THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 14, Issue of July 25, PP. 5706-5712, 1975

Printed in U.S.A.

Interactions between Actin, Myosin, and an Actin-binding

Protein from Rabbit Alveolar Macrophages

ALVEOLAR MACROPHAGE MYOSIN MG2+-ADENOSINE TRIPHOSPHATASE REQUIRES A COFACTOR FOR ACTIVATION BY ACTIN*

(Received for publication, September 30, 1974)

THOMAS P. STOSSEL$ AND JOHN H. HARTWIG

From the Division of Hematology-Oncology of the Children’s Hospital Medical Center, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02li5

SUMMARY

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-bind- ing protein (HARTWIG, J. H., AND STOSSEL, T. P. (1975) J. Biol Chem. 250,5696-5705) of rabbit alveolar macrophages.

Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor- activated macrophage actomyosin was as high as 0.6 pmol of Pijmg of myosin protein/min at 37”. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg*+ATPase activity of cofactor-activated muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage ex- tracts, was not influenced by the presence or absence of calcium ions. Therefore, as described for Acanfhamoeba casfeZ2anii myosin (POLLARD, T. D., AND KORN, E. D. (1973) J. Biol. Chem. 248, 46914697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participa- tion of calcium ions in the regulation of this activity.

In macrophage extracts containing 0.34 M sucrose, 0.5 m&r ATP, and 0.05 M KC1 at pH 7.0, the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KC1 at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2fATP.

* This work was supported by Grant HL-17742 from the United States Public Health Service.

$ Established Investigator, American Heart Association.

As part of our endeavor to understand the mechanism by which phagocytic cells ingest particulate objects, we have been studying their contractile proteins, and have purified actin and myosin from alveolar macrophages of the rabbit (1). The way in which these proteins might effect the pseudopod movements associated with ingestion is not known. One possibility is that actin filaments slide in parallel to myosin filaments, a force-generating mode utilized in skeletal muscle. This sliding is driven by energy de- rived from ATP hydrolysis which occurs on the myosin activated by F-actin (2). The control of this mechanism in vertebrate skeletal muscle involves reversible inhibition of the interaction between actin and myosin, which can be biochemically monitored by measuring the 1Llg2+ATPase activity of actomyosin at low ionic strength. The ultimate regulator of this activity in skeletal muscle is the calcium ion, which releases the inhibitory influence of muscle control proteins, tropomyosin and the troponins, on the activation of myosin Mg?+ATl’ase activity by actin (3). The relatively low Mg2+ATPase activities of most vertebrate cytoplasmic actomyosins have limited effective inquiry into the question of how this activity might be controlled in vertebrate nonmuscle cells.

A second possible mechanism for the extension and retraction of pseudopodia in phagocytes is the reversible polymerization of actin. This process has been invoked to explain the abrupt ap- pearance of microfilaments in platelets during aggregation (4) and the extension of the starfish sperm acrosomal process after exposure to egg jelly (5). Consistent with this theory is the ob- servation that actin in extracts of abnormal human polymorpho- nuclear leukocytes with defective locomotion and ingestion capacities polymerized incompletely in vitro (6).

The operation of either of these hypothetical systems in phago- cytes requires a transducer for transmitting a signal from the plasma membrane, the site of engagement of particles to be in- gested, to the contractile proteins. Actin has been observed in association with plasma membranes of some cells by morpho- logical (7) and biochemical (S-10) techniques, although the nature of the association is unknown. We discovered a protein in rabbit alveolar macrophages (1) with certain physical and chemi- cal properties resembling those of the peripheral water-soluble protein of the erythrocyte ghost called spectrin (11). The associa- tion of this protein with actin in alveolar macrophages and of

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erythrocyte spectrin with an actin-like protein (12), makes the actin-binding protein a potential candidate for this transducer

role. In this report, we describe some of the interactions between

macrophage actin, myosin, and the actin-binding protein. The properties analyzed included the binding of the proteins to one

another and the activation of the Mg2+ATPase activity of macrophage myosin at low ionic strength by rabbit skeletal

muscle and rabbit macrophage actin, which required the presence of an additional cofactor partially isolated from macrophage

extracts. The effect of this cofactor on the Mg2+ATPase activity

of rabbit skeletal muscle actomyosin and of calcium on the Mg2+- ATPase activity of the macrophage actomyosin-cofactor complex

was also examined. Portions of this work have been reported

previously in summary form (13, 14).

EXPERIMENTAL PROCEDURE

Rabbit skeletal muscle F-actin, myosin, troponin-tropomyosin, and tropomyosin, rabbit alveolar macrophage F-actin, myosin, and actin-binding protein were purified as described in the ac- companying report (1). A “soluble” extract of rabbit skeletal muscle myofibrils and a complex of tropomyosin plus troponins were prepared by the methods of Schaub et al. (15) and of Spudich and Watt (16), respectively. A precipitate, designated “P2,” was isolated as previously described from high speed extract super- natants of rabbit alveolar macrophages in 0.34 M sucrose, 1 mM EDTA, 0.5 mM ATP, 5 mM dithiothreitol, 20 mM Tris-maleate buffer, pH 7.0 (1) by addition of KC1 to a final concentration of 75 mM, stirring at 25” for 1.5 hours, and centrifugation at 12,000 X g for 10 min at 4”. A pellet, designated “Pa,” was collected from the remaining supernatant fluid by centrifugation at 80,000 X g for 3 hours. The pellets were dissolved either in 0.6 M KCl, 0.5 mM ATP, 5 mM dithiothreitol, 20 mM Tris-maleate buffer, pH 7.0 (KC1 solu- tion) or else in 0.6 M KI, 10 mM sodium thiosulfate, 5 mM ATP, 5 mM dithiothreitol, 20m~ Tris-maleate buffer, pH 7.0 (KI solution). Assays of ATPase activity, electron microscopy of negatively stained proteins, acrylamide gel electrophoresis with sodium dodecyl sulfate, and quantitative densitometry of stained poly- acrylamide gels were performed as described previously (1). To assay binding of macrophage F-actin and actin-binding protein, 0.3 mg of the purified proteins was added to 0.3 ml of 0.1 M KCl, 10 mM Tris-maleate buffer solution, pH 7.0, in Beckman micro- centrifuge tubes and incubated at 25” for 60 min. The tubes were centrifuged for 5 min in the Beckman microcentrifuge. The top 0.2 ml of fluid in the tubes was added to 0.8 ml of distilled water, dialyzed against distilled water, lyophilized, and subjected to electrophoresis on polyacrylamide gels with dodecyl sulfate.

RESULTS

Interactions between Purijied Macrophage and Muscle Actins and PuriJied Macrophage Myosin-As described in the accom- panying report, neither functional rabbit skeletal muscle nor rabbit alveolar macrophage actin which bound to purified rabbit

alveolar macrophage myosin activated the Mg2+ATPase activity

of macrophage myosin with high specific K+- and EDTA-acti- vated ATPase activity (1). As shown in Table 1,’ the presence or absence of calcium ions, additions of rabbit skeletal muscle tropomyosin, a mixture of troponins and tropomyosin, or of a soluble extract of rabbit skeletal muscle myofibrils failed to in- fluence markedly the Mgz+ATPase activity of macrophage acto- myosin.

Both rabbit skeletal muscle and rabbit alveolar macrophage F-actins activated the Mg*+ATPase activity of purified rabbit alveolar macrophage myosin if an additional factor, partially purified by gel filtration from the 20 to 50% saturated ammonium sulfate fraction of extract supernatants of alveolar macrophages

1 The abbreviation used is: EGTA, ethylene glycol his@-amino- ethyl ether)-N,N’-tetraacetic acid.

TABLE I

Effect of F-actin, other muscle proteins, and CaCL on the Mg2+ ATPase activity of rabbit alveolar macrophage myosin

The concentrations of myosin and actin protein in the ATPase assay were 0.03 mg/ml and 0.3 mg/ml, respectively.

Alveolar macrophage myosin + Muscle F-actin

+ CaCb (0.2 mM) + EGTA (1 mM)

+ Muscle tropomyosin 0.2 mg/ml 0.4 mg/ml 0.8 mg/ml

+ Muscle troponin-tropomyosin, 0.3 mg/ml + Soluble myofibril extract, 0.3 mg/ml

Mg*+ATPase activity

nmol of Pdmp of my&n

$rolein/min

13.8 18.8 17.0 21.2

18.8 19.0 19.0 13.5 13.4

VOLUME (ml)

FIG. 1. Gel filtration of the 20 to 50% saturated ammonium sulfate fraction of alveolar macrophage extract supernatant on Bio-Gel A-15m 200 to 400 mesh in 0.6 M KC1 solution. The elution profiles of absorbance at 290 nm (--) and of cofactor activity (o- - -0) are indicated. The Mg*+ATPase activity of rabbit alveolar macrophage myosin, 0.04 mg, plus rabbit skeletal muscle F-actin, 0.3 mg/ml, was assayed in the presence of added column fractions to determine cofactor activity.

or from P3 sediments dissolved in 0.6 M KC1 solution, was added to the actomyosin (Fig. 1). This material, designated “crude macrophage cofactor,” reproducibly eluted from Bio-Gel columns in the position of a globular protein of molecular weight 70,000. The peak of cofactor activity contained numerous protein bands, including a 70,000 molecular weight peptide, when subjected to electrophoresis on polyacrylamide gels in dodecyl sulfate. The activity was nondialyzable, soluble in 0.05 or 0.6 M KCl, and was rapidly inactivated by boiling, heating (56”, 30 min) and trypsin 1 mg/ml. It deteriorated rapidly during storage at 4”, and was not stabilized by 1 mM EDTA, 5 mM Mgz+ATP, 5 mM dithio- threitol, 0.1 mM 2-mercaptoethanol, 0.1 M NaF, pH 8 to 5, or rapid freezing. Therefore, attempts to purify the material further by gel filtration and ion exchange chromatography were not SUC-

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*I 6 + cofactor

1 2 3 4

Actin Protein (mg(ml)

FIG. 2. Effect of purified alveolar macrophage actin concen- tration on the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin in the absence (0 ) and presence (O ) of crude macrophage cofactor. The concentrations of myosin and cofactor protein in the assays were 0.05 mg/ml and 0.3 mg/ml, respectively.

1 1 I i

.l .2 .3 A Cofactor Protein

(mglml)

FIG. 3. Effect of crude alveolar macrophage cofactor concen- tration on the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin in the absence (0 ) and presence (0 ) of puri- fied alveolar macrophage actin. The concentrations of myosin and actin protein in the assays of 0.5 ml were 0.04 mg/ml and 0.8 mg/ml, respectively.

cessful. All active crude cofactor preparations contained a 70,000

molecular weight polypeptide when assayed by dodecyl sulfate polyacrylamide gel elcctrophoresis.

In the presence of crude cofactor, purified alveolar macrophage or rabbit skeletal muscle F-actin increased the Mg2+ATPase ac- tivity of purified alveolar macrophage mgosin as much as 22-fold, and the activation was in proportion to the quantity of F-actin added at limiting concentrations (Fig. 2). The cofactor had 110

Mg2+ATPase activity, either alone or in the presence of F-actin. As shown in Fig. 3, the activation of macrophage myosin NIg*+-

ATPase in the presence of a saturating concentration of macro- phage F-actin by crude cofactor was sigmoidal. On the basis of protein, the quantity of crude cofactor required for maximal activity was considerably less than that of actin. The degree of activation of Mgz+ATPase activity of macrophage actomyosin by cofactor was variable because of the instability of the cofactor activity. However, the Mg*+ATPase activity of purified macro- phage myosin in the presence of maximally effective fresh cofactor and actin concentrations was as high as 0.6 pmol of Pi/mg of myosin protein/min and was, therefore, higher than that reported

TABLE II

Effect of F-actin, crude macrophage cofactor, and calcium ions on the Mg=+ATPase activity of rabbit alveolar macrophage myosin

The proteins were in the assay system in the following concen- trations: myosin, 0.03 mg/ml; F-actin, 0.3 mg/ml; cofactor, 0.1 mg/ml .

Proteins Mg*ATPase activity

Alveolar macrophage myosin + Muscle F-actin + Cofactor + Muscle F-actin + Cofactor

+ CaCL (0.2 mM) + EGTA (1 mM)

nmol of P/mg of myosin protein/min

11 11 11

120 119 116

TABLE III

Effect of calcium ions and of muscle troponin + tropomyosin on the Mg2+ATPase activity of the PS sediment derived from extract

supernatants of rabbit alveolar macrophages

The concentrations of P3 and of troponin-tropomyosin in the assay system were 0.5 mg protein/ml and 0.6 mg/ml, respectively.

Additions to the Mg*ATPase assay system Mg* ATPase activity

None + 0.2 mM CaC12 + 1 mM CaClz + 5 mM CaClt + 10 mM CaCls + 1 mM EGTA + 5 mM EGTA + Troponin-tropomyosin

+ 1 mkr CaCL + 2 mM CaC12 + 1 mM EGTA

nnol of Pi/min/mg of gro1ein

17 17 18 18 17 19 22 10 11 10 10

for most vertebrate cytoplasmic actomyosins. The Mg2+ATPase activity of the alveolar macrophage actin-myosin-cofactor com- plex was not influenced by the presence or absence of calcium ions (Table II).

The crude cofactor preparations also increased the active Mgz+ATPase activity of rabbit skeletal muscle actomyosin (Ta- ble III). The crude macrophage cofactor also increased the Mg2+- ATPase activity of rabbit skeletal muscle actomyosin containing rabbit skeletal muscle troponins and tropomyosin, both in the presence and the absence of calcium ions (Fig. 4).

To determine whether proteolysis was responsible for the failure of F-actin to activate the Mgz+ATPase activity of macro- phage myosin, 1 mg of rabbit skeletal muscle myosin was added to an alveolar macrophage homogenate. Of a I’3 sediment ob- tained from the homogenate, 29 mg were dissolved in 0.6 M KI solution and applied to a column of Bio-Gel A-15m 200 to 400 mesh equilibrated with 0.6 M KI solution and 0.6 M KC1 solution, and eluted with 0.6 M KC1 solution. The fractions with K+- and EDTA-activated ATPase activity were collected, and the effect of rabbit skeletal muscle actin on the Mgz+ATPase activity of t.he pooled fractions was tested. Of the added muscle myosin, 80% was recovered as determined by the total Mg2+ATPase activity in the presence of muscle F-actin. The results of this experiment suggested that proteolysis did not account for the

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mg Cofactor Protein

FIG. 4. Effect of crude macrophage cofactor on the Mg2+ATPase of rabbit skeletal muscle actomyosin plus troponin-tropomyosin in the presence of 1 mM CaCh (0 --0 ) or 1 mM EGTA (0 -- 0 ). The assays of 1 ml contained 0.08 mg of myosin protein, 1.4 mg of F-actin protein, and 1.5 mg of troponin-tropomyosin.

cofactor requirement for activation of alveolar macrophage myo- sin by F-actin.

Interactions between Actin, Myosin, and Cofactor in Macrophage Extracts-The P2 sediment, of which the predominant proteins were actin and the actin-binding protein (Fig. 5), contained less than 10% of the total myosin of the extract supernatant, as de- termined by quantitative densitometry of stained polyacrylamide gels and by assay of Kf- and EDTA-activated ATPase activity. The P2 sediment had little to no Mg2+ATPase activity in 20 mM

KCl. The P3 pellet contained over 70% of the myosin found in the extract supernatant. Its specific Mg*+ATPase activity in 20 mM KC1 solution, 9.7 f 2.2 nmol of Pi/min/mg total protein, was similar to its K+- and EDTA-activated ATPase activity of 13.3 f 3.5 nmol of Pi/min/mg of total protein (mean f S.D., n = 7). By quantitative densitometry, the band corresponding to the myosin heavy chain constituted about 5% of the total stainable protein of P3 fractions analyzed by polyacrylamide gel electrophoresis with dodecyl sulfate. This finding suggested that the specific ATPase activity might be corrected to 0.194 and 0.266 pmol of Pi/mg of myosin protein/min, respectively, if the ATPase activity were entirely due to myosin. Since actin com- prises 14% of the stainable protein, the molar ratio of actin to myosin was 33 to 1. When the P3 sediment was dissolved in 0.6 M KC1 solution and chromatographed on Bio-Gel A-15m columns at 4”, Kf- and EDTA-activated and K+- and Ca2+- activated ATPase activities eluted in the position of myosin, but no Mgz+ATPase activity in 20 mM KC1 was recovered from the columns, unless fractions eluting with a K,, of 0.74 (crude co- factor) were combined with the myosin peak and F-actin was added. The myosin resolved by chromatography in cold 0.6 M

KC1 solutions in this manner was variably but minimally con- taminated with actin, and actin was found in fractions eluting close to the total volume of the columns (as determined by poly- acrylamide gel electrophoresis with dodecyl sulfate). This fact suggested that, despite the presence of KCl, most of the actin in the P3 fraction was depolymerized in the cold. If the P3 was dis- solved in 0.6 M KC1 solution at 25” and chromatographed at 25”, K+- and EDTA-activated, K+- and Ca2+-activated, and Mg2+-

FIG. 5. Appearance of P2 (left) and P3 (right) sediments after electrophoresis on 5% polyacrylamide gels with dodecyl sulfate. The molecular weights of the actin-binding protein (220,000), actin (45,000), and myosin heavy chain (200,000) are indicated.

ATPase activities eluted together with the bulk of the applied protein as a single peak in the void volume. These results sug- gested that a complex of F-actin, cofactor, and myosin was not resolved by chromatography, presumably because the actin re- mained highly polymerized at 25”, and that the actin polymers bound myosin and cofactor. All of the findings indicated that the entire Mgt+ATPase activity of the P3 fraction in 20 mM KC1 can be attributed to myosin activated by macrophage F-actin and cofactor.

The Mgz+ATPase activity of P3 was not influenced by the presence or absence of calcium ions (Table III), even when high concentrations of calcium were added to neutralize any effect of EDTA which was present in the extract supernatant. Further- more, when homogenates and extract supernatants were prepared with 1 mM CaC12-1 mM MgC12 instead of 1 mM EDTA in the homogenizing solution, P3 sediments prepared from extract supernatants had Mg2+ATPase activity in 20 mM KC1 that was unaffected by the presence or absence of added calcium ions.

Interactions between Actin, Myosin, and A&-binding Protein in Moxrophage Extracts-Quantitative densitometry of dodecyl sulfate polyacrylamide gels of the P2 sediment showed that 2.8% of the stainable protein was the actin-binding protein, and 80% of the stainable protein was act,in (Fig. 5). Over 70% of the total actin and actin-binding protein in the extract supernatant was found in the P2 sediment. On a molar basis the ratio of actin- binding protein to actin in the P2 sediment was approximately 1: 100. When the P2 pellet was suspended in 0.1 M KCI, 10 mM Tris-maleate, pH 7.0, stained with uranyl acetate and examined in the electron microscope, parallel arrays of actin filaments in

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FIG. 6. A and B, morphology of the P2 sediment in 0.1 M KCI, 10 mM Tris-maleate, pH 7.0, in the electron microscope. C, morphology of tmrified macronhaee a&in-binding urotein ~1~s Durified macroDhage actin incubated at 25” with 0.1 M KCl. 10 mM Tris-maleate buf- fer; pH 7.0. Magnifications: A, X 25,400; B and C,* X 180,000. -

bundles with interconnecting branches were observed (Fig. 6). No other filamentous structures were seen. In the P3 pellet, the actin-binding protein comprised only 0.8% of the total stainable protein (Fig. 5). The molar ratio of actin to actin-binding protein was, therefore, considerably higher than that in the P2 sediment.

Interactions between Purified Actin, Myosin, and Actin-binding Protein-In 0.6 M KCl, partially purified F-actin bound myosin in the absence but not the presence of Mg2+ATP, but did not bind to the actin-binding protein in either case (Fig. 7). Purified macrophage actin-binding protein caused purified macrophage actin to sediment at low speed in 0.1 M KCl. The actin filament arrays observed in the P2 sediments were also observed in the electron microscope when purified macrophage actin-binding protein was incubated with macrophage actin in 0.1 M KC1 (Fig. 6C).

DISCUSSION

Acanthamoeba castellanii myosin was found to require a novel cofactor for activation of its Mgz+ATPase by actin (17). The discovery, reported here, of a similar requirement for actin ac- tivation of rabbit alveolar macrophage myosin with much greater structural similarity to rabbit skeletal muscle myosin than Acanthamoeba myosin was unexpected. While it is premature to generalize, the finding of cofactor requirements for the activation

of Mg*+ATPase activities of myosins from two diverse sources raises the question as to whether such a requirement is common. The relatively poor activation of Mg*+ATPase activity of some vertebrate cytoplasmic myosins by actin reported in the past (U-20) is further evidence for this possibility.

The precise nature of the macrophage cofactor is unclear. As in the case of Acanthamoeba, the macrophage cofactor has not been completely purified, and both cofactors increased the Mg*+- ATPase activity of rabbit skeletal muscle myosin. However, the macrophage cofactor differed from Acanthamoeba cofactor in being unstable during storage, and, in this respect, it also differed from proteins which activate actomyosin of other cells such as tropomyosin, which is required for the activation of Limulus polyphemus myosin by actin (21), and from a factor derived from rabbit myofibrils which increases the Mg*+ATPase activity of skeletal muscle actomyosin (15). Neither of these proteins ac- tivated the Mg*+ATPase activity of rabbit alveolar macrophage actomyosin.

The Mg*+ATPase activity of alveolar macrophage actomyosin activated by macrophage cofactor was not influenced by the presence or absence of calcium ions. The Mg2+ATPase activity of P3, a relatively crude preparation derived from macrophage extracts containing actin, myosin, and cofactor, was also unaf- fected by the presence or absence of calcium ions. The absence of

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Fxo. 7. Experiment to assay binding of partially purified mus- cle actin to alveolar macrophage myosin and actin-binding pro- tein: effect of ATP. Myosin, isolated by a single gel filtration step from macrophage extracts in 0.6 M KI-KCl, actin, isolated by a single polymerization cycle from muscle acetone powder, and macrophage actin-binding protein, isolated by gel filtration, were incubated for 15 min at 25” in 0.6 M KCl, 10 mM Tris-maleate buf- fer, pH 7.0, with either 5 mM MgClz or 5 mM MgCls-5 mM ATP, and centrifuged at 150.000 X a for 1 hour. The supernatant fluids were dialyze; against ‘distilleh water, lyophilized, and analyzed by electrophoresis on 50/, polyacrylamide gels in sodium dodecyl sulfate. The gel on the left is of the incubation with Mg*+ATP, the gel on the right of the incubation with Mg2+. The molecular weights of the myosin heavy chain (200,000) and of the actin-bind- ing protein (220,000) are indicated.

a regulatory influence by calcium on Mgz+ATPase activities was also found when P3 was prepared without chelating agents, which can remove a subunit of molluscan myosins which confers cal- cium regulation on the molecule (22). The absence of calcium regulation in macrophage actomyosin does not appear to be a result of its inhibition by macrophage cofactor. Troponin-tropo- myosin imposed calcium regulation on the Mg*+ATPase activity of cofactor-activated muscle actomyosin. However, as was ob- served with the Mg*+ATPase of cofactor-activated Acanthamoeba actomyosin (23), troponin-tropomyosin inhibited the Mg2+ATP- ase activity of cofactor-activated alveolar macrophage actomyo- sin, and this inhibition was not released by calcium ions. Although tropomyosins have been isolated from nonmuscle cells (24-26), and crude actomyosin from diverse non-muscle cells has been reported to have calcium-regulated MgtfATPase activities (27-29), none of our findings indicated that calcium has any regulatory effect on the Mg2+ATPase activity of rabbit alveolar macrophage actomyosin activated by cofactor. However, it is theoretically possible that factors conferring such regulation were discarded or inactivated during preparation of the enzymes.

Extracellular divalent cations activate the ingestion of certain particulate objects by rabbit alveolar macrophages. However, calcium is less effective than magnesium, cobalt, and manganese ions (30). Furthermore, particles coated with an opsonin, an ac-

5711

tivated fragment of the third component of serum complement, are ingested by the cells in the absence of extracellular divalent cations (30). Therefore, the effects of extracellular calcium do not constitute critical evidence for the involvement of an intracellular calcium-sensitive actomyosin system in the cellular motility as- sociated with ingestion by alveolar macrophages.

A large fraction of the total macrophage actin, apparently bound to the macrophage actin-binding protein, sedimented from macrophage extracts at low centrifugal forces, which normally do not cause actin to sediment, when KC1 was added to these extracts at pH 7.0. The sediment had the same morphological appearance as macrophage G-actin allowed to polymerize in the presence of macrophage actin-binding protein. The actin fila- ments were aggregated and cross-linked in dense arrays, resem- bling the filament meshworks seen in macrophage pseudopodia.

Actin remaining in the supernatant fluid after centrifugation of the actin-actin-binding protein complex, bound and sedimented together with myosin when centrifuged at high speed. While the meaning of these findings is unclear, we suggest that if the actin- binding protein binds and aggregates actin filaments in response to changes in the ionic environment, the interaction between these proteins could influence the reversible assembly of actin filaments to produce and retract pseudopodia. Polypeptides resembling erythrocyte spectrins in molecular weight have recently been discovered in the acrosomal region of echinoderm sperm, and have been hypothesized to regulate the aggregation stage of actin in these organelles (31). Macrophage myosin, which did not bind to the actin-binding protein in 0.1 M KCl, might compete with this protein for binding with actin. Such competitive and possibly reciprocal binding could generate a mechanism for focal sliding or assembly of actin filaments, and play a role in macrophage movement and ingestion.

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T P Stossel and J H Hartwigtriphosphatase requires a cofactor for activation by actin.

alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine Interactions between actin, myosin, and an actin-binding protein from rabbit

1975, 250:5706-5712.J. Biol. Chem. 

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