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A contractile protein from leucocytes Its extraction and some of its properties
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BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35374 A CONTRACTILE P R O T E I N FROM LEUCOCYTES ITS EXTRACTION AND SOME OF ITS P R O P E R T I E S "
N O B U Y U K I SENDA, N O B U H I K O SHIBATA, N O R I Y U K I TATSUMI, K E I I C H I K O N D O AND K E I K O H A M A D A
Center for Adult Diseases, Osaka (Japan) (Received December 27th, 1968)
SUMMARY
I. A contractile protein was isolated from equine leucocytes. The protein exhibited superprecipitation at low ionic strength in presence of Mg2+ and ATP and possessed ATPase (EC 3.6.1.3) activity. The effect of divalent cations, Mg2+ and Ca 2+, on the ATPase activity resembled that on actomyosin from muscle. 2. Electron micrographs of the protein showed, at high ionic strength without ATP, the "arrowhead" structure characteristic of myosin B from striated muscle. Thick and thin filaments were observed at low ionic strength at a relatively high ATP level. 3. The possible role of the protein in the movement of leucocytes is discussed.
INTRODUCTION
Since 1948 the authors have reported the characteristic features of leucocytic motility both from the morphological and functional aspects 1-5. The migration velocity and chemo- and galvanotactic function of leucocytes were greatly accelerated by the addition of ATP to the surrounding medium. Arsenate, phloridzin and 2,4dinitrophenol inhibited the motile function of leucocytes which could be counteracted by the addition of ATP (ref. 6). These findings led us to consider that the motile form and function were coordinately controlled by ATP, and that leucocytes might have a mechanochemical system functioning with ATP as energy source. In a parallel study carried out from the phylogenetic point of view the constriction wave in leucocytes was found to be the same as that observed in Amoeba limax, Entamoeba histolytiea, Planaria gonocephala, Lumbricus trapezoides, and A plysia
kurodai 7. Abbreviation: EGTA, 1,2-bis(2-dicarboxymethylaminoethoxy)-ethane. * The work described in this paper was reported at the 2oth Annual Meeting of J a p a n Society for Cell Biology in 1967, and at the X I I t h Congress of the International Society of Hematology in New York, 1968.
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The fact that the motor organs of the above animals are governed by the muscular system led us to assume the presence of a contractile element in leucocytes and amoeba which functions circularly as well as longitudinally in their movement s and to try to isolate a contractile protein from leucocytes. This paper reports the isolation of a contractile protein having properties similar to those of actomyosin from muscle. The present finding is evidence supporting our previous hypothesis that the movement of leucocytes may be based on the reaction of contractile protein with ATP. EXPERIMENTAL PROCEDURE
Isolation of leucocytes from whole blood. From flesh equine arterial blood leucocytes were isolated by a modification of the method reported by TAKAHASHI 9, a s follows. IO.O g of EDTA (tetrasodium salt), 8.5 g of NaC1 and 3.6 g of cysteine were dissolved in I 1 of distilled water into which 9 1 of the blood were mixed. This was kept on ice during transport from a slaughter house. About i h after the blood sample was obtained the mixture was warmed to room temperature and was left to stand for 2 h to separate into two layers. The supernatant layer was removed by a syphon and then filtered through two layers of gauze in order to remove the platelets. The following procedures were all conducted at 0-4 ° . The filtrate was centrifuged for 8 rain at 250 × g, and the supernatant layer was again centrifuged at io ooo × g for 8 min to obtain platelet-free plasma. The sediment obtained by centrifugation at 250 × g was suspended in 3 vol. of platelet-free plasma and the suspension was again filtered through gauze, followed by centrifugation at 250 × g for 7 mill. By repeating this procedure twice, platelets were removed through the gauze and the red cells in the lower layer of the sediment by centrifugation. The upper half of the sediment thus obtained was washed at least twice with io vol. isotonic NaC1 and again centrifuged at 250 × g for 7 min to obtain the buffy coat of leucocytes. The volume of the huffy coat collected was about 20 ml from a starting volume of 18 1 of blood. The leucocytes counted as i.o. lO4-1.3. IO4/ram3 in whole blood at the beginning of the procedure were concentrated to i.o. lO6-1.3 • IOn/ram 3 in the huffy coat at the final step of isolation. Contamination with red cells in the huffy coat was less than 5 %, and that of platelets was negligible. About 9 o°/o of the collected leucocytes was usually found to be neutrophiles. Extraction of contractile protein from leucocytes. All procedures were conducted at about 4 °. The buffy coat was first mixed with i vol. o.i M KC1 and then with i vol. of the extract solution containing 1.6 M KC1, 2 mM ATP, 3 mM eysteine, o.oi M Na2CO 3 and 0.04 M NaHCO 3, whose pH was adjusted to 7.0 by the addition of I M acetic acid. The leucocytes were homogenized for 2 rain in a Waring blendor and a Potter-Elvehjem homogenizer. The resulting highly viscous homogenate was stirred with a magnetic stirrer for 3 h, and, after the addition of 2 mM ATP, stirred for another 9 h. This procedure reduced the viscosity of the homogenate. The homogenate was then centrifuged at 33 ooo × g for i h to remove cell fragments and corpuscular materials, leaving a pink supernatant fluid of relatively high viscosity. In order to isolate the nucleoprotein considered to be contaminating the supernatant fluid, an equivalent volume of a solution at pH 7.o containing 2 mM MgC12, o.5 mM ATP and 5 mM 1,2-bis(2-dicarboxymethylaminoethoxy)ethane (EGTA) was added according 13iochim. Biophys. Acta, ISI (1969) 191-2oo
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to the method of HOFFMANN-BERLING1°. The resulting sediment, called Fraction A and having a maximal absorption at 260 m/,, was removed b y centrifugation at io ooo × g for 20 min. I t was later found that Fraction A dissolved in 0.6 M KC1 did not exhibit superprecipitation. The supernatant fluid obtained b y centrifugation was added to 5 vol. water to reduce the ionic strength to 0.05, and the resultant flocculent precipitate was collected 2-3 h later b y centrifugation at 3000 X g for 20 min. Further, in order to remove Mg 2+, the precipitate was washed with 50 vol. 0.05 M KC1 containing I mM EDTA whose pH was adjusted to 7.0 b y Tris. After metalchelated and free EDTA had been removed b y washing with IOO vol. 0.05 M KCI solution, the sediment was dissolved in an appropriate volume of 2.4 M KC1 solution whose ionic strength was finally adjusted to 0.6 with water. The contaminated corpuscular materials were removed b y centrifugation at 33 ooo x g for 2o mill. Precipitation and dissolution of the above supernatant fluid in KC1 solution of final ionic strength of either 0.05 or 0.6 was repeated at least twice. The protein in 0.6 M KC1 was kept at o ° (Fraction B), and used in testing for the properties a contractile protein should demonstrate. The foregoing extraction procedure was completed within 24 h and the tests were conducted within 2 days after the extraction. The protein in Fraction B extracted from 20 ml of buffy coat of leucocytes usually amounted to about 5 ° mg. The amount of protein was determined b y the method of LOWRY et al. 11 A T P a s e activity. ATPase (EC 3.6.1.3) activity of contractile protein was measured by determining the amount of Pi liberated from ATP b y the method of MARSH12. Superprecipitation. The ionic strength of the reaction mixture used for observation of superprecipitation was adjusted to 0.06 b y mixing Fraction B into the appropriate reaction medium indicated in the text. The superprecipitation was followed with a Hitachi UV-VIS spectrophotometer by measuring the change in absorbance at 660 m/, in the reaction mixture after addition of ATP at room temperature according to the method developed by EBASH113. In some experiments the superprecipitation in the reaction mixture containing Fraction B was also followed with time macroscopically and b y means of the phase-contrast microscope after the addition of ATP. Viscosimetric determination. This was performed in an Ostwald viscosimeter with 5-ml capacity at 25 °. The relative viscosity was measured at p H 7.0 on Fraction B in 0.6 M KC1 (~rel) and after addition of 0.05 ml of a solution containing 0.5 M MgC12 and o.I M A T P in 0.6 M KC1 ('Y]relATP)- The sensitivity towards ATP was calcula{ed (in °/o) according to PORTZEHL et al. 14 by the formula l o g e ~ r e l - - l o g e ~ r e l ATP
lOge~rel A T P
X IOO
Ultracentrifuge. A Hitachi Model UCA-I analytical ultracentrifuge was employed for the sedimentation-velocity measurement. Electron microphotographs. Micrographs of Fraction B were taken b y the negative-staining technique of HUXLEY as. One drop of the ice-cold preparation of Fraction B was placed on a microgrid coated with collodion carbon. The preparation was negatively stained with I~o uranyl acetate, and examined in a Nihon Denshi Model J E M 6C electron microscope with an acceleration voltage of 80 kV. Biochim. Biophys. Acta, 181 (1969) 191-2oo
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Fig. I. S u p e r p r e c i p i t a t i o n of contractile p r o t e i n ( F r a c t i o n ]3) f r o m leucocytes. T h e u p p e r series of p i c t u r e s h o w s t h e c o n t r a c t i o n of p r e c i p i t a t e d p r o t e i n in t h e presence of o.5 m M A T P a t 2o °. T h e lower series s h o w s t h e s e d i m e n t a t i o n w i t h o u t a d d e d A T P . P r o t e i n c o n c e n t r a t i o n , 1.2 5 m g / m l ; ionic s t r e n g t h , o.o6; MgC12, 5 m M ; T r i s - m a l e a t e buffer, 2o m M (pH 7.0); t o t a l vol., 3.o ml.
Chemicals. All chemicals used for this study were analytical grade. Tris-ATP was obtained from Sigma Chemical Co., U.S.A., and EGTA from the Dojin Igaku Institute. The water used in preparing solutions was redistilled in glass. RESULTS
Superpr ecipitation It is a well-known characteristic of actomyosin, the contractile protein of muscle, to undergo superprecipitation at low ionic strength in the presence of Mg 2+ by the addition of a low concentration of ATP. A marked superprecipitation was observed in the reaction mixture with low ionic strength (0.06) containing 5 mM MgCI~, 20 mM Tris-maleate buffer (pH 7.0) and Fraction B in the presence of 0.5 mM ATP (Fig. I). The superprecipitation reached its maximal level in about 15 min after the addition of ATP. Fig. 2 shows the phase-contrast micrograph of Fraction B in the superprecipitation under the same
Fig. 2. P h a s e - c o n t r a s t m i c r o g r a p h s of contractile p r o t e i n (Fraction B) f r o m leucocytes. T h e exp e r i m e n t a l c o n d i t i o n s are t h e s a m e as for Fig. 1. R i g h t , s u p e r p r e c i p i t a t e d p r o t e i n after t h e a d d i tion of A T P . Left, w i t h o u t a d d i t i o n of A T P . × 9o0.
Biochim. Biophys. Acta, i 8 i (1969) 191-2oo
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Fig. 3. Effects of Ca ~+ and E D T A on the superprecipitation at 20 ° of contractile protein (Fraction B) from leucocytes. Protein concentration, 0.8o m g / m l ; ionic strength, 0.o6; T r i s - m a l e a t e buffer, 2o mM (pH 7.o) ; ATP, o.5 mM ; total vol. 3.o ml. The reaction was s t a r t e d b y the addition of ATP. G - - C ) , w i t h added 0. 5 mM E D T A ; x - - x , with added o. 5 mM E G T A and 5 mM MgC12; ~ - - - - - - A , in presence of 0. 5 mM CaC12 a n d 5 mM MgC1v Fig. 4. Effects of added Mg ~+ on the superprecipitation at 20 ° of contractile protein (Fraction B from leucocytes. Protein concentration, o.52 m g / m l ; ionic strength, o.06; EGTA, 0. 5 mM; T r i s maleate buffer, 2o mM (pH 7.o) ; ATP, o.1 mM; total vol. 3.o ml. The reaction was s t a r t e d b y the addition of ATP. Final concentrations of added MgCI~ were 5 ° .uM and o. i mM ( × - - × ), 5 mM (Q 0 ) , IO and 20 mM ( ~ - - ~ ) and 3 ° mM ( C ) - - - - - - © ) . C)--C), with no added MgC12; 0 - - 0 , with I mM E D T A .
condition. The synerated feature was similar to that observed by RICE et al. 16 in skeletal myosin B. The rapid increase in superprecipitation was measured by the increase in absorbance at 660 m/~, as shown in Fig. 3. The marked superprecipitation was induced by adding o.5 mM ATP in presence of 5 mM MgC12 and 0.5 mM CaC12. The addition of 0.5 mM EGTA, a relatively specific chelater of Ca ~+, had no effect on the degree of superprecipitation, indicating non-participation of Ca z+, whereas the addition of 0.5 mM EDTA to the reaction mixture with no added Mg z+ and Ca ~+ resulted in complete inhibition of the superprecipitation by ATP. Fig. 4 shows the effect on the superprecipitation of adding Mg ~+ to the reaction mixture containing Fraction B, 0.5 mM EGTA, 2o mM Tris-maleate buffer (pH 7.0) and o.I mM ATP. Up to 20 mM of added MgCI~ the superprecipitation was enhanced, then was gradually inhibited by increasing concentration of Mg 2+. The occurrence of a considerable degree of superprecipitation without addition of Mg 2+ could possibly be due to a trace of contaminating Mg z+ during the preparation of Fraction B. The requirement of Mg 2+ but not Ca z+ for the superprecipitation by ATP of Fraction B is the same as that reported with synthetic actomyosin from striated muscle, although the optimal concentration of Mg2+ in the striated muscle is considerably lower than that in our experiments. A T P a s e activity
If Fraction B contains a contractile protein similar to actomyosin from muscle, it should exhibit ATPase activity affected b y Mg ~+, Ca 2+ and ionic strength. Biochim. Biophys. Acta, 181 (1969) 191-2oo
N. SENDA et al.
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Fig. 5. A T P a s e a c t i v i t y of contractile p r o t e i n (Fraction B) f r o m leucocytes. P r o t e i n c o n c e n t r a tion, 0.30 m g / m l ; T r i s - m a l e a t e buffer, 20 m M (pH 7.o) ; A T P , i m M ; E D T A , (when added) I raM; total vol. 3.o ml. (A) Effect of KC1 in presence of 5 m M MgCl v (B) Effect of Mg *+. (C) Effect of Ca *+ a t two different ionic s t r e n g t h s , 0.06 ( ) a n d 0.60 ( - - - - - - ) . T h e reaction w a s s t a r t e d b y t h e a d d i t i o n of A T P , a n d t h e A T P a s e a c t i v i t y w a s d e t e r m i n e d b y m e a s u r i n g t h e Pl liberated for IO m i n at 20 °.
The Mg2+-dependent ATPase activity of Fraction B under conditions shown in Fig. 5A became lower as the ionic strength was increased. The activity was 0.023 and O.Ol7 mole Pi liberated per mg protein per min at ionic strengths of 0.06 and 0.6, respectively. As shown in the dotted line in Fig. 5B, the ATPase activity of Fraction B was inhibited by increasing concentration of Mg 2+ at high ionic strength, whereas at low ionic strength, as shown by the solid line, the activity was apparently activated with increase in concentration of Mg 2+. As shown in Fig. 5C ATPase was activated by Ca *+ both at high and low ionic strengths, though in lesser degree at low strength. In actomyosin from skeletal muscle, HASSELBACH17 reported that the Mg 2~dependent ATPase was inhibited as the ionic strength was raised, the activity being about 0.26 and o.oi/~mole Pi liberated per mg protein per min at ionic strengths of o.o7-o.1 and 0.6, respectively, in the presence of I mM Mg 2+ and 0. 9 mM ATP. The Ca2+-dependent ATPase activity in the presence of o.oi M Ca 2+ and 0. 9 mM ATP was about 0.35 and o.15 #mole Pt liberated per mg protein per rain at ionic strengths of o.o7-o.1 and 0.6, respectively. Ca 2+, however, activated ATPase activity of actomyosin from skeletal muscle and Fraction B in high and low ionic strengths, although the enzymic activity of the former was high in low ionic strength compared with that in the higher one, but in the latter the situation was reversed (0.040 and 0.085 #mole Pi liberated per mg protein per rain at low and high ionic strengths in the presence of o.oi M Ca2+). The same was true for actomyosin from smooth muscle ls,19. It seems reasonable to postulate that Fraction B exhibited ATPase (EC 3.6.1.3) activity of the actomyosin type which was activated b y Mg 2+ and Ca ~+ at low ionic strength, and also ATPase activity of the myosin type which was inhibited b y Mg 2÷ and activated by Ca ~+ at high ionic strength, under conditions in which actomyosin is dissociated into myosin and actin.
Change in viscosity by addition of A T P The relative viscosity of Fraction B was decreased by the addition of ATP and Mg 2+ at high ionic strength. This was followed b y a gradual increase towards the initial leve] through the hydrolysis of ATP (Fig. 6). The sensitivity towards ATP computed according to the formula of PORTZEHL et al. 14 was 52.60/0 . The value was Biochim. Biophys. ~lcta, 181 (1969) 191-2oo
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Fig. 6. Effect of ATP on the viscosity at 25 ° of contractile protein (Fraction B) from leucocytes. Protein concentration, 3.Io mg/ml; ionic strength, 0.60; Tris-maleate buffer, 2o mM (pH 7.o). For details see text. Fig. 7. Ultracentrifugal pattern of contractile protein (Fraction B) from leucocytes at .54 200 rev./ min in a solution containing 7.4 mg protein per ml, 0.6 M KC1, 5 mM MgC1v 5 mM ATP and 20 mM histidine buffer, pH 7.0. Sedimentation proceeds from left to right. The photograph was taken at 22 min after top speed was reached.
very low compared with those ranging from 112 to 165% obtained by PORTZEHL et al. ~4 for actomyosin from skeletal muscle, but the value of 52.6% found for Fraction B was comparable with the values, ranging from 4 ° to 64%, presented by NEEDHAM AND CAWKWELLTM for actomyosin from smooth muscle of uterus. This result again indicates a similarity in properties of Fraction B and actomyosin. Ultracentrifugal pattern The ultracentrifugal pattern of Fraction B in 20 mM histidine buffer (pH 7.o) with ionic strength of o.6 at 2o ° shows two peaks (Fig. 7) corresponding to 6 S and 27 S in the presence of ATP and Mg2+. The former peak corresponds fairly well to that of myosin z0 and the latter to that of F-actin from smooth muscle 21.
Fig. 8. Electron micrographs of the filamentous structures of contractile protein (Fraction B from leucocytes at high ionic strength. The preparation contained o.55 mg of protein per ml, 0.6 M KCI, 5 mM MgC12 and 20 mM Tris-maleate buffer, pH 7.o. The arrowhead filamentous structure ( ~ ) may be seen. Fig. 9. Electron micrograph of contractile protein (Fraction B) from leucocytes at low ionic strength. The preparation contained o.55 mg of protein per ml, o,o6 M KCI, 5 mM MgCI~ ,5 mM ATP and 20 mM Tris-maleate buffer, pH 7,o, Thick and thin filaments (M and A respectively) are visible.
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These results strongly suggest that Fraction B contained a contractile protein from leucocytes and that it had characteristics similar to those of actomyosin of muscle.
Electron-microscopic findings Fraction B at high and low ionic strengths was studied by electron microscopy. Fig. 8 shows a typical negatively stained micrograph of Fraction B at high ionic strength. The figure shows the composite filaments with almost the same type of polarized arrowhead structure as that of myosin B from muscle 15. This structure could be a complex of myosin molecules attached to an actin filament similar to that of myosin B from muscle, since on the addition of 5 mM ATP the structure disappeared and only the actin-like filament was seen. For Fig. 9 Fraction B was diluted with the same buffer containing added 5 mM ATP to reduce the concentration of KC1 to 0.o6 M, and the solution was left standing for 30 min at room temperature. In this solution, the protein showed a clearing response and it was negatively stained. In this clearing medium thick and thin filaments were seen of about 15o and 7 °/~ in diameter, respectively. It m a y be that the thick filaments correspond to myosin aggregates and the thin ones to F-actin. DISCUSSION
From the above experimental findings it m a y be concluded that Fraction B contains a contractile protein from leucocytes, a so-called actomyosin, although the protein is not yet completely purified. The protein had various biophysical and biochemical properties characteristic of actomyosin from muscle. Although the procedure employed for the extraction of the contractile protem from leucocytes was not greatly different from that for myosin B from striated muscle, which requires Mg 2+ and Ca 2+ for superprecipitation, a contractile protein which we extracted from leucocytes did not show any Ca 2+ sensitivity for superprecipitation. The reason is not yet known. Since "native tropomyosin", in the presence of which Ca 2+ effects the interaction of myosin and actin, was shown to be labile in chicken gizzard ~z, the absence of Ca ~+ sensitivity for superprecipitation of Fraction B m a y be due to the inactivation of a native tropomyosin during extraction. Some reports have been published on the presence of contractile protein in free cells and on its physiological significance. HOFFMANN-BERLING23 in 1954 succeeded in extracting an actomyosin-like protein from fibroblasts and presented convincing evidence that the motility of fibroblasts was due to the contraction of the protein under the influence of ATP. In 1956 he extracted a similar protein from Yoshida sarcoma cells and Jensen sarcoma cells 1°. In the tissue culture of sarcoma these cells also possessed some motility. In platelets too, a contractile protein called thrombosthenin was extracted by BETTEX-GALLANDAND LUSCHER 24, and the significance of this protein was discussed in the light of the observation that platelets showed a pseudopod-like protuberance and attached themselves to fibrin fibers. The motility of these cells, however, is much less than that of leucocytes, and the superprecipitation or contractility also seems to be less than that of a contractile protein extracted by us from leucocytes. The ATPase activity of actomyosin-like protein both of platelets and Yoshida sarcoma cells was about 5 times less active than that from leucocytes. t~iochim. Biophys. Acta, 181 (1969) 19I-2oo
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Recently, PUSZKII~et al. ~s isolated a protein with characteristics similar to actomyosin from whole brain of rat and cat, and YOUNG AND NELSON ze, using viscosimetric analysis, demonstrated that the contractile apparatus of the mammalian spermatozoa contained at least two proteins, flactin (resembling muscle actin) and spermosin which interacted to form a "complex" deformable in the presence of ATP and divalent cations. HATANO AND TAZAWA~7 reported the presence of myosin B in Myxomycete plasmodium whose ATPase activity was as high as that of myosin B from rabbit striated muscle, and discussed the role of the contractile protein in protoplasmic streaming. PUSZKIN et al. ~ suggested that the contractile protein in brain probably functioned in conformational changes transmitted to membranes. The interaction of flactin and spermosin was considered to be intimately involved in the conversion of chemical energy into mechanical action. There have been some reports on the extraction of contractile protein from nuclei 28 or mitochondria *°. However, these actomyosin-like proteins seem to be very low in their activities of superprecipitation or contractility. As mentioned in the INTRODUCTION,we have considered that leucocytes mu~t contain a contractile protein to achieve their vigorous movement. From this point of view, an a t t e m p t was made to extract a contractile protein from leucocytes. The actomyosin-like protein we extracted m a y contain material from nuclei or mitochondria in leucocytes, but most of the protein could possibly have originated from the parts participating in the movement of leucocytes according to our previous cytological observation on the motility of the cells and with respect to the activity of contractility of the protein extracted. REFERENCES I K. FUKUSHIMA, N. SENDA, H. INuI, H. MIURA, Y. TAMAI AND Y. MURAKAMI, Med. J. Osaka Univ., 4 (1953) 195. 2 K. FUKUSHIMA, N. SENDA, H. MIURA, S. ISHIGAMI AND Y. MURAKAMI, Med. J. Osaka Univ., 5 (1954) I. 3 K. FUKUSrtlMA, N. SENDA, H. INUI, M. YONEDA AND Y. MURAKAMI, Med. J. Osaka Univ., 5 (1954) 57. 4 N. SENDA, Sang, 25 (1954) 7o7 • 5 N. SENDA, Rev. Hematol., 9 (1954) 418. 6 K. FUKUSHIMA, N. SENDA, S. ISI~IGAm, J. ENDO, M. I s a l I , Y. MURAKAraI, K. NISHIAN AND Y. UEDA, Med. J. Osaka Univ., 5 (1954) 231. 7 N. SENDA AND H. TAmURA, Proc. 8th Intern. Congr. Haematol., Tokyo, z96o, V01.2, P a n Pacific Press, T o k y o , 1962, p. 826. 8 N. SENDA AND H. TAMURA, Ann. Rep. Center Adult Diseases, Osaka, i (1961) I. 9 H. TAKAItASm, Nagoya J. Med. Sei., 24 (1961) I. lO H. HOFFMANN-DEaLING, Bioehim. Biophys. Acta, 19 (1956) 453. 1i O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 12 B. B. MARSH, Biochim. Biophys. Acta, 32 (1959) 357. 13 S. EBASHI, J. Biochem. Tokyo, 5 ° (1961) 236. 14 H. PORTZEHL, G. SCHRAMM AND H. H. WEBER, Z. Naturforsch., 5b (195 o) 61. 15 H. E. HUXLEY, J . Mol. Biol., 7 (1963) 281. 16 R. V. RICE, H. ASAI AND M. MORALES, Proc. Natl. Aead. Sei. U.S., 5 ° (1963) 549. 17 W. HASSELBACH, Z. Naturforseh., 7 b (1952) 163. 18 D. M. NEEDHAM AND J. M. CAWKWELL, Bioehem. J., 63 (1956)337. 19 R. H. SCHIRMER, Biochem. Z., 343 (1965) 269. 2o P. JOHNSON AND A. J. ROWE, Bioehem. J., 74 (196o) 432. 21 J. H u Y s , Arch. Intern. Physiol. Biochem., 69 (1961) 667. 22 S. EBASHI, H. IWAKURA, H. NAKAJIMA, R. NAKAMURA AND Y. Ooi, Bioehem. Z., 345 (1966) 2oi.
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H. HOFFMANN-BF,RLING, Biochim. Biophys. Acta, 14 (1954) 172. M. BETTEX-GALLAND AND E. F. LUSCHER, Biochim. Biophys. Acta, 49 (1961) 536. S. PUSZKIN, S. BERL, E. PUSZKIN AND D. D. CLARKE, Science, 161 (1968) 17o. L. G. YOUNG AND L. NELSON, Exptl. Cell Res., 51 (1968) 34. S. HATANO AND M. TAZAWA, Biochim. Biophys. Acta, 154 (1968) 507. T. OHNISHI, H. KAWAMURA AND Y. TANAKA, J. Biochem. Tokyo, 56 (1964) 6. S. A. NEIFAKH, J. A. AVRAMOR, V. S. GAISKOKI, T. n . KAZAKORA, N. K. MONAKHOR, V . S . REPIN, V. V. TURORSKI AND I. M. VASSILETZ, Biochim. Biophys. Acta, IOO (1965) 329.
Biochim. Biophys. Acta, 181 (1969) 191-2oo
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35374 A CONTRACTILE P R O T E I N FROM LEUCOCYTES ITS EXTRACTION AND SOME OF ITS P R O P E R T I E S "
N O B U Y U K I SENDA, N O B U H I K O SHIBATA, N O R I Y U K I TATSUMI, K E I I C H I K O N D O AND K E I K O H A M A D A
Center for Adult Diseases, Osaka (Japan) (Received December 27th, 1968)
SUMMARY
I. A contractile protein was isolated from equine leucocytes. The protein exhibited superprecipitation at low ionic strength in presence of Mg2+ and ATP and possessed ATPase (EC 3.6.1.3) activity. The effect of divalent cations, Mg2+ and Ca 2+, on the ATPase activity resembled that on actomyosin from muscle. 2. Electron micrographs of the protein showed, at high ionic strength without ATP, the "arrowhead" structure characteristic of myosin B from striated muscle. Thick and thin filaments were observed at low ionic strength at a relatively high ATP level. 3. The possible role of the protein in the movement of leucocytes is discussed.
INTRODUCTION
Since 1948 the authors have reported the characteristic features of leucocytic motility both from the morphological and functional aspects 1-5. The migration velocity and chemo- and galvanotactic function of leucocytes were greatly accelerated by the addition of ATP to the surrounding medium. Arsenate, phloridzin and 2,4dinitrophenol inhibited the motile function of leucocytes which could be counteracted by the addition of ATP (ref. 6). These findings led us to consider that the motile form and function were coordinately controlled by ATP, and that leucocytes might have a mechanochemical system functioning with ATP as energy source. In a parallel study carried out from the phylogenetic point of view the constriction wave in leucocytes was found to be the same as that observed in Amoeba limax, Entamoeba histolytiea, Planaria gonocephala, Lumbricus trapezoides, and A plysia
kurodai 7. Abbreviation: EGTA, 1,2-bis(2-dicarboxymethylaminoethoxy)-ethane. * The work described in this paper was reported at the 2oth Annual Meeting of J a p a n Society for Cell Biology in 1967, and at the X I I t h Congress of the International Society of Hematology in New York, 1968.
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The fact that the motor organs of the above animals are governed by the muscular system led us to assume the presence of a contractile element in leucocytes and amoeba which functions circularly as well as longitudinally in their movement s and to try to isolate a contractile protein from leucocytes. This paper reports the isolation of a contractile protein having properties similar to those of actomyosin from muscle. The present finding is evidence supporting our previous hypothesis that the movement of leucocytes may be based on the reaction of contractile protein with ATP. EXPERIMENTAL PROCEDURE
Isolation of leucocytes from whole blood. From flesh equine arterial blood leucocytes were isolated by a modification of the method reported by TAKAHASHI 9, a s follows. IO.O g of EDTA (tetrasodium salt), 8.5 g of NaC1 and 3.6 g of cysteine were dissolved in I 1 of distilled water into which 9 1 of the blood were mixed. This was kept on ice during transport from a slaughter house. About i h after the blood sample was obtained the mixture was warmed to room temperature and was left to stand for 2 h to separate into two layers. The supernatant layer was removed by a syphon and then filtered through two layers of gauze in order to remove the platelets. The following procedures were all conducted at 0-4 ° . The filtrate was centrifuged for 8 rain at 250 × g, and the supernatant layer was again centrifuged at io ooo × g for 8 min to obtain platelet-free plasma. The sediment obtained by centrifugation at 250 × g was suspended in 3 vol. of platelet-free plasma and the suspension was again filtered through gauze, followed by centrifugation at 250 × g for 7 mill. By repeating this procedure twice, platelets were removed through the gauze and the red cells in the lower layer of the sediment by centrifugation. The upper half of the sediment thus obtained was washed at least twice with io vol. isotonic NaC1 and again centrifuged at 250 × g for 7 min to obtain the buffy coat of leucocytes. The volume of the huffy coat collected was about 20 ml from a starting volume of 18 1 of blood. The leucocytes counted as i.o. lO4-1.3. IO4/ram3 in whole blood at the beginning of the procedure were concentrated to i.o. lO6-1.3 • IOn/ram 3 in the huffy coat at the final step of isolation. Contamination with red cells in the huffy coat was less than 5 %, and that of platelets was negligible. About 9 o°/o of the collected leucocytes was usually found to be neutrophiles. Extraction of contractile protein from leucocytes. All procedures were conducted at about 4 °. The buffy coat was first mixed with i vol. o.i M KC1 and then with i vol. of the extract solution containing 1.6 M KC1, 2 mM ATP, 3 mM eysteine, o.oi M Na2CO 3 and 0.04 M NaHCO 3, whose pH was adjusted to 7.0 by the addition of I M acetic acid. The leucocytes were homogenized for 2 rain in a Waring blendor and a Potter-Elvehjem homogenizer. The resulting highly viscous homogenate was stirred with a magnetic stirrer for 3 h, and, after the addition of 2 mM ATP, stirred for another 9 h. This procedure reduced the viscosity of the homogenate. The homogenate was then centrifuged at 33 ooo × g for i h to remove cell fragments and corpuscular materials, leaving a pink supernatant fluid of relatively high viscosity. In order to isolate the nucleoprotein considered to be contaminating the supernatant fluid, an equivalent volume of a solution at pH 7.o containing 2 mM MgC12, o.5 mM ATP and 5 mM 1,2-bis(2-dicarboxymethylaminoethoxy)ethane (EGTA) was added according 13iochim. Biophys. Acta, ISI (1969) 191-2oo
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to the method of HOFFMANN-BERLING1°. The resulting sediment, called Fraction A and having a maximal absorption at 260 m/,, was removed b y centrifugation at io ooo × g for 20 min. I t was later found that Fraction A dissolved in 0.6 M KC1 did not exhibit superprecipitation. The supernatant fluid obtained b y centrifugation was added to 5 vol. water to reduce the ionic strength to 0.05, and the resultant flocculent precipitate was collected 2-3 h later b y centrifugation at 3000 X g for 20 min. Further, in order to remove Mg 2+, the precipitate was washed with 50 vol. 0.05 M KC1 containing I mM EDTA whose pH was adjusted to 7.0 b y Tris. After metalchelated and free EDTA had been removed b y washing with IOO vol. 0.05 M KCI solution, the sediment was dissolved in an appropriate volume of 2.4 M KC1 solution whose ionic strength was finally adjusted to 0.6 with water. The contaminated corpuscular materials were removed b y centrifugation at 33 ooo x g for 2o mill. Precipitation and dissolution of the above supernatant fluid in KC1 solution of final ionic strength of either 0.05 or 0.6 was repeated at least twice. The protein in 0.6 M KC1 was kept at o ° (Fraction B), and used in testing for the properties a contractile protein should demonstrate. The foregoing extraction procedure was completed within 24 h and the tests were conducted within 2 days after the extraction. The protein in Fraction B extracted from 20 ml of buffy coat of leucocytes usually amounted to about 5 ° mg. The amount of protein was determined b y the method of LOWRY et al. 11 A T P a s e activity. ATPase (EC 3.6.1.3) activity of contractile protein was measured by determining the amount of Pi liberated from ATP b y the method of MARSH12. Superprecipitation. The ionic strength of the reaction mixture used for observation of superprecipitation was adjusted to 0.06 b y mixing Fraction B into the appropriate reaction medium indicated in the text. The superprecipitation was followed with a Hitachi UV-VIS spectrophotometer by measuring the change in absorbance at 660 m/, in the reaction mixture after addition of ATP at room temperature according to the method developed by EBASH113. In some experiments the superprecipitation in the reaction mixture containing Fraction B was also followed with time macroscopically and b y means of the phase-contrast microscope after the addition of ATP. Viscosimetric determination. This was performed in an Ostwald viscosimeter with 5-ml capacity at 25 °. The relative viscosity was measured at p H 7.0 on Fraction B in 0.6 M KC1 (~rel) and after addition of 0.05 ml of a solution containing 0.5 M MgC12 and o.I M A T P in 0.6 M KC1 ('Y]relATP)- The sensitivity towards ATP was calcula{ed (in °/o) according to PORTZEHL et al. 14 by the formula l o g e ~ r e l - - l o g e ~ r e l ATP
lOge~rel A T P
X IOO
Ultracentrifuge. A Hitachi Model UCA-I analytical ultracentrifuge was employed for the sedimentation-velocity measurement. Electron microphotographs. Micrographs of Fraction B were taken b y the negative-staining technique of HUXLEY as. One drop of the ice-cold preparation of Fraction B was placed on a microgrid coated with collodion carbon. The preparation was negatively stained with I~o uranyl acetate, and examined in a Nihon Denshi Model J E M 6C electron microscope with an acceleration voltage of 80 kV. Biochim. Biophys. Acta, 181 (1969) 191-2oo
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Fig. I. S u p e r p r e c i p i t a t i o n of contractile p r o t e i n ( F r a c t i o n ]3) f r o m leucocytes. T h e u p p e r series of p i c t u r e s h o w s t h e c o n t r a c t i o n of p r e c i p i t a t e d p r o t e i n in t h e presence of o.5 m M A T P a t 2o °. T h e lower series s h o w s t h e s e d i m e n t a t i o n w i t h o u t a d d e d A T P . P r o t e i n c o n c e n t r a t i o n , 1.2 5 m g / m l ; ionic s t r e n g t h , o.o6; MgC12, 5 m M ; T r i s - m a l e a t e buffer, 2o m M (pH 7.0); t o t a l vol., 3.o ml.
Chemicals. All chemicals used for this study were analytical grade. Tris-ATP was obtained from Sigma Chemical Co., U.S.A., and EGTA from the Dojin Igaku Institute. The water used in preparing solutions was redistilled in glass. RESULTS
Superpr ecipitation It is a well-known characteristic of actomyosin, the contractile protein of muscle, to undergo superprecipitation at low ionic strength in the presence of Mg 2+ by the addition of a low concentration of ATP. A marked superprecipitation was observed in the reaction mixture with low ionic strength (0.06) containing 5 mM MgCI~, 20 mM Tris-maleate buffer (pH 7.0) and Fraction B in the presence of 0.5 mM ATP (Fig. I). The superprecipitation reached its maximal level in about 15 min after the addition of ATP. Fig. 2 shows the phase-contrast micrograph of Fraction B in the superprecipitation under the same
Fig. 2. P h a s e - c o n t r a s t m i c r o g r a p h s of contractile p r o t e i n (Fraction B) f r o m leucocytes. T h e exp e r i m e n t a l c o n d i t i o n s are t h e s a m e as for Fig. 1. R i g h t , s u p e r p r e c i p i t a t e d p r o t e i n after t h e a d d i tion of A T P . Left, w i t h o u t a d d i t i o n of A T P . × 9o0.
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195
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CONTRACTILE PROTEIN FROM LEUCOCYTES
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Fig. 3. Effects of Ca ~+ and E D T A on the superprecipitation at 20 ° of contractile protein (Fraction B) from leucocytes. Protein concentration, 0.8o m g / m l ; ionic strength, 0.o6; T r i s - m a l e a t e buffer, 2o mM (pH 7.o) ; ATP, o.5 mM ; total vol. 3.o ml. The reaction was s t a r t e d b y the addition of ATP. G - - C ) , w i t h added 0. 5 mM E D T A ; x - - x , with added o. 5 mM E G T A and 5 mM MgC12; ~ - - - - - - A , in presence of 0. 5 mM CaC12 a n d 5 mM MgC1v Fig. 4. Effects of added Mg ~+ on the superprecipitation at 20 ° of contractile protein (Fraction B from leucocytes. Protein concentration, o.52 m g / m l ; ionic strength, o.06; EGTA, 0. 5 mM; T r i s maleate buffer, 2o mM (pH 7.o) ; ATP, o.1 mM; total vol. 3.o ml. The reaction was s t a r t e d b y the addition of ATP. Final concentrations of added MgCI~ were 5 ° .uM and o. i mM ( × - - × ), 5 mM (Q 0 ) , IO and 20 mM ( ~ - - ~ ) and 3 ° mM ( C ) - - - - - - © ) . C)--C), with no added MgC12; 0 - - 0 , with I mM E D T A .
condition. The synerated feature was similar to that observed by RICE et al. 16 in skeletal myosin B. The rapid increase in superprecipitation was measured by the increase in absorbance at 660 m/~, as shown in Fig. 3. The marked superprecipitation was induced by adding o.5 mM ATP in presence of 5 mM MgC12 and 0.5 mM CaC12. The addition of 0.5 mM EGTA, a relatively specific chelater of Ca ~+, had no effect on the degree of superprecipitation, indicating non-participation of Ca z+, whereas the addition of 0.5 mM EDTA to the reaction mixture with no added Mg z+ and Ca ~+ resulted in complete inhibition of the superprecipitation by ATP. Fig. 4 shows the effect on the superprecipitation of adding Mg ~+ to the reaction mixture containing Fraction B, 0.5 mM EGTA, 2o mM Tris-maleate buffer (pH 7.0) and o.I mM ATP. Up to 20 mM of added MgCI~ the superprecipitation was enhanced, then was gradually inhibited by increasing concentration of Mg 2+. The occurrence of a considerable degree of superprecipitation without addition of Mg 2+ could possibly be due to a trace of contaminating Mg z+ during the preparation of Fraction B. The requirement of Mg 2+ but not Ca z+ for the superprecipitation by ATP of Fraction B is the same as that reported with synthetic actomyosin from striated muscle, although the optimal concentration of Mg2+ in the striated muscle is considerably lower than that in our experiments. A T P a s e activity
If Fraction B contains a contractile protein similar to actomyosin from muscle, it should exhibit ATPase activity affected b y Mg ~+, Ca 2+ and ionic strength. Biochim. Biophys. Acta, 181 (1969) 191-2oo
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Fig. 5. A T P a s e a c t i v i t y of contractile p r o t e i n (Fraction B) f r o m leucocytes. P r o t e i n c o n c e n t r a tion, 0.30 m g / m l ; T r i s - m a l e a t e buffer, 20 m M (pH 7.o) ; A T P , i m M ; E D T A , (when added) I raM; total vol. 3.o ml. (A) Effect of KC1 in presence of 5 m M MgCl v (B) Effect of Mg *+. (C) Effect of Ca *+ a t two different ionic s t r e n g t h s , 0.06 ( ) a n d 0.60 ( - - - - - - ) . T h e reaction w a s s t a r t e d b y t h e a d d i t i o n of A T P , a n d t h e A T P a s e a c t i v i t y w a s d e t e r m i n e d b y m e a s u r i n g t h e Pl liberated for IO m i n at 20 °.
The Mg2+-dependent ATPase activity of Fraction B under conditions shown in Fig. 5A became lower as the ionic strength was increased. The activity was 0.023 and O.Ol7 mole Pi liberated per mg protein per min at ionic strengths of 0.06 and 0.6, respectively. As shown in the dotted line in Fig. 5B, the ATPase activity of Fraction B was inhibited by increasing concentration of Mg 2+ at high ionic strength, whereas at low ionic strength, as shown by the solid line, the activity was apparently activated with increase in concentration of Mg 2+. As shown in Fig. 5C ATPase was activated by Ca *+ both at high and low ionic strengths, though in lesser degree at low strength. In actomyosin from skeletal muscle, HASSELBACH17 reported that the Mg 2~dependent ATPase was inhibited as the ionic strength was raised, the activity being about 0.26 and o.oi/~mole Pi liberated per mg protein per min at ionic strengths of o.o7-o.1 and 0.6, respectively, in the presence of I mM Mg 2+ and 0. 9 mM ATP. The Ca2+-dependent ATPase activity in the presence of o.oi M Ca 2+ and 0. 9 mM ATP was about 0.35 and o.15 #mole Pt liberated per mg protein per rain at ionic strengths of o.o7-o.1 and 0.6, respectively. Ca 2+, however, activated ATPase activity of actomyosin from skeletal muscle and Fraction B in high and low ionic strengths, although the enzymic activity of the former was high in low ionic strength compared with that in the higher one, but in the latter the situation was reversed (0.040 and 0.085 #mole Pi liberated per mg protein per rain at low and high ionic strengths in the presence of o.oi M Ca2+). The same was true for actomyosin from smooth muscle ls,19. It seems reasonable to postulate that Fraction B exhibited ATPase (EC 3.6.1.3) activity of the actomyosin type which was activated b y Mg 2+ and Ca ~+ at low ionic strength, and also ATPase activity of the myosin type which was inhibited b y Mg 2÷ and activated by Ca ~+ at high ionic strength, under conditions in which actomyosin is dissociated into myosin and actin.
Change in viscosity by addition of A T P The relative viscosity of Fraction B was decreased by the addition of ATP and Mg 2+ at high ionic strength. This was followed b y a gradual increase towards the initial leve] through the hydrolysis of ATP (Fig. 6). The sensitivity towards ATP computed according to the formula of PORTZEHL et al. 14 was 52.60/0 . The value was Biochim. Biophys. ~lcta, 181 (1969) 191-2oo
CONTRACTILE PROTEIN FROM LEUCOCYTES
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1,50ATIo_c~ 1.40 41,
l °r 1'20t
0
30
60 90 Time (rain)
12J0
Fig. 6. Effect of ATP on the viscosity at 25 ° of contractile protein (Fraction B) from leucocytes. Protein concentration, 3.Io mg/ml; ionic strength, 0.60; Tris-maleate buffer, 2o mM (pH 7.o). For details see text. Fig. 7. Ultracentrifugal pattern of contractile protein (Fraction B) from leucocytes at .54 200 rev./ min in a solution containing 7.4 mg protein per ml, 0.6 M KC1, 5 mM MgC1v 5 mM ATP and 20 mM histidine buffer, pH 7.0. Sedimentation proceeds from left to right. The photograph was taken at 22 min after top speed was reached.
very low compared with those ranging from 112 to 165% obtained by PORTZEHL et al. ~4 for actomyosin from skeletal muscle, but the value of 52.6% found for Fraction B was comparable with the values, ranging from 4 ° to 64%, presented by NEEDHAM AND CAWKWELLTM for actomyosin from smooth muscle of uterus. This result again indicates a similarity in properties of Fraction B and actomyosin. Ultracentrifugal pattern The ultracentrifugal pattern of Fraction B in 20 mM histidine buffer (pH 7.o) with ionic strength of o.6 at 2o ° shows two peaks (Fig. 7) corresponding to 6 S and 27 S in the presence of ATP and Mg2+. The former peak corresponds fairly well to that of myosin z0 and the latter to that of F-actin from smooth muscle 21.
Fig. 8. Electron micrographs of the filamentous structures of contractile protein (Fraction B from leucocytes at high ionic strength. The preparation contained o.55 mg of protein per ml, 0.6 M KCI, 5 mM MgC12 and 20 mM Tris-maleate buffer, pH 7.o. The arrowhead filamentous structure ( ~ ) may be seen. Fig. 9. Electron micrograph of contractile protein (Fraction B) from leucocytes at low ionic strength. The preparation contained o.55 mg of protein per ml, o,o6 M KCI, 5 mM MgCI~ ,5 mM ATP and 20 mM Tris-maleate buffer, pH 7,o, Thick and thin filaments (M and A respectively) are visible.
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These results strongly suggest that Fraction B contained a contractile protein from leucocytes and that it had characteristics similar to those of actomyosin of muscle.
Electron-microscopic findings Fraction B at high and low ionic strengths was studied by electron microscopy. Fig. 8 shows a typical negatively stained micrograph of Fraction B at high ionic strength. The figure shows the composite filaments with almost the same type of polarized arrowhead structure as that of myosin B from muscle 15. This structure could be a complex of myosin molecules attached to an actin filament similar to that of myosin B from muscle, since on the addition of 5 mM ATP the structure disappeared and only the actin-like filament was seen. For Fig. 9 Fraction B was diluted with the same buffer containing added 5 mM ATP to reduce the concentration of KC1 to 0.o6 M, and the solution was left standing for 30 min at room temperature. In this solution, the protein showed a clearing response and it was negatively stained. In this clearing medium thick and thin filaments were seen of about 15o and 7 °/~ in diameter, respectively. It m a y be that the thick filaments correspond to myosin aggregates and the thin ones to F-actin. DISCUSSION
From the above experimental findings it m a y be concluded that Fraction B contains a contractile protein from leucocytes, a so-called actomyosin, although the protein is not yet completely purified. The protein had various biophysical and biochemical properties characteristic of actomyosin from muscle. Although the procedure employed for the extraction of the contractile protem from leucocytes was not greatly different from that for myosin B from striated muscle, which requires Mg 2+ and Ca 2+ for superprecipitation, a contractile protein which we extracted from leucocytes did not show any Ca 2+ sensitivity for superprecipitation. The reason is not yet known. Since "native tropomyosin", in the presence of which Ca 2+ effects the interaction of myosin and actin, was shown to be labile in chicken gizzard ~z, the absence of Ca ~+ sensitivity for superprecipitation of Fraction B m a y be due to the inactivation of a native tropomyosin during extraction. Some reports have been published on the presence of contractile protein in free cells and on its physiological significance. HOFFMANN-BERLING23 in 1954 succeeded in extracting an actomyosin-like protein from fibroblasts and presented convincing evidence that the motility of fibroblasts was due to the contraction of the protein under the influence of ATP. In 1956 he extracted a similar protein from Yoshida sarcoma cells and Jensen sarcoma cells 1°. In the tissue culture of sarcoma these cells also possessed some motility. In platelets too, a contractile protein called thrombosthenin was extracted by BETTEX-GALLANDAND LUSCHER 24, and the significance of this protein was discussed in the light of the observation that platelets showed a pseudopod-like protuberance and attached themselves to fibrin fibers. The motility of these cells, however, is much less than that of leucocytes, and the superprecipitation or contractility also seems to be less than that of a contractile protein extracted by us from leucocytes. The ATPase activity of actomyosin-like protein both of platelets and Yoshida sarcoma cells was about 5 times less active than that from leucocytes. t~iochim. Biophys. Acta, 181 (1969) 19I-2oo
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Recently, PUSZKII~et al. ~s isolated a protein with characteristics similar to actomyosin from whole brain of rat and cat, and YOUNG AND NELSON ze, using viscosimetric analysis, demonstrated that the contractile apparatus of the mammalian spermatozoa contained at least two proteins, flactin (resembling muscle actin) and spermosin which interacted to form a "complex" deformable in the presence of ATP and divalent cations. HATANO AND TAZAWA~7 reported the presence of myosin B in Myxomycete plasmodium whose ATPase activity was as high as that of myosin B from rabbit striated muscle, and discussed the role of the contractile protein in protoplasmic streaming. PUSZKIN et al. ~ suggested that the contractile protein in brain probably functioned in conformational changes transmitted to membranes. The interaction of flactin and spermosin was considered to be intimately involved in the conversion of chemical energy into mechanical action. There have been some reports on the extraction of contractile protein from nuclei 28 or mitochondria *°. However, these actomyosin-like proteins seem to be very low in their activities of superprecipitation or contractility. As mentioned in the INTRODUCTION,we have considered that leucocytes mu~t contain a contractile protein to achieve their vigorous movement. From this point of view, an a t t e m p t was made to extract a contractile protein from leucocytes. The actomyosin-like protein we extracted m a y contain material from nuclei or mitochondria in leucocytes, but most of the protein could possibly have originated from the parts participating in the movement of leucocytes according to our previous cytological observation on the motility of the cells and with respect to the activity of contractility of the protein extracted. REFERENCES I K. FUKUSHIMA, N. SENDA, H. INuI, H. MIURA, Y. TAMAI AND Y. MURAKAMI, Med. J. Osaka Univ., 4 (1953) 195. 2 K. FUKUSHIMA, N. SENDA, H. MIURA, S. ISHIGAMI AND Y. MURAKAMI, Med. J. Osaka Univ., 5 (1954) I. 3 K. FUKUSrtlMA, N. SENDA, H. INUI, M. YONEDA AND Y. MURAKAMI, Med. J. Osaka Univ., 5 (1954) 57. 4 N. SENDA, Sang, 25 (1954) 7o7 • 5 N. SENDA, Rev. Hematol., 9 (1954) 418. 6 K. FUKUSHIMA, N. SENDA, S. ISI~IGAm, J. ENDO, M. I s a l I , Y. MURAKAraI, K. NISHIAN AND Y. UEDA, Med. J. Osaka Univ., 5 (1954) 231. 7 N. SENDA AND H. TAmURA, Proc. 8th Intern. Congr. Haematol., Tokyo, z96o, V01.2, P a n Pacific Press, T o k y o , 1962, p. 826. 8 N. SENDA AND H. TAMURA, Ann. Rep. Center Adult Diseases, Osaka, i (1961) I. 9 H. TAKAItASm, Nagoya J. Med. Sei., 24 (1961) I. lO H. HOFFMANN-DEaLING, Bioehim. Biophys. Acta, 19 (1956) 453. 1i O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 12 B. B. MARSH, Biochim. Biophys. Acta, 32 (1959) 357. 13 S. EBASHI, J. Biochem. Tokyo, 5 ° (1961) 236. 14 H. PORTZEHL, G. SCHRAMM AND H. H. WEBER, Z. Naturforsch., 5b (195 o) 61. 15 H. E. HUXLEY, J . Mol. Biol., 7 (1963) 281. 16 R. V. RICE, H. ASAI AND M. MORALES, Proc. Natl. Aead. Sei. U.S., 5 ° (1963) 549. 17 W. HASSELBACH, Z. Naturforseh., 7 b (1952) 163. 18 D. M. NEEDHAM AND J. M. CAWKWELL, Bioehem. J., 63 (1956)337. 19 R. H. SCHIRMER, Biochem. Z., 343 (1965) 269. 2o P. JOHNSON AND A. J. ROWE, Bioehem. J., 74 (196o) 432. 21 J. H u Y s , Arch. Intern. Physiol. Biochem., 69 (1961) 667. 22 S. EBASHI, H. IWAKURA, H. NAKAJIMA, R. NAKAMURA AND Y. Ooi, Bioehem. Z., 345 (1966) 2oi.
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H. HOFFMANN-BF,RLING, Biochim. Biophys. Acta, 14 (1954) 172. M. BETTEX-GALLAND AND E. F. LUSCHER, Biochim. Biophys. Acta, 49 (1961) 536. S. PUSZKIN, S. BERL, E. PUSZKIN AND D. D. CLARKE, Science, 161 (1968) 17o. L. G. YOUNG AND L. NELSON, Exptl. Cell Res., 51 (1968) 34. S. HATANO AND M. TAZAWA, Biochim. Biophys. Acta, 154 (1968) 507. T. OHNISHI, H. KAWAMURA AND Y. TANAKA, J. Biochem. Tokyo, 56 (1964) 6. S. A. NEIFAKH, J. A. AVRAMOR, V. S. GAISKOKI, T. n . KAZAKORA, N. K. MONAKHOR, V . S . REPIN, V. V. TURORSKI AND I. M. VASSILETZ, Biochim. Biophys. Acta, IOO (1965) 329.
Biochim. Biophys. Acta, 181 (1969) 191-2oo