Effect of α-actinin on actin structure viscosity studies

Biochimica et Biophysica Acta, 669 ( 1981) 1-6

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Elsevier/North-Holland Biomedical Press BBA 38677 EFFECT OF ot-ACTININ ON ACTIN STRUCTURE VISCOSITY STUDIES * INDERJIT SINGH a,**, DARREL E. GOLL b, ***, RICHARD M. ROBSON a and M.H. STROMER a a Muscle Biology Group, Departments of Animal Science, Biochemistry and Biophysics and Food Technology, cooperating, Iowa State University, Ames, 1.4 50011, and b Muscle Biology Group, University of Arizona, Tucson, AZ 85721 (U.S.A.)

(Received December 22nd, 1980) Key words: a-Actinin; F-actin; A TP; Viscosity

The effect of ATP on ability of a-actinin to increase viscosity of F-actin was measured in three different solutions: 100 mM KCI; 100 mM KCI/I mM Mg2+; and Mg2+ alone at concentrations of 1 - 6 mM. When ATP and Mg2÷ are added at equimolar ratios or at added [ATP] to added [Mg 2+ ] greater than equimolar, a-actinin has no effect on F-actin viscosity in the absence of KCI. ATP decreases viscosity of a-actinin/F-aetin mixtures by 20% even in the presence of KCI, evidently because ATP affects the a-actinin-F-actin interaction. Molar ratios of 1 a-actinin to 49 a~tins increase specific viscosity of F-actin approx. 2-fold at 37°C in the presence of 1 mM ATP, so ATP does not prevent the a-actinin-F-actin interaction.

Introduction The physiological function of a-actinin remains unclear even though highly purified a-actinin can be obtained in reasonable quantities [ 1 4 ] and the a-actinin molecule has been thoroughly characterized [4]. It has been shown that a-actinin binds only to F-actin among the known muscle proteins [5-8] and that a-actinin causes gelation of F-actin solutions [5]. This gelation may be related to the ostensible ability of a-actinin to cross-link actin filaments. The locations of actin and a-actinin in various nonmuscle cells have also led to the suggestion that a-actinin acts to bind actin filaments to the plasma membrane [9-16]. a-Actinin was discovered in skeletal muscle extracts because it increased the rate of ATP-induced turbidity response [ 17-21 ] and the Mg2÷-modified ATPase * Journal Paper No. J-9744 of the Iowa Agriculture and Home Economics Experiment Station, Projects 2025, 2127 and 2361. ** Present address: The John F. Kennedy Institute, Johns Hopkins Medical School, 707 N. Broadway, Baltimore, MD 21205, U.S.A. *** To whom correspondence should be addressed.

activity [1,2, 2 2 - 2 4 ] of reconstituted actomyosin suspensions. The physiological significance of a-actinin's ability to alter these two in vitro measures of contraction has not been studied extensively. Because a-aetinin seems to bind only to actin, it must exert its effects on aclomyosin through the actin component of this complex. Therefore, we have studied the effect of a-actinin on the ATPase activity (Singh, I., Goll, D.E. and Robson, R.M., unpublished results) and rate of exchange of bound ADP of F-actin (CraigSchmidt, M., Robson, R.M., GoU, D.E. and Stromer, M.H., unpublished results) to determine whether a-actinin causes measurable changes in these two indicators of actin structure. Because ATP itself seems to be involved in regulating polymerization of actin into filaments [25-28] and because a-actinin also affects rate of formation of actin filaments, it was necessary first to ascertain whether ATP would affect binding of a-actinin to actin filaments. Hence, we also have studied the effects of ATP on ability of a.actinin to increase the viscosity of F-actin solutions. The results reported in this paper show that, although ATP decreases viscosity of a-actinin/F-actin mixtures slightly, ATP does not prevent the a-actinin-F-actin interaction.

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Materials and Methods Actin was made from porcine semitendinosus, biceps femoris, and semimembranosus muscles as described previously [1] for rabbit skeletal muscle, except that the initial polymerization was done in 0.5 M KC1 to remove tropomyosin [29]. a-Actinin was extracted from porcine skeletal muscle and was purified using DEAE-cellulose and hydroxyapatite chromatography [4]. a-Actinin also was made from frozen bovine hearts obtained from Pel-Freez Biochemicals, Inc., Rogers, AR. The hearts were thawed just before use by placing them in a 2°C room for 24h, and cardiac a-actinin was extracted and purified as described previously [20,30]. Although skeletal and cardiac a-actinins differ slightly in amino acid composition [20] no differences in ability to increase F-actin viscosity and ATPase activity or in ability to increase the release of bound nucleotide from actin were found between porcine skeletal and bovine cardiac a-actinins in our studies (Singh, I., Goll, D.E. and Robson, R.M. and Craig-Schmidt, M., Robson, R.M., Goll, D.E. and Stromer, M.H., unpublished results). All protein preparations were routinely monitored for purity by using sodium dodecyl sulfate polyacrylamide gel electrophoresis [4,31 ]. Protein concentrations were determined by the biuret method [32,33]. Viscosity measurements were made with Cannon viscometers having flow times of 175-185 s for water at 37°C. At the protein concentrations used in this study, density differences between protein solutions and their solvents were negligible, especially when compared with the difficulties inherent in measuring viscosity of highly thixotropic substances such as F-actin. Therefore, no corrections were made for these small density differences. Specific viscosities were calculated as described earlier [7] with no kinetic energy corrections. Water bath temperatures were controlled to within 0.05°C. In all experiments, unpolymerized actin (G-actin) was added to the viscometer, followed sequentially by a-actinin (when added), ATP (when added), and 0.1 M Tris • acetate, pH 7.5. These ingredients were mixed in the viscometer. G-actin was then polymerized to F-actin by addition of KC1 or MgC12 or both. Flow times were recorded immediately and repeatedly to estimate changes due to the thixotropic nature of the F-actin

mixtures. Changes due to thixotropy were small compared with the differences in specific viscosity reported in this paper. All experiments were done in 20 mM Tris • acetate, pH 7.5, at 37°C with different amounts of KC1, Mg2÷, or ATP. Disodium ATP was obtained from Sigma Chemical Co. and was treated with Dowex 50 ion-exchange resin in the hydrogen form, followed by neutralization with solid Tris to convert it to the Tris salt. Results As has been reported previously [3,7,34], a-actinin at 37°C causes a 2-3.fold increase in specific viscosity of F-actin (Table I). Adding 1 mM ATP to the G-actin before formation of F-actin has no effect on viscosity of the resulting F-actin solution if polymerization is initiated by adding KC1 to a final concentration of 100 mM (Table I). If, however, polymerization is initiated by adding Mg2+ to a final concentration of 1 mM, viscosity of the resulting F-actin solution is decreased by approx. 14% (Table I). This small decrease in specific viscosity probably results from binding of some of the added Mg2÷ by ATP (ATP has an apparent affinity constant, Kb, of l0 s 10 6 M -1 for Mg2÷, depending on ionic conditions, pH, etc.). Strzelecka-Golaszewska et al. [35] have shown that actin molecules contain two classes of binding sites: a single site for binding divalent cations with an apparent affinity constant of approx, l0 s M-t, and five sites that will bind either monovalent or divalent cations with apparent affinity constants on the order of ( 5 - 6 ) • 103 M-1 for the divalent cations. Because only the second class of lower affinity sites seems to be involved in polymerization .of monomeric actin into filamentous actin [35], ATP competes very favorably with actin for the Mg2÷ needed for such polymerization. Hence, adding ATP to G-actin solutions containing added Mg2÷ with no added KCI reduces the amount of Mg2÷ available to actin for polymerization and would shift the equilibrium among G-actin monomers and F-actin filaments [ 3 6 38] in the direction of unpolymerized G-actin. Although the free Mg2÷ concentration would be very low in the presence of ATP, the presence in all solutions of 20 mM Tris, which is mostly cationic at pH 7.5, is sufficient to cause G-actin to polymerize even in the presence of added ATP.

TABLE I EFFECT OF a-ACTININAND ATP ON VISCOSITYOF F-ACTINPOLYMERIZEDBY DIFFERENT AGENTS G-aetin was added to the viscometer either alone or with a-actinin and ATP as indicated, and 0.5 ml of 0.10 M Tris-acetate, pH 7.5, was added. The G-actin was then polymerized by addition of 2 M KCI or 0.1 M MgC12 or both. Final conditions: 1.0 mg actin/ml, 20 mM Tris-acetate, pH 7.5, KC1 and MgCI2 as indicated when added, 1 mM ATP, 0.10 mg ~-actinin/ml, 37°C. Final volume was 2.5 ml. Figures are mean specific viscosities -+S.E.of the number of determinations given in parentheses. Proteins and nucleotides present

Actin Actin + ATP Actin + ~-actinin Actin + a-actinin and ATP

Agents used to polymerizeactin 100 mM KC1

1 mM MgC12

100 mM KC1+ 1 mM MgC12

1.07 ±0.04 (8) 1.13 ± 0.04 (8) 2.60 ± 0.35 (6) 2.06 ± 0.18 (6)

1.10 ± 0.03 (16) 0.95 ± 0.02 (16) 2.32 ± 0.24 (13) 1.09 ± 0.06 (13)

1.tl 1.15 2.70 2.12

Although ATP has no direct effect on viscosity of F-actin solutions in the absence of a-actinin, 1 mM ATP decreases viscosity of a-actinin/F-actin mixtures approx. 21% when actin polymerization occurs in the presence of 100 mM KCI (Table I). It seems unlikely that ATP's effect under these conditions would be due to binding of cations (Table I). Hence, these results suggest that free ATP has a direct effect on the a-actinin-F-actin interaction. The actin ATPase results, which will be described in a different paper (Singh, I., Goll, D.E. and Robson, R.M., unpublished results), also indicate that ATP affects the a-actininF-actin interaction directly. When actin polymerization occurs in the presence of 1 mM Mg2+ with no added KC1, ATP causes an even larger decrease in viscosity of a-actinin/F-actin mixtures than it does when actin polymerization occurs in the presence of 100 mM KCI (Table I). Part of the effect of ATP on the a-actinin-F-actin interaction in the presence of I mM Mg2÷ may originate from shifting of the G-actin to F-actin equilibrium discussed in the preceding paragraph, so that a-actinin cross-links relatively short F-aetin filaments. The very large effect of ATP on viscosity of a-actinin/F-actin mixtures in the presence of Mg2÷ with no added KC1, however, suggests that chelation of Mg2÷ by ATP decreases the ability of a-actinin to cross-link or gelate F-actin f'flaments.Adding 100 mM KC1 in addition to 1 mM Mg2+ eliminates this large effect of ATP on viscosity of a-actinin/F-actin mixtures. Even in the presence of 100 mM KC1 plus 1 mM Mg2÷, however, ATP reduced

± 0.03 (9) ± 0.02 (9) ± 0.23 (6) ± 0.12 (6)

viscosity of a-actinin/F-actin mixtures approx. 21% (Table I, third column). Because ATP decreases viscosity of a-actinin/F-. actin mixtures so markedly in the presence of 1 mM Mg2÷ with no added KC1 and because both ATP and Mg2÷ are needed to study the effects of a-actinin on the release of actin nucleotide (Craig-Schmidt, M., Robson, R.M., GoU, D.E. and Stromer, M.H., unpublished results) and on actin ATPase activity (Singh, I., GoU, D.E. and Robson, R.M., unpublished results), a series of experiments was done to determine the effects of different levels of ATP and Mg2÷ on viscosity of F-actin and a-actinin/F-actin mixtures (Table II). The results of these experiments generally support the idea that free Mg2+ uncomplexed with ATP is needed for a-actinin to cross-link or gelate F-actin solutions in the absence of 100 mM KCI. Increasing Mg2÷ concentration from 1 to 6 mM in the absence of ATP increases viscosity of both F-actin and a-actinin/ F-actin mixtures (Table II). The effect of increasing Mg2+ concentration on viscosity of F-actin alone may originate partly from a shift of the G-actin to F-actin equilibrium toward F-actin. Because specific viscosity of actin in 100 mM KC1/1 mM Mg2÷, which should shift the G- to F.actin equilibrium strongly toward aggregation, was 22% lower than specific viscosity of actin in 6 mM Mg2+ alone (cf. Tables I and II), high Mg2÷ concentrations may also support formation of longer and fewer F-actin filaments in addition to shifting the G- to F-actin equilibrium toward aggrega. tion. The effect of increasing Mg2÷ concentration on

TABLE II EFFECT OF Mg2+ AND ATP ON VISCOSITY OF F-ACTIN AND a-ACTININ/F-ACTIN SOLUTIONS G-actin was added to the viscometer either alone or with a-actinin and ATP as indicated, and 0.5 ml 0.10 M Tris • acetate, pH 7.5 was added. The G-actin was then polymerized by addition of 0.1 M MgC12. Final conditions: 1.0 mg actin/ml, 20 mM Tris-acetate, pH 7.5, 0.10 mg a-actirdn/ml when added, ATP and MgC12 as indicated, 37°C. Final volume was 2.5 ml. Figures are specific viscosities. Protein and nucleotides present

Actin Actin + 1 mM ATP Actin + 2 mM ATP Actin + 4 mM ATP Actin + 6 mM ATP Actin + a-actinin Actin + a-actinin + 1 mM ATP Actin + a-actinin + 2 mM ATP Actin + a-actinin + 4 mM ATP Actin + ,~-actinin + 6 mM ATP

MgCI~ concentration (raM) 1

2

4

6

0.86 0.83 0.80 0.84 0.90 1.57 0.86 0.80 0.84 0.86

0.92 0.83 0.83 0.83 0.89 1.89 1.01 0.83 0.84 0.83

1.05 1.00 0.87 0.88 0.89 2 .53 1.70 1.23 0.96 1.00

1.43 1.15 1.00 0.92 0.86 * 2.07 2.08 1.11 1.10

* This solution was so viscous that it would not flow through the viscometer.

viscosity o f a-actinin/F-actin mixtures probably is due b o t h to a-actinin cross-linking o f longer F-actin filaments and to promotion b y higher Mg2÷ concentrations of the a-actinin-F-actin interaction. ATP greatly decreases viscosity o f a-actinin/F-actin mixtures in the presence o f Mg2÷ and the absence o f KC1 (Table II), just as was shown in Table I. This effect o f ATP seems related to ATP's ability to bind Mg 2÷ (Table II). As added Mg2÷ increases above added A T P , the viscosity o f a-actinin/F-acfin mixtures increases (Table II). This increase probably is in response to the presence of free Mg2÷ that is uncomplexed because the ability o f ATP to complex Mg 2÷ has been saturated. The results in Table II also indicate that Mg-ATP complex decreases viscosity o f a-actinin/F-actin mixtures because specific viscosities o f a-actinin/F-actin mixtures containing ATP and Mg 2÷ were always slightly lower than specific viscosities o f a-actinin/F-actin mixtures containing only added Mg2÷, even when the free Mg 2÷ concentrations presumably are approximately equal (cf., specific viscosity o f mixture at 2 mM Mg2÷/1 mM ATP with mixture at 1 mM Mg; o f mixture at 6 mM Mg2+/ 2 mM ATP with mixture at 4 mM Mg 2÷, etc. in Table II). It is clear from the results in Tables I and II, how-

ever, that ATP itself does not negate ability o f a-actinin to increase viscosity o f F-actin solutions. Discussion Results of the experiments described in this paper lead to three general conclusions. First, studies done with added Mg 2÷ and the presence or absence of 100 mM KC1 suggest that cations associated with polymerization of G-actin to F-actin filaments are also required for cross-linking or gelation of F-actin filaments by o~-actinin. These cations evidently must be uncomplexed and present in as large or larger concentrations than needed to induce polymerization o f G- to F-actin because ATP addition has a larger effect on viscosity of a-actinin/F-actin mixtures than on viscosity of F-actin solutions when Mg 2+ was present without 100 mM KC1. The role o f these cations in the a-actinin-F-actin interaction is not understood. The 20 mM Tris present in our experiments should provide electrostatic shielding for the 318 net excess negative charges (uncorrected for asparagine and glutamine) [20,21] o f the a-actinin molecule and the 13 net excess negative charges (corrected for asparagine and glutamine) of the actin

molecule. Moreover, it seems likely that, once formed, the a-actinin-F-actin complex is stabilized by hydrophobic rather than electrostatic forces because this complex is disrupted by very low ionic strength [1,18] or by pH values above neutrality [1,4] and not byhigh ionic strength [1]. This requirement for cations also may explain the failure of a-actinin to bind to G-actin in our earlier studies done in the absence of such cations [7]. Second, studies done in the presence of 100 mM KCI with or without added Mg2+ show that ATP decreases the viscosity of a-actinin/F-actin mixtures approx. 20%. This ATP-induced decrease in viscosity seems to be due to an effect of ATP on the c~-actininF-actin interaction or on properties resulting from this interaction. The Mg-ATP complex also decreases viscosity of a-actinin/F-actin mixtures slightly. The thixotropic and highly polydisperse nature of F-actin solutions and ct-actinin/F.actin mixtures makes it very difficult to ascertain the exact cause of the ATP-induced decrease in viscosity. It is unlikely that this decrease is due to ATP breaking or increasing flexibility of F-actin filaments because ATP concentrations up to 6 mM had no effect on viscosity of F-actin alone, even when 1 mM Mg2+ was present without KC1. Because the binding constants of K +, Mg2+, and ATP for c~-actinin alone have not been determined, it is not known whether binding of these cations or ATP to a-actinin would affect the a-actinin-F-actin interaction. Third, although ATP reduces viscosity of a-actinin/F-actin mixtures, it does not prevent the a-actininF-actin interaction. 0.1 part a-actinin to 1.0 part F-actin, by weight (molar ratio of one 206 000 dalton a-actinin molecule to forty nine 42000 dalton actins), increases the specific viscosity of F-actin solutions approximately 2-fold, even in the presence of ATP. Therefore, the effects of a-actinin on actin can be studied in the presence of ATP. Indeed, because ATP decreases the viscosity of a-actinin/F-actin mixtures, the a-actinin-F-actin interaction in the presence of ATP may be different from this interaction in the absence of ATP. The a-actinin-F-actin complex almost certainly is formed and functions physiologically in the presence of ATP in vivo.

Acknowledgements We are grateful to Mary Bremner, Jackie Harvey, Darlene Markley, Diane Rath and Jean Fatka for expert technical help and to Joan Andersen for help with the manuscript. This work was supported in part by grants from the National Institutes of Health (HL15679 and AM-19864), the Muscular Dystrophy Association of America, the Iowa Heart Association, the Arizona Agricultural Experiment Station (Projects 20 and 28) and by a grantqn-aid from the American Heart Association (79-948) with funds contributed in part by the Iowa Affiliate.

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