Eisenberg, E.D. Korn, Characterization of
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L. Ciszak et al. / Biochimica et Biophysica Acta 1451 (1999) 141^152 cytoplasmic actin isolated from Acanthamoeba castellanii by new method, J. Biol. Chem. 251 (1976) 4778^4786. D.J. Gordon, J.L. Boyer, E.D. Korn, Comparative biochemistry of non-muscle actins, J. Biol. Chem. 252 (1977) 8300^ 8309. A. Weber, V.T. Nachmias, C.R. Pennise, M. Pring, D. Safer, Interaction of thymosin L 4 with muscle and platelet actin: implications for actin sequestration in resting platelets, Biochemistry 31 (1992) 6179^6185. K. Zechel, Isolation of polymerization-competent cytoplasmic actin by a¤nity chromatography on immobilized DNase I using formamide as eluant, Eur. J. Biochem. 110 (1980) 343^348. M. Malicka-Blaszkiewicz, J.S. Roth, Some factors a¡ecting the interaction between actin in leukemic L 1210 cells and DNase I, Biochem. Biophys. Res. Commun. 102 (1981) 594^ 601. M. Kunitz, Crystalline deoxyribonuclease. I. Isolation and general properties. Spectrophotometric method for the measurement of deoxyribonuclease activity, J. Gen. Physiol. 33 (1950) 349^362. M. Malicka-Blaszkiewicz, J.S. Roth, Evidence for the presence of DNase-actin complex in L 1210 leukemia cells, FEBS Lett. 153 (1983) 235^239. M. Malicka-Blaszkiewicz, Aktyna i DNaza w komo¨rkach eukarioto¨w, in: Acta Universitatis Wratislaviensis No. 1048 (monograph), Editorial O¤ce of University of Wroclaw, 1988. I.H. Segel, Biochemical Calculations, John Wiley and Sons, New York, 1975. T. Kouyama, K. Mihashi, Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin, Eur. J. Biochem. 114 (1981) 33^38. U. Laemmli, Cleavage of structural proteins during assembly of the head bacteriophage T 4, Nature 227 (1970) 680^685. W. Wray, T. Boulikas, V.P. Wray, R. Hancock, Silver staining of proteins in polyacrylamide gels, Anal. Biochem. 118 (1981) 197^203.
[42] P.H. O'Farrell, High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem. 250 (1975) 4007^4021. [43] H. Towbin, T. Staehelin, T. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350^4354. [44] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265^275. [45] J. Dieckho¡, H.G. Mannherz, The interaction of 5P-nucleotidase puri¢ed from chicken gizzard and actin, and the reversible loss of the inhibitory capacity of actin on deoxyribonuclease I, Biochim. Biophys. Acta 829 (1985) 209^220. [46] M. Malicka-Blaszkiewicz, I. Majcher, D. Nowak, DNase Ilike enzyme from the carp liver-inhibition by muscle and endogenous actin, Int. J. Biochem. 26 (1994) 1147^1155. [47] B. Jaberg, Preparation and characterization of actin from liver, Z. Naturforsch. 38c (1983) 829^833. [48] T. Miron, M. Wilchek, B. Geiger, Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle, Eur. J. Biochem. 178 (1988) 543^553. [49] K. Rouhnau, E. Schro«er, A. Wegner, Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle, Eur. J. Biochem. 170 (1988) 583^587. [50] T.E. Morgan, R.O. Lockerbie, L.S. Minamide, M.D. Browning, J.R. Bamburg, Isolation and characterization of a regulated form of actin depolymerizing factor, J. Cell Biol. 122 (1993) 623^633. [51] H.G. Mannherz, R.S. Goody, M. Konrad, E. Nowak, The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin, Eur. J. Biochem. 104 (1980) 367^379. [52] A. Krawczenko, L. Ciszak, M. Malicka-Blaszkiewicz, Does a natural DNase-actin complex exist in the carp liver cytosol?, Acta Biochim. Pol. 41/2 (1994) 113^116. [53] T.E. Bunton, Expression of actin and desmin in experimentally induced hepatic lesions and neoplasms from medaka (Oryzias latipes), Carcinogenesis 16 (1995) 1059^1063.
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Carp liver actin: isolation, polymerization and interaction with deoxyribonuclease I (DNase I) Lidia Ciszak 1;a , Agnieszka Krawczenko 1;a , Bernhard Polzar b , Hans Georg Mannherz b , Maria Malicka-Blaszkiewicz a; * a b
Department of Cell Pathology, Institute of Biochemistry and Molecular Biology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland Cytoskeletal Laboratory, Department of Anatomy and Embryology, Ruhr-University, Universita«tsstrasse 150, D-44780 Bochum, Germany Received 12 April 1999; received in revised form 3 June 1999; accepted 11 June 1999
Abstract 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 L-actin isoform as verified by immunoblotting using isotype specific antibodies. Its isoelectric point (pI) was slightly higher than the pI of rabbit skeletal muscle K-actin. Polymerization of purified carp liver actin by 2 mM MgCl2 or CaCl2 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 (Kb ) of carp liver actin to DNase I was calculated to be 1.85U108 M31 which is about 5-fold lower than the affinity of rabbit skeletal muscle K-actin to DNase I. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Actin; DNase I; Actin-DNase I interaction; Actin polymerization; Carp liver actin
1. Introduction Actin is one of the most abundant proteins in nature occurring in all eucaryotic cells [1^3]. In striated muscle cells, actin is the main constituent of the thin ¢laments. In non-muscle cells, actin is one of the three major components of the cytoskeleton and is
* Corresponding author. Fax: +48 (71) 3252930; E-mail: [email protected] 1 Present address: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland.
involved in many cellular functions including cell motility, contractile ring formation during cytokinesis and the maintenance of cell shape. Actin plays a very substantial role in organelle movement [4], secretion [5], neurotransmitter release [5,6], the regulation of the epithelial Na -K channel [7,8], and in signal transduction [9]. In hepatocytes actin ¢laments participate in the processes of bile formation like the transport of secretory vesicles, in exocytosis and contraction of the canalicular walls [10]. For all these functions of actin, a dynamic conversion between monomeric (G-actin) and ¢lamentous actin (F-actin) is necessary [1,2]. Intracellularly, actin polymerization is regulated by a large number of
0167-4889 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 8 9 ( 9 9 ) 0 0 0 8 3 - X
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actin-binding proteins [1,2]. Some of these proteins are able to stabilize monomeric actin by direct complex formation and thus sequester it from the polymeric pool. Deoxyribonuclease I from bovine pancreas (DNase I) has been shown to interact both with monomeric and with ¢lamentous actin [11^13]. Both proteins form a 1:1 complex with concomitant inhibition of the DNA degrading activity of DNase I and the ability of actin to form high molecular mass polymers [12^14]. The biological role of this interaction is still unclear. The study of the interaction between various DNase I-like enzymes and di¡erent actin isoforms might help to determine whether one of the many functions of actin is to control the degradative activity of DNase I-like enzymes towards cellular DNA. Due to di¡erences in sequence and isoelectric points, a number of actin isoforms have been distinguished [15^18], which in mammals are distributed in a tissue speci¢c manner: muscle (K-cardiac, K-skeletal, K-vascular and Q-smooth muscle actin) and nonmuscle (L- and Q-cytoplasmic) actins. In spite of the documented very high conservation among actin genes and their encoded proteins, recent results have indicated that there exist functional di¡erences among the actin isoforms [19^22]. Puri¢ed isoforms and actins isolated from di¡erent species di¡er in their thermodynamic stability, polymerization characteristics, binding of Ca2 ions and ATP and their a¤nity for particular actin-binding proteins like for instance DNase I [19,22]. Little information is available on the actins occurring in tissues and organs of the lower vertebrates. Particularly, information on the properties and stability of actin in ¢sh cells is lacking. Several investigators have puri¢ed actin from the back muscle of the milk¢sh [23] and from the liver and back muscle of the electric ¢sh [24]. Electron microscopic and immuno£uorescence studies have allowed to localize actin micro¢laments in microridges of the oral mucosal epithelium of carp [25] and in horizontal cells in carp retina [26]. In contrast, detailed information on the actin content in ¢sh liver, its stability and ability to interact with other actin binding proteins like DNase I is not available. Therefore, the aim of this study was to isolate and characterize actin from the carp liver. Special attention was given to its ability to interact with bovine pancreatic DNase I.
2. Materials and methods 2.1. Isolation procedures for actins Rabbit skeletal muscle K-actin was puri¢ed from acetone powder according to Spudich and Watt [27]. To purify ¢sh liver actin, we used carp (Cyprinus carpio). Fresh livers were homogenized in 3 vols. of freshly made 10 mM Tris-HCl bu¡er, pH 7.4, containing 0.1 mM ATP, 1 mM DTT, 0.1 mM CaCl2 and 0.25 M sucrose (bu¡er A), as described previously by Malicka-Blaszkiewicz [28,29], except that 0.1 mM PMSF, 0.5 mg/1 aprotinin and 1 mg/1 leupeptin were added to the homogenization bu¡er. Homogenates were centrifuged at 105 000Ug for 1 h at 4³C. The supernatant served as the cytosolic fraction and was used immediately or kept frozen at 370³C for further analysis. The cytosol sample was dialyzed for 24 h against bu¡er A without sucrose containing 0.1 mM PMSF before it was loaded on a DEAE-cellulose column (2.5U36 cm) according to Gordon et al. [30]. Proteins adsorbed were eluted from the column with increasing concentration of NaCl (linear gradient from 0.1 to 0.5 M) in bu¡er A without sucrose. Actin containing fractions were identi¢ed by SDS-PAGE, pooled, concentrated by pressure dialysis and loaded on a Sephacryl S-300 column (1.6U92 cm) equilibrated with bu¡er A without sucrose. Proteins were eluted with the same bu¡er. All operations were done at 4³C. Finally a cycle of polymerization and depolymerization was employed to further purify the actin obtained by gel ¢ltration [30,31]. To this aim, the pooled fractions obtained after Sephacryl S-300 were pressure concentrated to 2 ml in the cold (using Amicon with PM 10 membrane) and incubated for 2 h at room temperature precipitated with an equal volume of 2 M potassium phosphate bu¡er, pH 7.6, containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT, and then centrifuged for 30 min at 20 000Ug at 15³C [32]. The supernatant was discarded, the loose precipitate was suspended in 2 ml of polymerizing bu¡er (5 mM Tris-HCl, pH 7.6, containing 0.5 mM ATP, 2 mM MgCl2 , 0.5 mM DTT, 0.2 mM CaCl2 , 10 WM EDTA) and centrifuged for 2 h at 100 000Ug at 15³C. For further experiments, the actin pellet was homogenized in bu¡er A without su-
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crose and the F-actin was depolymerized by dialysis against the same bu¡er for 24 h at 4³C. 2.1.1. A¤nity chromatography on DNase I agarose Alternatively, we performed DNase I a¤nity chromatography essentially as described by Zechel [33]. DNase I was immobilized on vinylsulfone-agarose (Mini Leak, Kem-En-Tec, Copenhagen, Denmark) following the instructions of the supplier. DNase Iagarose was mixed gently overnight at 4³C with the actin samples using a rotary shaker and then centrifuged at 15 000 rpm for 15 min in order to remove unbound protein. DNase I-agarose with adsorbed proteins was poured into a column, which was washed successively with bu¡ers according to the procedure of Zechel [33]. The proteins strongly adsorbed to DNase I-agarose were eluted with bu¡er A containing 40% formamide plus 20% glycerol in order to minimize the denaturation of actin. The fractions collected were immediately diluted 1/2 with bu¡er G (2 mM Tris-HCl, pH 7.9, 0.2 mM CaCl2 , 0.2 mM ATP, 0.2 mM DTE). The actin containing fractions were combined and concentrated by pressure ¢ltration in an Amicon cell using a PM 10 membrane. The formamide was removed by repeated addition of bu¡er G. All operations were done at 4³C. 2.2. Enzymatic tests DNase I activity was determined spectrophotometrically as described earlier by Malicka-Blaszkiewicz and Roth [34]. Highly polymerized calf thymus DNA was used in all experiments as substrate. In some experiments DNase I activity was measured by the hyperchromicity assay of Kunitz [35] at 260 nm and room temperature. 0.2 Wg DNase I was added to the cuvette containing 10 mM Tris-HCl, pH 8.0, 1 mM MgCl2 , 0.1 mM CaCl2 , 1 mM NaN3 and 50 Wg DNA. Activity was expressed in Kunitz units; one Kunitz unit corresponds to an increase in absorbance of 0.001 min31 at 260 nm. The actin content was measured as inhibitor of bovine pancreatic DNase I (Sigma) under standard DNase assay conditions [29,34,36,37] and expressed as units of DNase I inhibitor per mg of protein. One unit of actin was de¢ned as the amount which decreases the activity of 20 ng of DNase I by 10%. In the part of experiments the actin content was
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measured as an inhibitor of DNase I under Kunitz assay conditions [35]. Various actin concentrations were incubated with 0.2 Wg DNase I for 15 min at room temperature and then transferred to ice to stop the reaction. 2.3. Determination of the binding constant of carp liver actin to bovine DNase I The binding constant of carp liver actin to bovine DNase I was determined according to Dixon [38]. DNase I (at a constant concentration) and increasing concentrations of actin were added sequentially to cuvettes containing 40 mM HEPES-OH, pH 7.0, 5 mM MgCl2 , 0.1 mM CaCl2 and incubated for 30 s at room temperature. The reaction was started by addition of substrate (calf thymus DNA, Sigma). The initial velocity of the reaction was measured for 200 s at 260 nm. Both enzyme and substrate concentrations were selected such that the increase in reaction products was directly proportional to the amount of inhibitor (actin). The measurements were performed in the presence of two substrate concentrations: 30 Wg and 70 Wg DNA. The dissociation constant Kd and the binding constant Kb were calculated from double-reciprocal plots of the dependence of the enzymatic activity on the inhibitor concentration (1/v against actin concentration [I]). The measurements were performed on a diode spectrophotometer HP 8452 A (Hewlett-Packard, Palo Alto, CA, USA). 2.4. Fluorescence measurements Polymerization of actin from the carp liver cytosol was determined by the £uorescence increase of pyrene-labeled K-actin. Carp liver actin was mixed with 1.4 WM pyrene-labeled rabbit skeletal muscle actin [39] at room temperature under di¡erent bu¡er conditions. The £uorescence intensity was measured at 407 nm after excitation at 365 nm at room temperature on a spectro£uorometer Labmath Shimadzu RF-5001 PC V2.00. The identical procedure was used to examine the e¡ect of bovine DNase I on the carp liver polymerization. Carp liver actin was mixed with 1.4 WM pyrenyl-actin and incubated for 15 min at room temperature with bovine pancreatic DNase I at various
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molar ratios. Filament elongation was started by addition of 1 M potassium phosphate bu¡er, pH 7.6, containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT. The control sample did not contain DNase I. 2.5. Electrophoretic procedures and immunoblotting SDS-PAGE was performed according to Laemmli [40] in 10% gels, at 30 mA per slab, using 0.1% SDS in electrode bu¡ers. Gels were stained with Coomassie brilliant blue R-250 or the silver method [41]. Isoelectric focusing was performed essentially as described by O'Farrell [42] in cylindrical gels cast into glass tubes (75 mmU1.5 mm) in the presence of 9.5 M urea, 2% Nonidet P40, 3.3% acrylamide and 2% ampholine mixture (pH 3^5/5^7). The gels were prefocused for 30 min at 4³C at 250 V before the proteins samples were layered onto the cathodic end of the gels. At the end of the isoelectric focusing, the gels were extruded from the tubes and washed for 10 min in 0.125 M Tris-HCl bu¡er, pH 6.8, containing 10% glycerol, 4.9 mM DTT and 2% SDS in order to remove the Ampholines. The gels were subsequently used for protein separation by SDS-PAGE or kept frozen at 370³C for further analysis. pI values of actin from the carp liver cytosol was calculated based on the comparison with isoelectric focusing of muscle K-actin. The transfer of proteins to nitrocellulose sheets was achieved according to Towbin et al. [43]. For immunoblotting we used a monoclonal antibody (Sigma) directed against L-cytoplasmic actin. Immunoreactivity was shown by the avidin-biotin peroxidase (Sigma) technique, using 4-chloro-1-naphthol as substrate. The presence of actin was also veri¢ed by using a goat antiserum directed against skeletal muscle K-actin. Immunoreactivity was detected as described for the monoclonal antibodies. 2.6. Determination of protein concentration Protein concentrations were determined either by following the procedure given by Lowry et al. [44] or spectrophotometrically at 280 nm or 290 nm (when ATP was present in the used bu¡er). The concentration of carp liver actin was calculated using A1% 290nm = 6.3 [45].
3. Results 3.1. Isolation of actin from carp liver cytosol The procedure developed by Gordon et al. [30,31] to purify actin from non-muscle cells was also applied to isolate actin from carp liver. Fractions of each puri¢cation step were analyzed by SDS-PAGE and for actin content measured as an inhibitor of DNase I. 3.1.1. Ion exchange chromatography The cytosol of carp liver was loaded on a DEAEcellulose column and eluted as described in Section 2 (Fig. 1a). DNase I inhibitory activity eluted in the two peaks (peak I and peak II) at 0.19 and 0.26 M NaCl probably due to the presence of complexes of actin with actin binding proteins. As shown in Fig. 1a, the fractions of the peak II revealed higher inhibitory activity than the fractions of peak I. Therefore the fractions of peak II were used for further actin puri¢cation. 3.1.2. Gel ¢ltration The combined fractions of peak II were concentrated by dialysis against sucrose and chromatographed on Sephacryl S-300 using the conditions described in Section 2. The elution pro¢le is shown in Fig. 1b. The proteins were separated into two distinct peaks, whereas DNase I inhibitory activity was eluted in one symmetrical peak within the descending arm of the ¢rst protein peak. The fractions with highest inhibitory activity (59 to 68) were collected and subjected to further analysis. SDS-PAGE analysis of the collected fractions showed the presence of only a few protein bands with a predominance of two bands: one with a mobility equal to skeletal muscle K-actin and another one with a mobility corresponding to Mr of 21 kDa (compare Fig. 2, lanes 4, 5 and 7). 3.1.3. Polymerization of actin In order to further purify the actin present in the fractions obtained after Sephacryl S-300 ¢ltration, the pooled material (fractions 59^68, Fig. 1b), was subjected to a polymerization-depolymerization cycle according to Gordon et al. [30,31]. However, no pellet was seen under applied conditions after centrifu-
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Fig. 1. Puri¢cation of carp liver actin. (a) Ion exchange chromatography. DEAE-cellulose column (2.5U36 cm) was equilibrated with bu¡er A without sucrose containing 0.1 M NaCl. Elution was carried out with the same bu¡er and then with linear NaCl concentration gradient (0.1^0.5 M). 4 ml fractions were collected at a £ow rate 60 ml/h. 1 g of protein was applied to the column. +, absorption at 290 nm; a, inhibition capacity of DNase I. (b) Gel ¢ltration. Sephacryl S-300 column (1.6U92 cm) was equilibrated with bu¡er A without sucrose and elution was carried out with the same bu¡er. 65 mg of protein of the pooled fractions of peak II was applied to the column. 2 ml fractions were collected at a £ow rate 10 ml/h. +, absorption at 290 nm ; a, inhibition capacity of DNase I.
gation at 100 000Ug. Therefore we concluded that carp liver actin had not polymerized after addition of 2 mM MgCl2 and 0.1 M KCl to bu¡er A without sucrose. In order to dissociate possible complexes of actin with actin binding proteins (AbPs), which might have inhibited its polymerization, we applied a procedure described by Weber et al. [32], i.e., we incubated the pooled fractions in 2 M potassium bu¡er, pH 7.6, containing 4 mM ATP, 2 mM MgCl2 and 2 mM DTT to induce polymerization of the carp liver actin. After centrifugation only one protein band was seen after SDS-PAGE of equal mobility to K-actin (Fig. 2, lanes 5 and 6). 3.1.4. A¤nity chromatography on DNase I agarose Alternatively, a¤nity chromatography using agarose immobilized DNase I was employed to purify carp liver cytosolic actin. For this purpose we used the pooled fractions collected from peak I and peak
Fig. 2. Electrophoretic analysis of samples obtained during puri¢cation of carp liver actin. Slab SDS-PAGE was performed according to Laemmli [40] on 10% gels. The proteins were stained with Coomassie brilliant blue. Lanes: 1, carp liver cytosol, 55 Wg; 2, pooled fractions of peak I (147^156, Fig. 1a), 80 Wg; 3, pooled fractions of peak II (169^184, Fig. 1a), 75 Wg; 4, pooled fractions (59^68, Fig. 1b), 70 Wg; 5, standard K-rabbit skeletal muscle actin, 10 Wg; 6, actin puri¢ed from the carp liver, 15 Wg; 7, marker proteins, 10 Wg.
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Using DNase I a¤nity chromatography we obtained two actin preparations: preparation A (obtained from peak I) and preparation B (from peak II). SDS-PAGE analysis of the preparation A showed the presence of only one protein band with a mobility identical to skeletal muscle K-actin, whereas in the preparation B a few additional protein bands were seen (Fig. 3a, lanes 1^3). For further analysis we used the actin obtained from preparation A.
Fig. 3. Electrophoretic analysis and immunoblotting of the actin preparations obtained after a¤nity chromatography on DNase I agarose. Slab SDS-PAGE was performed in the same conditions as in Fig. 2. (a) The proteins were stained with Coomassie brilliant blue. (b) Immunoblotting. Murine monoclonal antibodies directed against L-cytoplasmic actin were used. Lanes; 1, 1P, preparation A, 8 Wg; 2, 2P, preparation B, 4.5 Wg; 3, 3P, standard K-rabbit skeletal muscle actin, 7 Wg; 4, 4P, marker proteins, 10 Wg.
II of the DEAE-cellulose column (see Fig. 1a). The elution pro¢le was analyzed for DNase I inhibitor activity measured in the DNase assay conditions according to Kunitz [35]. The actin present in peak I as well as in peak II was strongly adsorbed on DNase Iagarose: DNase I inhibitory activity was only associated with the fractions eluted with 40% formamide.
Fig. 4. Two-dimensional PAGE. Isoelectric focusing according to O'Farrell [42], SDS-PAGE in 10% gels according to Laemmli [40]. (a) Puri¢ed carp liver actin, 10 Wg (stained with silver method, [41]); (b) standard K-rabbit skeletal muscle actin, 7 Wg (stained with Coomassie brilliant blue).
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3.2. Characterization of carp liver actin 3.2.1. Two-dimensional PAGE Two-dimensional PAGE was applied to examine the homogeneity, isoform composition and isoelectric point (pI) of carp liver actin. The results obtained were compared with rabbit skeletal muscle K-actin. No di¡erences were found in the protein pattern after two-dimensional PAGE of the actin obtained after the polymerization-depolymerization cycle and for both preparations puri¢ed by DNase I-agarose a¤nity chromatography. Only one protein spot was seen after two-dimensional PAGE with a pI slightly higher than the pI of skeletal muscle K-actin, i.e., approx. 5.4 (compare Fig. 4a and b). 3.2.2. Immunoblotting using isoform speci¢c antibodies The 2D gel electrophoresis indicated the presence of only one actin variant in carp liver. Therefore the identity of the puri¢ed band with actin was veri¢ed by immunoblotting using polyclonal antibodies directed against K-actin (Fig. 5a) and a monoclonal antibody directed against cytoplasmic L-actin (Fig. 5b). As can be seen, carp liver actin interacted weakly with the polyclonal antibodies directed against K-actin, but strongly with the monoclonal antibody against cytoplasmic L-actin (compare Fig. 5a, lane 2 and Fig. 5b, lane 2P). In contrast, skeletal
Fig. 5. Immunoblotting of carp liver actin; comparison with Kactin. The proteins were separated in SDS-PAGE [40] in 10% gels, transferred to nitrocellulose sheets [43] and incubated with the antibodies. Goat antiserum directed against rabbit skeletal muscle K-actin (a) and murine monoclonal antibodies directed against L-cytoplasmic actin (b) were used. Lanes: 1, 1P, standard K-rabbit skeletal muscle actin, 7 Wg; 2, 2P, actin from the carp liver, 15 Wg.
Fig. 6. E¡ect of bu¡er composition on polymerization of carp liver actin. Actin (3.25 WM, preparation A from a¤nity chromatography on DNase I-agarose) was mixed with 1.4U1036 M of K-actin conjugated with N-(1-pyrenyl)iodoacetamide. Fluorescence intensity was measured at 407 nm after excitation at 365 nm at room temperature. The following bu¡ers were used to start the ¢lament elongation: E, bu¡er G+2 mM MgCl2 + phalloidin (5 WM); O, bu¡er G+2 mM CaCl2 ; a, bu¡er G+2 mM MgCl2 ; +, 1 M potassium phosphate bu¡er pH 7.6 containing 4 mM ATP; 2 mM MgCl2 ; 2 mM DTT.
muscle actin was only recognized by the antibodies directed against K-actin (compare Fig. 5a, lane 1 and Fig. 5b, lane 1P). This result was obtained irrespective of the ¢nal puri¢cation procedure (Fig. 3b, lane 1P^ 3P). Based on this comparison we concluded that the puri¢ed carp liver actin is a cytoplasmic L-actin variant. 3.2.3. Fluorescence measurements of carp liver actin polymerization Optimal conditions for carp liver actin polymerization were examined spectro£uorometrically after addition of 4% of pyrene-labeled K-actin. First, we measured the £uorescence increase obtained at room temperature in the presence of bu¡er G (2 mM TrisHCl, pH 7.9, 0.2 mM CaCl2 , 0.2 mM ATP, 0.2 mM DTE) supplemented by either 2 mM MgCl2 or 2 mM CaCl2 . As can be seen from Fig. 6, carp liver actin did not polymerize under these ionic conditions. Only after addition of phalloidin and 2 mM MgCl2 we observed an increase in £uorescence intensity. However, we found that carp liver actin polymerized in the presence of 1 M potassium phosphate bu¡er,
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Fig. 7. Inhibition of the standard DNase I from bovine pancreas by carp liver actin. (a) Actin was puri¢ed by polymerization-depolymerization cycle. Inhibition was performed in standard assay conditions [34], in the presence of 0.75 nM DNase I. (b) Actin puri¢ed by a¤nity chromatography on DNase I-agarose was used. Inhibition was carried out in the DNase assay conditions of Kunitz [35], in the presence of 6.2 nM DNase I.
pH 7.6, containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT [32]. The formation of regular micro¢laments by puri¢ed carp liver actin under these conditions was observed in electron microscopy (data not shown).
that about 80 nM carp liver actin was required for decrease the DNase I activity by 50% (at a molar ratio of actin to DNase I of about 13, see Fig. 7b). Thus both actin preparations exhibited a similar abil-
3.3. Interaction of carp liver actin with bovine pancreatic DNase I 3.3.1. Inhibition of DNase I by carp liver actin Actins isolated from non-muscle cells di¡er in their a¤nity to DNase I [22]. We examined the inhibitory e¡ect of carp liver actin on puri¢ed bovine pancreatic DNase I using two di¡erent actin preparations: (i) the actin obtained after the polymerization-depolymerization cycle and (ii) the actin obtained by a¤nity chromatography on DNase I-agarose (preparation A). In the case of actin obtained by polymerization and depolymerization cycle DNase I inhibition was measured under standard assay conditions [29,34,36]. As shown in Fig. 7a 12 nM actin was required to decrease the activity of 0.75 nM DNase I by 50% (molar ratio of 16). When using the actin obtained after a¤nity chromatography on DNase I-agarose (preparation A), the inhibitory activity was measured immediately after removal of the formamide by ultra¢ltration (see Section 2). The inhibition was measured using assay conditions according to Kunitz [35]. We found
Fig. 8. E¡ect of DNase I on polymerization of actin from carp liver cytosol. Carp liver actin was mixed with 1.4U1036 M pyrene-labeled K-actin and incubated for 15 min at room temperature with standard DNase I in various molar ratio of actin to DNase I. Filament elongation was started by simultaneous addition of 1 M potassium phosphate bu¡er pH 7.6 containing 4 mM ATP, 2 mM MgCl2 , 2 mM DTT. Fluorescence intensity was measured at 407 nm after excitation at 365 nm at room temperature. E, actin without DNase I (3.25U1036 M); O, actin plus DNase I mixed in molar ratio of actin to DNase I 18:1; a, molar ratio of actin to DNase I 8:1; b, molar ratio of actin to DNase I 3:1.
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Fig. 9. Determination of the binding constant of bovine pancreas DNase I to carp liver actin. (a) Inhibition of DNase I by actin. DNase I was added to the test solution (see Section 2) to give a ¢nal concentration of 3.8U1039 M, mixed with increasing concentrations of inhibitor (carp liver actin) and incubated for 30 s. Reaction was started by addition of 30 Wg DNA and carried out at room temperature. Identical measurements were done for 70 Wg DNA (data not shown). a, DNase I without actin; O, DNase I plus 2.1U1039 M actin; b, DNase I plus 3.3U1039 M actin; +, DNase I plus 6.6U1039 M actin; E, DNase I plus 8.7U1039 M actin. (b) Dixon plots analysis of the dependence of DNase I activity on actin concentration. The actin concentration (I0 ) was calculated usa, data obtained in the presence of 70 Wg DNA. ing A1% 290nm = 6.3 [45]. b, data obtained in the presence of 30 Wg DNA;
ity to inhibit DNase I activity. These results con¢rmed previous observations [46] which had demonstrated that actin from carp liver cytosol is able to inhibit bovine DNase I. 3.3.2. E¡ect of DNase I on the polymerization of carp liver actin The e¡ect of DNase I on the ability of carp liver actin to polymerize was analyzed by the changes of the £uorescence intensity in the presence of pyrenelabeled K-actin. Actin (preparation A obtained after a¤nity chromatography on DNase I-agarose) was incubated with bovine DNase I in the molar ratios of actin to DNase I of 18:1; 8:1 and 3:1. A pronounced inhibitory e¡ect of DNase I on carp liver actin polymerization was observed (Fig. 8). Filament elongation was completely inhibited, when the molar ratio of actin to DNase I was 3:1. These results indicated that actin from carp liver cytosol interacts with bovine pancreatic DNase I leading to the formation of a stable complex with concomitant inhibition of DNA degrading activity of DNase I and ability of carp liver actin to polymerize. 3.3.3. Determination of the binding constant of bovine DNase I to actin The actin dependent DNase I inhibition was em-
ployed for computation of the binding constant according to Dixon (see Section 2). Actin puri¢ed by polymerization-depolymerization was used in these experiments. From the data given in Fig. 9a double-reciprocal plots were constructed relating the dependence of the DNase I activity on inhibitor concentration (1/v against actin concentration [I]) in the presence of 30 Wg and 70 Wg DNA, Fig. 9b. It can be seen that straight lines were obtained, which intercepted at a common point above the negative [I] axis. The dissociation constant Kd was estimated to be 5.4U1039 M resulting in a Kb of carp liver L-actin to bovine DNase I of 1.85U108 M31 . 4. Discussion Here we describe the puri¢cation of actin from the carp liver cytosol. The isolated actin was used to examine some of its properties. The puri¢cation procedure given by Gordon et al. [30,31] was particularly useful to isolate actin from these cells. By this procedure actin was obtained in two peaks after ion exchange chromatography most probably being due to the occurrence of actin in complexes with other cytosolic AbPs. After the subsequent gel ¢ltration on Sephacryl S-300 and a cycle of de- and polymeriza-
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tion a highly pure actin preparation was obtained (see also the SDS-PAGE of Fig. 2, lanes 1 and 4). The obtained actin preparation was solely composed of the cytoplasmic L-isoform indicating that carp liver contains this L-variant as only actin isoform. Our results are in agreement with data from Zechel and Weber [24] demonstrating the presence of only L-actin in the liver of the electric ¢sh Torpedo marmorata. The isoelectric points of both ¢sh liver L-actins were slightly higher than of skeletal muscle K-actin. In contrast, mammalian livers were shown to contain both L- and Q-cytoplasmic actins [31,47]. We have con¢rmed the presence of L-actin in the carp hepatocytes also by immunogold labeling at the ultrastructural level. It was localized in the cytoplasm and nuclei (data not shown). Although the exact amino acid composition of carp liver actin has not yet been determined, ¢sh liver will represent a reliable source for future studies of the isotype speci¢c properties of cytoplasmic L-actin. Polymerization of puri¢ed carp liver actin was only induced in the presence of the high ionic strength phosphate bu¡er as described by Weber et al. [32]. We did not obtain its polymerization under standard ionic conditions (like in 10 mM Tris-HCl, pH 7.4, 0.1 mM ATP, 1 mM DTT, 0.1 mM CaCl2 , 2 mM MgCl2 and 100 mM KCl) except when phalloidin was present indicating its ability to bind this drug. It is probable that the occurrence of AbPs might have inhibited its polymerization. The inhibitor may be the observed 21 kDa protein, whose presence was shown by SDS-PAGE (see Fig. 2, lane 4). Miron et al. [48] have reported the presence of an inhibitor of Mr = 25 kDa in many chicken tissues including liver. Rouhnau and coworkers [49] puri¢ed a polymerization inhibiting protein of 21 kDa from chicken gizzard smooth muscle. It is also probable that actin depolymerizing or sequestering proteins were present in the preparation obtained after gel ¢ltration, such as co¢lin (Mr = 19 kDa) or actin depolymerizing factor (Mr = 18.5 kDa) [22,50]. A¤nity chromatography on DNase I-agarose was also applied for further actin puri¢cation using both peaks obtained after ion-exchange chromatography on DEAE-cellulose. Actin present in both peaks strongly adsorbed onto DNase I-agarose from which it was eluted using a bu¡er containing 40% form-
amide and 20% glycerol. We obtained two actin preparations, A and B from the protein peaks I and II, respectively, initially separated by ion-exchange chromatography (see Fig. 1a). SDS-PAGE analysis showed that in preparation B a few additional bands occurred (see Fig. 3a, lane 2). It is interesting that these higher Mr bands (corresponding to Mr from 80 kDa to 130 kDa) interact similarly as the 43 kDa band (actin) with the monoclonal antibodies directed against L-cytoplasmic actin (see Fig. 3b, lane 2P). We have shown that puri¢ed carp liver actin inhibited DNase I to 50% when added in a 13^16-fold molar excess. A molar binding ratio of skeletal muscle K-actin to bovine pancreatic DNase I of 1:1 was reported earlier by Mannherz et al. [51]. The discrepancy may have been due to partial denaturation of carp liver actin during elution from DNase Iagarose or an intrinsic property of this cytoplasmic actin. Indeed, the ability of cytoplasmic actins from di¡erent sources to inhibit DNase I was reported to be very labile and variable [29,34]. The estimated the binding constant of carp liver actin to DNase I (Kb ) was calculated to be 1.85U108 M31 . This is the ¢rst determination of the binding constant of cytoplasmic L-actin to bovine pancreatic DNase I. Previously, the binding constant of K-rabbit skeletal muscle actin to DNase I was shown to range from 5 to 10U108 M31 [51] indicating an approx. 5-fold lower a¤nity of carp liver actin to bovine pancreatic DNase I. We have suggested previously that one of the many functions of non-muscle actin may be to inhibit intracellular DNase I-like activities [28,29,36,37, 46]. Here we demonstrated that carp liver L-actin is indeed able to inhibit bovine pancreatic DNase I. In parallel experiments we have isolated a DNase I-like activity from carp liver cytosol (data not shown). The enzymatic activity of the endogenous carp liver DNase I-like enzyme was inhibited by both K-rabbit skeletal muscle and carp liver actin (to be published). The presence of natural complex of carp liver actin with carp liver DNase was suggested by Krawczenko et al. [52]. Experiments using DNase I-like enzymes and its inhibitor (actin) from the same source may help to understand the role of this interaction in biological systems. In this respect carp liver may be an advantageous system, since it contains high concentrations of both proteins. Such studies may also be of value in understanding some clinical aspects of liver
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pathology like hepatic ¢brosis [53] where actin may serve as functional markers. Acknowledgements The studies were supported by a research grant from the University of Wroclaw (Wroclaw, Poland). The ¢nancial support from the Deutsche Akademischer Austauschdienst (DAAD, Bonn, Germany) and the Deutsche Forschungsgemeinschaft (Bonn, Germany) is gratefully acknowledged.
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