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A contractile protein possessing Ca2+ sensitivity (natural actomyosin) from leucocytes. Its extraction and some of its properties
BIOCHIMICA ET BIOPHYSICA ACTA
565
m~A 4~ 237 A CONTRACTILE PROTEIN
P O S S E S S I N G Ca 2+ S E N S I T I V I T Y ( N A T U R A L
ACTOMYOSIN) F R O M L E U C O C Y T E S ITS E X T R A C T I O N AND SOME OF ITS P R O P E R T I E S " N(~BUHII,~() SHIBATA, NORIYUKI TATSUMI, KAZUHIKO TANAKA, \-()SHIM[ OI,~AMURA AND NOBUYUKI SENDA Ccldcl'.fi~r Adult Diseases, Osaka (Japan)
(Received June 281h, 197f)
SUMMARY I. Methods have been developed for tile isolation of contractile protein from equine leucocytes resembling n a t u r a l actomyosin of the smooth nmscle. 2. This protein exhibited ATPase (EC 3.6.1.3) a c t i v i t y and u n d e r w e n t superprecipitation at low ionic strength in the presence of ATP, Mg 2+ a n d Ca"+, b u t revealed no superprecipitation in the absence of either Ca 2+ or Mg ~-+ even in the presence of ATP. Superprecipitation of this protein was enhanced b y an increase of Ca ~+ conc e n t r a t i o n in a m e d i u m c o n t a i n i n g Mg 2+ and its i n t e n s i t y was closely associated with e n h a n c e m e n t of the Mg~+-dependent ATPase (EC 3.6.1.3) activity of this protein by an increase of Ca 2+. The free Ca 2+ concentration at which the Mg~+-dependent ATPase a c t i v i t y of the protein was half activated was a b o u t I- io 4 M. Superprecipit a t i o n of this protein was e n h a n c e d b y an increase of Mg 2+ c o n c e n t r a t i o n in a m e d i u m c o n t a i n i n g Ca 2÷. I n this case, too, the superprecipitation was closely associated with e n h a n c e m e n t of the ATPase a c t i v i t y of this protein t33: increasing Mg 2+ with a c o n s t a n t Ca ~- concentration. The ATPase a c t i v i t y was half activated in the presence of a b o u t 3 mM Mg 2+. 3. Electron micrographs of the protein showed thick a n d thin filaments at low ionic strength a n d a relatively high A T P level conditions in which muscular actomyosin was dissociated into myosin aggregates a n d fibrous actin. At a relatively low A T P level, b o t h thin a n d thick filaments were aggregated with each other, as in actomvosin from the muscle, which seemed to be subject to superprecipitation.
INTR~ ) D U C T I O N
On the basis of the s t u d y of the motile form and function of leucocytes, we have suggested t h a t leucocvtes are most likely to possess a motile organ, a mechanochemical system coupled with ATP l, of which the essential substance m a y be a k i n d of actoAbbreviations: I';GTA, t,2dois-(2-dicarboxymethylarninoethoxy)ethane; KIU, trypsin l(allikrein inhibi~corvunit. * An outline of this paper was reported at the 8th Annual Meeting of the Japan Society of the l{cticuloendothelial System Research in ()saka, Japan, 197o. Biochim. l~i@hys. Acta, 256 (i972) 565 576
566
x. SlIIBAT.\ c! al.
myosin-like contractile p r o t e i n similar to t h a t of tile muscle". A contractile protein was first isolated b y us from equine leucocytes a in I967. The protein had various biophysical and biochemical properties characteristic of actomyosin from the muscle. However, it did not show a n y Ca 2+ s e n s i t i v i t y in superprecipitation, unlike n a t u r a l a c t o m y o s i n or nlyosin B from muscle. This finding posed the problem of whether " n a t i v e t r o p o n l y o s i n " , consisting of t r o p o m y o s i n and t r o p o n i n < s, in the presence of which Ca e+ effects the interaction of myosin and actin from the muscle, was subs t a n t i a l l y absent in leucocytes or i n a c t i v a t e d during the e x t r a c t i o n process. Thus, an a t t e m p t was m a d e to e x t r a c t a contractile protein from leucocytes possessing Ca e~- s e n s i t i v i t y silnilar to n a t u r a l actomyosin. EXP/~;RIMENTAL P R O C E D U R E
Isolation of leucocytes from whole blood Leucocytes were collected from fresh equine arterial blood. The isolation procedure of leucocytes from whole b l o o d was the same as t h a t e m p l o y e d previously except for some points 3. The modifications were the use of blood suspending solution containing d i t h i o t h r e i t o l and Trasylol< ~ of a strong inhibitor for protease. T h a t is, IO.O g of E D T A ( t e t r a s o d i u m salt), &5 g of NaC1, 3.6 g of cysteine, 2 m g of dithiothreitol a n d IO 5 K I U (I K I U corresponds to o.I 4/~g of Trasylol) of Trasylol dissolved in 1o ml of o.15 M saline solution were dissolved in I 1 of distilled w a t e r into which 9 1 of the blood was mixed. The w)tume of leucocyte buffy coat collected from a s t a r t i n g volmne of i~ t of b l o o d was 40-5 ° m l . As in the previous e x p e r i m e n t a, ttle leucocytes (I.O- IO4-i.3 • lO 4, m m a in whole blood) were c o n c e n t r a t e d to I.O. IoG-I.3 • Io6/mm a in the b u l l y coat at the final step of isolation. C o n t a m i n a t i o n with red cells in the huffy coat was less t h a n 5 °o, and t h a t of p l a t e l e t s was negligible. A b o u t 9o % of the collected leucocytes were usually counted to be neutrophiles on the smear p r e p a r a t i o n .
Extraction of contractile protein possessing Ca 2+ sensitivity from leucocytes All procedures were c o n d u c t e d at a b o u t 4 ' . The buffy coat of leucocytes was first m i x e d with ¼ vol. of T r a s y l o l in o.15 M NaCI (lO 5 K I U in IO mI), 2 mM dithiothreitol and 2.5 vo]. of tlle e x t r a c t i o n solution containing I mM A T P and 20 mM histidine buffer (pH 7.0). The final ionic s t r e n g t h of tllis m i x t u r e was about 0.o5. The leucocytes were homogenized in a Virtis homogenizer at half the maxinaum speed and, further, in a P o t t e r - E l v e h j e m homogenizer for 30 sec each. This homogenization procedure was v e r y i m p o r t a n t for o b t a i n i n g contractile protein possessing significant sensitivity for Ca 2÷ in s u p e r p r e c i p i t a t i o n , so it was carried out v e r y carefully. The contractile protein p r e p a r e d without the above care with regard to the homogenation d e m o n s t r a t e d only a p a r t i a l Ca `,+ sensitivity, although it was never devoid of Ca "~+ sensitivity. The resulting homogenate was not as viscous as tile leucocyte h o m o g e n a t e p r e p a r e d b y the previous procedure a. The fiomogenate was then centrifuged at 4oo00 7< g for I h to remove cell fragments and corpuscular material, leaving a pink s u p e r n a t a n t fluid. This s u p e r n a t a n t was dialysed for 2 d a y s against a b o u t 25 vol. of 5 mM histidine buffer (pH 7.o), containing 0.05 M KC1 and 2 mM ditlliothreitol in order to remove ATP. The resultant flocculent p r e c i p i t a t e was collected b y centrifugation at 3000 :>,~,~ for 20 rain. The sediment was dissolved in an tJMchbn
lTi,ptu,<~. At/a, 256 (T072) 505 570
C~)NTRACT1LE PROTEIN FROM LEUC()CYTES
5(i7
a p p r o p r i a t e volume of 2. 4 M KCI solution coutaining 2 mM d i t h i o t h r e i t o l whose ionic s t r e n g t h was finally a d j u s t e d to o.6 with water, The indissoluble m a t e r i a l was r e m o v e d b y centrifugation at 1oooo 7< g for I o rain. The s u p e r n a t a n t was again dialvsed for about I(~ h against a b o u t Ioo vol. of 5 mM histidine buffer (pH 7.o) containing o.24 M KC1 and 2 mM dithiothreitol. T h e r e s u l t a n t flocculent p r e c i p i t a t e was collected bv centrifugation at 3ooo z: g for I5 rain. The sediment was again dissolved in o.(i M KC1 (pH 7.o) containing 2 mM dithiothreitol. The h a r d l y dissolved m a t e r i a l was r e m o v e d b y centrifugation at 33ooo ~, g for 3 o rain. P r e c i p i t a t i o n and dissolution ~f the above s u p e r n a t a n t fluid was r e p e a t e d at least twice in KCI solution containing 2 mM d i t h i o t h r e i t o l at the final ionic strength of o.o6 and o.6, respectively. The p r o t e i n dissolved in o.0 M KC1 was k e p t at o ' a n d used for e x p e r i m e n t s within a d a y after the extraction. The protein e x t r a c t e d from 4 ° ml of the buffy coat of leucocytes u s u a l l y a m o u n t e d to a h o u t I6o mg. The a m o u n t of protein was d e t e r m i n e d b y the lnethod of LOWRY el al. 8 using bovine serum a l b u m i n as s t a n d a r d . .1 T P a s c a c t i v i ( v
The effect of Ca ~+ on the Mg'~+-dependent A T P a s e (EC 3.6.i.3) a c t i v i t y was d e t e r m i n e d in the coupled s y s t e m with p y r u v a t e kinase as an A T P generating s y s t e m b y m e a s u r i n g t h e time dependence of p y r u v a t e liberation according to the m e t h o d of RZYXA~I~ d alY. The reaction m e d i u m was composed of 0.4-0. 7 mg protein per ml, 0.6 #g p y r u v a t e kinase per ml, • m M p h o s p h o e n o l p y r u v a t e , I o mM T r i s - m a l e a t e (pH 7.o), I o mM MgC12, different a m o u n t s of Ca 2~ set u p bv C a - E G T A buffer (E(iTA, raM), 0.06 M KC1 and o.I mM ATP. The reaction was s t a r t e d b y an a d d i t i o n of A T P and was carried out at 3 7 . At a p p r o p r i a t e times, o.5 ml of the reaction m i x t u r e was t r a n s f e r r e d to a solution containing o.2 ml of 2.5 mM 2 , 4 - d i n i t r o p h e n y l h y d r a z i n in 3 M HCI to stop the reaction. This coupled system was not influenced b y the Mg ~+ c o n c e n t r a t i o n in the m e d i u m over the range from 0.5 to 2o raM. The effect of Mg 2+ on t h e A T P a s e (EC 3.6.I.3) a c t i v i t y u n d e r a constant Ca ~+ concentration was likewise determined. In the d e t e r m i n a t i o n of the A T P a s e a c t i v i t y of the reaction m e d i u m w i t h ~ mM A T P as s u b s t r a t e , the reaction was i n i t i a t e d b y adding A T P and was s t o p p e d b y a d d ing 5 %, trichloroacetie acid at a p p r o p r i a t e times. In this case, the coupling s y s t e m consisting of p h o s p h o e n o l p y r u v a t e a n d p y r u v a t e kinase was o m i t t e d . Then, the a m o u n t of Pi l i b e r a t e d was d e t e r m i n e d b y the m e t h o d of MARSIt 1°. ~uperprecipitation
The ionic s t r e n g t h of the reaction m i x t u r e used for observation of s u p e r p r e c i p i t a tion was brought to 0.06 b y m i x i n g the protein solution into an a p p r o p r i a t e reaction m e d i u m i n d i c a t e d in the text. The s u p e r p r e c i p i t a t i o n was followed with a H i t a c h i UV-VIS s p e c t o r o p h o t o m e t e r b y measuring the change in absorbanee at 660 nm in the reaction m i x t u r e on a d d i t i o n of A T P at room t e m p e r a t u r e following the m e t h o d developed by EBASH111. In some e x p e r i m e n t s the s u p e r p r e c i p i t a t i o n in the reaction m i x t u r e containing t h e protein was also macroscopically studied. I "i s c o s i m e t r i c d e t e r m i n a t i o n
This was p e r f o r m e d in an Ostwald viscosimeter with I ml c a p a c i t y at 25 :'. The r e l a t i v e viscosity was m e a s u r e d at p H 7.0 first in 0.6 M KC1 (~re0 and then after Biochim. Biopkys. Acta, _,5(i (1972) 565 ~576
568
x . S H I B A T A t'[ a[.
a d d i t i o n of o.oi nil of a solution containing 0.5 M MgC12 and o.~ M A T P in o.0 M KC1 (t]rel ATP). The s e n s i t i v i t y to A T P was calculated (in %) according to PoR'rzmm el al. 12 with the fornmla: logdb~, - l o g ~ q ~ A T P Iog~q~jATP
X IOO
( ~ltracentrif~tgc A H i t a c h i Model UCA-I a n a l y t i c a l ultracentrifuge was e m p l o y e d for the s e d i m e n t a t i o n - v e l o c i t y measurements.
Eleclronmicrophotographs Micrographs of the protein were t a k e n b y the n e g a t i v e - s t a i n i n g technique of HvxLI~v '3. One drop of an ice-cold p r e p a r a t i o n of the protein was placed on a microgrid coated with collodion carbon. The p r e p a r a t i o n was n e g a t i v e l y stained with 1 % u r a n y t acetate, and e x a m i n e d in a Nihon I)enshi Model J E M 6(; electronmicroscope with an acceleration voltage of 80 kV.
Chemicals The chenficals used were a n a l y t i c a l grade. Trasylot, was o b t a i n e d from B a y e r Leverkusen, G e r m a n y a n d E G T A of a Ca2+-chelating agent from Dojin I g a k u Institute, J a p a n . Various free Ca 2+ concentrations were set up b y means of C a - E G T A buffer p r e p a r e d in the m a n n e r described by P(mTZmtL el aI. '4. The w a t e r used in p r e p a r i n g the solution was redistilled in a glass vessel. RESULTS
Nuperprecipitation I t is a well-known characteristic of n a t u r a l a c t o m y o s i n , the contractile protein of the muscle, to undergo s u p e r p r e c i p i t a t i o n at low ionic strength in the presence of not only Mg 2+ and A T P b u t also Ca 2+ (ref. rx). As shown in Fig. ~, the e x t r a c t e d protein from leucocytes showed a m a r k e d s u p e r p r e c i p i t a t i o n on a d d i t i o n of o.x mM A T P in a reaction m i x t u r e with low ionic s t r e n g t h (0.06) of I o mM T r i s - m a l e a t e buffer (pH 7.o) containing i o mM MgC12 and x. xo-4 M Ca "2+ (Fig. I, right), b u t failed to d e m o n s t r a t e s u p e r p r e c i p i t a t i o n in the m e d i u m w i t h o u t free Ca 2+ (addition of x mM E G T A alone instead of I mM Ca-EGTA buffer) (Fig. I, middle). T h a t is, i m m e d i a t e l y after the a d d i t i o n of ATP, t u r b i d i t y of the reaction m i x t u r e r a p i d l y increased in tbe vessel containing I . i o -4 M Ca 2+ (Fig. i , u p p e r right), whereas it decreased to exhibit clearing when Ca 2+ was e l i m i n a t e d by the a d d i t i o n of o n l y E G T A i n s t e a d of C a - E G T A buffer (Fig. I., u p p e r middle). ~.o rain after the a d d i t i o n of ATP, the protein showed a m a r k e d s u p e r p r e c i p i t a t i o n (Fig. I, lower right), b u t it k e p t clearing in the m e d i u m not containing free Ca ~+ (Fig. i, lower middle) a n d no s u p e r p r e c i p i t a t i o n was observed in the control (left) containing no a d d e d ATP. I t was e v i d e n t t h a t the degree of s u p e r p r e c i p i t a t i o n which was m e a s u r e d b y an increase in absorbance at 660 nm (Fig. 2) was associated with the concentration of free Ca ~+ in the medium. T h a t is, a s u p e r p r e c i p i t a t i o n occurred significantly at conc e n t r a t i o n s a b o v e i . IO * M Ca "-~- and increased up to i . Io -4 M Ca z+, whereas the 13i(,cki,z. tHophys..qela. 25~ (t()72) 5~)5 57~
569
CONTRACTILE PROTEIN FI~,()3I LEUCOCYTES
l:ig. ~. S u p e r p r e c i p i t a t i o n of t h e n a t u r a l a c t o m y o s i n - l i k e c o n t r a c t i l e p r o t e i n e x t r a c t e d from leucocytes. T h e u p p e r series s h o w s c h a n g e s in t u r b i d i t y j u s t after an a d d i t i o n of ATP. T h e lower simws s e d i m e n t a t i o n of t h e p r o t e i n 2o rain after an a d d i t i o n of ATP. T h e left vessels in each series are controls for each c o n t a i n i n g no a d d e d ATP. T h e middle vessels in each series c o n t a i n i n151 lCGTA i n s t e a d of C a - E G T A buffer. The controls c o n t a i n I - lo -4 M Ca 2+. P r o t e i n c o n c e n t r a t i o n , 1.75 m g / m l ; ionic s t r e n g t h , o.o6; MgC1 z, io raM; Ca e~ c o n c e n t r a t i o n , i. IO - 4 M b y ~ m M Cat : ( ; T A buffer (pH 7 . o ) ; T r i s m a l e a t e buffer, io m M (pH 7.o). ATt ~ c o n c e n t r a t i o n used is o.l raM. Total vol., 1. 5 1111; t e m p . , zo:.
elimination
o f C a 2+ in a m e d i u m
containing
_~.o m M
EGTA
resulted
in a c o m p l e t e
inhibition of superprecipitation. Fig. 3 shows the effect on superprecipitation of t h e e x t r a c t e d p r o t e i n f o l l o w i n g t h e a d d i t i o n of MgC12 t o a r e a c t i o n m i x t u r e w i t h l o w i o n i c s t r e n g t h (o.o6) c o n t a i n i n g i o h i M T r i s - m a l e a t e b u f f e r ( p H 7.o), o . z m M A T P a n d z. I o -~ M C a ~+. U p t o 2o m M MgCI,, a d d e d , t h e s u p e r p r e c i p i t a t i o n was enhanced. In a medium containing 5 mM E D T A , o.~ m M A T P a n d T. - o -~ M C a 2+, b u t n o M g 2~-, t h e p r o t e i n s h o w e d n o s u p e r precipitation.
!~iochi~t. IHophyx. Acta, -'56 (I972) 565-576
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X. SHIBATA £~
al.
From the above results, it was evident that the presently extracted protein required not only Mg ~+ and A T P but also Ca ~+ for superprecipitation.
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Fig. 2. Fffects of Ca 2÷ on s u p e r p r e c i p i t a t i o n a t 2o' o[ t h e e x t r a c t e d p r o t e i n . P r o t e i n c o n c e n t r a t i o n , o.65 m g / m l ; ionic s t r e n g t h , o.o6; T r i s - m a l e a t e buffer, To mM (pH 7.o); ATP, o.[ raM; p h o s p h o enolpyruvate, ~ raM; p y r u v a t e kinase, Eo p g ; MgCli, [o raM; t o t a l vol., i. 5 ml. The r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n of ATP. Free Ca ~ c o n c e n t r a t i o n w a s s e t u p b y C a - E G T A buffer, E G T A c o n c e n t r a t i o n b e i n g 2 raM. C o n c e n t r a t i o n s of Ca % in t h e r e a c t i o n m e d i u m were I • Io -4 M (X x ) , T-to SM ( O - - O ) , 5 " t o * M ( / ~ - - - ~ ) , t . ~ o 6M ( Q - - - Q ) , i - [ o ~3I (A ~k) a n d n e a r l y zero o b t a i n e d w i t h o n l y P;GTA w i t h o u t a d d e d CaC12 ( ~ - - - < ). Fig. 3. Effects of a d d e d Mg 2~ on t h e s u p e r p r e c i p i t a t i o n at 20 ° of t he e x t r a c t e d p r o t e i n . P r o t e i n c o n c e n t r a t i o n , o.45 m g / m l ; ionic s t r e n g t h , o.o6: T r i s - m a l e a t e buffer, To mM (pH 7.o); ATP, o.t raM; p h o s p h o e n o l p y r u v a t e , I raM; p y r u v a t e kinase, l o fig; Ca 2+ c o n c e n t r a t i o n , ~- i o -~ M (Ca-EGTA buffer); t o t a l vol., L 5 ml. The r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n of ATP. I q n a l concentration of a d d e d MgC12 were 20 mM (L" (~), ro nl3I ( x - - x ) , 5 mM (O -O), 2.5 h i m ( p j _ _ , A ) , [ nl3f ( ( -- (9), 0. 5 ii1~[ ( x - - x ) . ~ - - - ~ , w i t h [ mM ED TA .
.4 TPase activity F w m the above results, it is reasonable to expect that there m a y b e s o m e correlations between the activation of the Mg2+-dependent ATPase a c t i v i t y or the ATPase activity under a constant Ca 2+ concentration of the protein and the enhancement of superprecipitation due to an elevated concentration of Ca2. or Mg 2+ in the reaction medium. The Mg2+-dependent ATPase a c t i v i t y was activated b y an elevation of Ca"+ concentration in a m e d i u m containing the ATP-generating system consisting of phosphoenolpyruvate, pyruvate kinase and Mg 2+. It increased rapidly at i . i o * M reaching a m a x i m u m at I. i o 4 M (Fig. 4), showing correlation with the enhancement of the superprecipitation. That is, the ATPase activity was o.o2o 7 and o.o283 # m o l e pyruvate per mg protein per min w i t h o u t added Ca z+ and at I. Io 4 M Ca e+, respectively. The free Ca 2+ concentration at which the Mg"+-dependent ATPase activity of the protein was half activated was about ~ • i o ~ M. This value was almost the same as that of muscular actomyosin with Ca 2+ sensitivity 'a and actomvosin reconstituted
t3~ochhm Hiophy,< .4cla, 256 (t972)
565 576
571
CONTRACTILE PI{OTEIN FROM LEUCOCYTES
(ref. 16) from myosin and the actin/tropomyosin-troponin complex of the heart or skeletal muscle with regard to the above organs. 0.0350 c
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Fig. 4" E f f e c t s of 171e+ c o n c e n t r a t i o n o n t h e M g ~ + - d e p e n d e n t A T P a s e a c t i v i t y of t h e e x t r a c t e d protein. I';xperimental c o n d i t i o n s are t h e s a m e as t h o s e in Fig. 2, except t h a t the t e m p e r a t u r e is 37 ° a n d t h e r e a c t i o n t i m e is 5 rain. Vig. 5. E f f e c t s of a d d e d Mg 2+ o n t h e A T P a s c a c t i v i t y u n d e r a c o n s t a n t Ca 2~ c o n c e n t r a t i o n ot t h e e x t r a c t e d p r o t e i n . E x p 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 t h o s e in ]rig. 3, e x c e p t t h a t the t e m p e r a t u r e is 37 ° a n d t h e r e a c t i o n t i m e is 5 rain.
Up to 20 mM added MgCI~, the ATPase activity of the protein was also activated by an increase of Mg ~+ concentration in a medium containing the ATP generating system and a constant amount of Ca2+ (Fig. 5). The ATPase activity was O.OLO7 and 0.0338 ffmole pyruvate per nag protein per rain at medium concentrations of 0.5 mM and 20 mM MgC12, respectively. This effect of Mg~+ on tim ATPase activity was similar to that seen with an arterial contractile protein of the smooth musclOL The extracted protein showed ATPase activity activated by Ca2+ and inhibited by Mg 2+ at high concentration of KC1. At low KC1 concentration, the ATPase activity of the protein was slightly activated by Ca~+ and Mg 2+ (Table I). The Ca"+-activated ATPase activity of actomyosin from striated muscle was to be greater at low ionic strength than at high ionic strength is, but in the extracted TABLE I ATPase ACTIVITY OF NATURAL ACTOMYOSIN L I K E CONTRACTILE PRO T E I N FROM LEUCOCYTJ~;S AT DIFFERENT
ATPase
KCI
activity
CONCENTRATIONS
was measured
under the following conditions:
IO m M T r i s - m a l e a t e
buffer
(pH 7.o) w i t h o r w i t h o u t i m*I MgC12 or Ca.C12, I mM ATP at 37°; p r o t e i n c o n c n . , i . I o m g / m l ; r e a c t i o n t i m e , 5 nlin.
A'Cl conch. (M)
0.06 0.60
Split P (ffmoles/mg protein per rain) No metal ions added
,$lgCl 2 added
CaCl 2 added
0.0201 0-0293
0.0205 0.0268
0.0268 0.0647
Biochim. Biophys. Acta, 256 (I972) 5 6 5 - 5 7 6
N. SHI/3AT.\ d[ ell.
572
1)rotein from leucocytes, the A T P a s e a c t i v i t y was higher at high ionic s t r e n g t h than at low. The same thing was true for actomyosin from the smooth muscle L%'°. I t seems reasonable to postulate that the contractile protein extracted from leucocytes e x h i b i t e d A T P a s e a c t i v i t y of the actomvosin t y p e which was a c t i v a t e d by Mg "~+a n d Ca 2+ at low ionic strength, a n d also A T P a s e a c t i v i t y of the myosin t y p e which was i n h i b i t e d b y Mg 2+ and a c t i v a t e d by ('a ")+ at high ionic strength, under conditions in wlfich actomvosin is dissociated into myosin a n d actin.
Change in viscosi O, by addition of A T P The relative viscosity of the protein was decreased b y the addition of A T P a n d 3Ig ~+ at high ionic strength. This was followed b y a g r a d u a l increase in viscosity towards the initial level, p r o b a b l y being b r o u g h t a b o u t by the hydrolysis of A T P (Fig. 6). The s e n s i t i v i t y t o w a r d s A T P c o m p u t e d according to the formula of PoIcrzl,: uJ et al? z was 19o %. This value was ahnost the same as t h a t o b t a i n e d for a c t o m v o s i n from s t r i a t e d musclO 2 or smooth musclO 7.
A nalvlical ultracentr@,gal pattern The ultracentrifugal p a t t e r n of the protein in I o mM T r i s - m a l e a t e buffer (pH 7.0) with ionic strength of 0.6 at 20 shows two peaks (Fig. 7) corresponding to 5.8 S a n d 37 S in the presence of A T P and Mg 2+. The former peak corresponded fairly well to t h a t of myosin from s t r i a t e d 2~ a n d smooth musclO 7,2° and the l a t t e r n e a r l y to t h a t of F - a c t i n from the smooth muscle 22 or from p l a s m o d i u m "3.
200 ATP
/*/
1.50
1.00 0 -20
0
30
60
90
120
150
180
210
rime (rain)
FiR.(i. Effect o f A T P o n t l l e ~ i s c o s i t y of the p r o t e i n from l e u c o c y t e s a t 25 . P rot e i n c o n c e n t r a t i o n , 4.o m g / m l ; ionic s t r e n g t h , o.0; T r i s - m a l e a t e buffer, 1o mM (pH 7.o). For d e t a i l s see t e x t . [:ig. 7" U l t r a c e n t r i f u g a l p a t t e r n of the e x t r a c t e d p r o t e i n . The e e n t r i f u g a t i o n was carri('(l out a t 542oo r e v . / m i n in io h i m T r i s - m a l e a t e buffer (pH 7.o) c o n t a i n i n g 6.5 m g p r o t e i n pe r ml, o.0 M KC1, IO mM 3IgCl 2, a n d 5 mM ATP. S e d i m e n t a t i o n proceeds from left to right. The p h o t o g r a p h was t a k e n at z5 nlin a f t e r t o p speed was reached.
Electronmicroscopic fi~zdit~gs The protein at low ionic s t r e n g t h was s t u d i e d b y electronmicroscopy I rain after an a d d i t i o n of the protein solution. The protein dissolved in o.6 M KC1 was a d d e d to IO mM T r i s - m a l e a t e buffer (pH 7.o) containing IO mM MgCI2 and either 5 mM A T P and I mM E G T A or o.I mM A T P and o.I mM CaC12. The final KCI concentration in these p r e p a r a t i o n s was o.o6 M. t3iochim. 13iophy,< Hera, 256 (1972) 565 .576
CONTRACTILE PROTEIN FROM I.EUCOCYTEg
57.3
Fig. 8. F.lectron micrograph of the extracted protein in clearing phase at low ionic strength. The p r e p a r a t i o n contained o. I5 mg of protein per ml, 0.06 M 1<(21, io mM MgC12, i mM EGTA, 5 mM ATP and :o mM Tris maleate buffer, p H 7.0.
In the preparation containing 5 mM ATP and I mM EGTA, the protein showed a clearing response. In the electron micrograph of this preparation, thick (1~I) and thin (A) filaments were seen about I5o A and 8o .~ in diameter, respectively (Fig. 8). It is suggested that the former corresponds to myosin aggregates and the latter to F-actin filaments. In the preparation containing o.I mM ATP and o.I mM CaC12, electron micrographs of negatively stained material showed the formation of m a n y large aggregates, apparently due to joining together both myosin aggregates and F-actin filaments, as observed in the superprecipitation of skeletal actomvosin ~4,2~ (Fig. 9)DISCUSSI()N
From the above experimental findings, it may be concluded that the protein under investigation corresponds to a so-called natural actomyosin-like contractile protein of leucocytes. The viscosity of the extracted protein was somewhat lower than that of actomyosin from striated muscle reported by MOSISrA~;i~TS2a. In this point, there arises an argument that the extracted protein should not be called natural actomvosin. Reconstitution of myosin- and actin-like protein obtained separately from the extracted protein of leucocytes demonstrated the superprecipitation characteristic of synthetic actomyosin2L This finding, together with biopl~ysical and biochemical features Biochim. Biophys. Acta, 256 (I97 z) 565-576
574
X. sH1z~;x'ra el a /
Fig. 9. Electron micrograph of the extracted protein superprecipitated at low ionic strength. The preparation contained o.15 mg of protein per ml, o.o6 M KC1, io mM Mg('l,,, o.~ mM Ca('l,,, o.I mM ATP and [o mM Tris maleate buffer (pH 7.o) of the protein described a l r e a d y in results, m a y indicate t h a t the e x t r a c t e d protein a c t u a l l y corresponds to n a t u r a l a c t o m y o s i n from muscle. In order to e x t r a c t the n a t u r a l actomyosin-like protein from leucocytes, leucocytes were homogenized in a solution with low ionic s t r e n g t h containing A T P and histidine buffer (pH 7.o) i n s t e a d of Weber-F2dsall's solution 40 with high ionic s t r e n g t h used for the e x t r a c t i o n of skeletal myosin B. This procedure was based on the finding t h a t the actomyosin-like contractile protein e x t r a c t e d previously bv us from leucocytes e x h i b i t e d biophysical and biochemical features similar to those of carotid myosin B, except for the absence of Ca `'.+s e n s i t i v i t y 3. S u p e r p r e c i p i t a t i o n of the carotid myosin B was accelerated b y increasing the concentration of a d d e d l~'Ig2+ (ref. I7). The contractile protein with Ca 2+ sensitivity from carotids was capable of being e x t r a c t e d with a low ionic s t r e n g t h solution containing A T P and histidine buffer (pH 7.o) ~,°s. F u r t h e r m o r e , the Ca'~÷-sensitizing protein contained in n a t u r a l actomyosin, a socalled troponin, is r e p o r t e d to be v e r y labile to proteinase digestion 2" and, since the ATPase of myosin is an S H e n z y m e 30, Trasylol, a n a t u r a l proteinase inhibitor, and dithiothreitol, an SH protector, were a d d e d to the above e x t r a c t i n g solution. The protein thus p r e p a r e d e x h i b i t e d the Ca 2+ s e n s i t i v i t y in s u p e r p r e c i p i t a t i o n (Figs. I and 2) and the Mg2+-dependent A T P a s e a c t i v i t y (Fig. 4), as well as biophysical a n d biochemical properties characteristic of myosin B or n a t u r a l actoinyosin from muscle. In n a t u r a l actomvosin or myosin B from s t r i a t e d muscle the o p t i m a l Mg -°+ Biochi~z. l~io[~h.vs. ,4cla, 256 (I97 z) 565 -576
COXTRACT1LE PROTEIN
FROM LEUCOCYTES
375
concentration necessary for superprecipitation is considerably lowat, whereas it was very high in the extracted protein from leucocytes (Fig. 3), just as observed in carotid smooth musclelL Furthermore, as described in 1~1~.svl:rs, the extracted protein demonstrated features resembling actomyosin from smooth muscle in the behaviour of the ATPase and analytical ultracentrifugal pattern. Thus, the presently extracted contractile protein from leucocytes seems to be similar to natural actomyosin from smooth muscle rather than that from striated muscle. In the analytical ultracentrifugal pattern (Fig. 7), the s°2o.w value of the retarding peak of the extracted protein was almost the same as that of the retarding peak of an actomyosin-like protein extracted previously from leucocytesa, but the value of the foregoing peak in Fig. 7 was somewhat greater than that of the foregoing peak of the protein prepared by the previous method a. This increase in s value of the foregoing component observed in the extracted protein having Ca '2+ sensitivity, may be explained by the assumption that the F-actin of the protein may be endowed with a native tropomyosin-like substance, The contractile protein has been extracted, so far, not only from muscle but also from free cells, for example from platelets 32, plasmodium as, fibroblasts a4, sarcoma cellsa~, and leucocytes3, dc.. These contractile proteins, however, showed requirement for Mg -°+, ATP and no Ca 2+ unlike natural actomyosin from muscle, which has a Ca"+ requirement for superprecipitation. Therefore, it has been posed that the question of whether or not the contractile protein of free cells other than muscle is different from that of muscle is decided on the basis of a need for a Ca`)+ concentration in the superprecipitation. From the nature of tire contractile protein presently extracted from leucocytes, it is suggested that even the contractile protein of free cells may be susceptible to Ca ')+ as observed in the contractile protein from the muscle. H.\T.xx~ 3" demonstrated that the movement of plasmodial fragments was subject to the specific effect of Ca ~'+ following caffeine treatment, although the contractile protein possessing ( ' a " ' sensitivity for the superprecipitation had not been isolated from plasmodium. From findings obtained in morphological, physiological and phylogenetic studies concerning the motility of leucocytes, we have reported that the mntile form and function of leucocvtes are coordinately controlled by ATP and that leucocx'tes po.~sess a motor organ consisting of an actomyosin-like contractile protein acting as a mechanochemical system coupled with ATP", a7 a~. In this paper, it has become evident that the contractile protein of leucocytes, as well as of the natural act.myosin of smooth muscle, exhibits the contraction and relaxation regulated by concentrations of free Ca2 ~ in the presence of ATP and Mg 2.. It is conceivable that the motor organ of leucocytes consists of natural actomyosin-like contractile protein similar to that of muscle and that the movement of leucocvtes is achieved by contraction and relaxation of the natural actomvosin-like contractile protein.
i 1(. IeUKUSIIIMA, N. SENDA, S. ISHIGAMr, J. ]~NDO, -'~f. ISHII, Y. MURAKAMI, l(. NISHIAN AND Y. UEDA, Alcd..[. Osaka Univ., 5 (I954) 23I. 2 N. ~ENDA AND ~{. TAMURA, A*z~Z~t. Rep. Center Adult Dis., Osaka, I (1961) I. 3 N. ~F. NDA, N. ~HIBATA, N. TATSUMI, I{. KONDO AND K. HAMADA, Biochim. Biophys. Ac/a, lSI (I969) 191. 4 "~- I'~BASHI AND 1~'. FBASH1, J. Biochem. Tokyo, 55 (1964) 604. 5 ~. tLm~sHi .~No A. KODA51A, J. Biochem. Tokyo, 60 (1966) 733.
Biochim. Biophys. Acla, 256 (1972) 5 6 5 - 5 7 6
5 7 ()
s . SHIBAT_\ v[ al
0 12. ~3¢ERLF;, l~eport, lt~l..~i),rnlS, on Nea~ .4~pects (it Trasvlol Therapy, .llzt~¢ick, 196~', \'~]. 3 I". 1£. S c h a t t a u e r V e r l a g , S t u t t , ~ a r t N e w Y o r k . 197 o, p. 51 . 7 N. BACK AND H. ~'ILI£ENS, R e p o r t , 1~1..b'yll~p. 0n Ne*e~ ~4specIs ~![ J'razvl~d Therapy, Met,te~h 2968, Vol. 3, F. 1 , [ . . q c h a t t a u e r V e r l a g . S t u t t g a r t N e w Y o r k , ~97 o, p. 63. s (). H. l . o w ~ v , N. R o s E m ~ o v ( m , A. L. l:am~ xxl) R. J. NA,X'DALL, .]. HiOl. Ck~'rJ~., 193 (m511 -'r~5 9 A. ~[. RT,:VNAI~D, L. 1". t I a s s , 1). 1). JACO>SVN axl) 1'. I). BOVl,:l{,.]. Hiol. Cke~u., 236 (rO~)rl 2277 r o B. B. 3la~asl~, Biockim. I¢iopkys..-Iota, 3-' (r959) 357I ~ S. I,:UASWr, .[. t3iochem. Tokyo, 5 ° ( t 9 6 I ) 230. I2 1[. t)ORTZFHL, (). SHRAMM AND H. l~. \¥EBP;R, Z. Natu~'fc~rsch., 5 b (195 o) ¢,~. 13 H. P;. HUXLEY, .]. :]l(}l. 141ol., 7 (I963) 2,SI. 14 If. I)O~aTZFmI., 1~. C. ('.XLmV]~L AND J. ('. lU?]~{;(;, Bioehim. 1No/~kvs..tela, 79 (I964) 581. 15 ;\. ~VEBEI~ AND %. \~'INIC1;I~, .[. t~iol. Che~?., 236 (196~) 319,S. ~6 .X.M. I(ATZ, D. 1. ]{EPKF AND 13. 1~. ('OHEN, Circltlatio~z Res., ~9 (I906) lO02. ~7 N. SHIBATA AND T. YAMAGAIUI, ,]. ,]rap. Cvll..~l~giol., {} (1969) ~76. I8 \¥. HASSELBACH, Z. Natltrforsck., 7 b 0 9 5 2 ) I03. 19 D. M. NE~DHA~ ANt) J. M. CAWKWELL, f]iocke~z. J., 63 (~956) 3372o R. H . SCHrR,~Et¢, Biochem. Z., 343 (t965) 269. 2~ P. J o m ~ s o N AN~) A. J. R o w a , Hiochem..1., 74 (196o) 432. 22 L. LASZT AND (;. HAMOIa, 14iochim. Biophys. Acla, .50 (196o) 43 o. ; 3 S. HATANO, T. TOTSUKA AND F. ()OSA\vA, Biochim. t3iophys..4eta, 14o (r967) ~o9. 24 N. IKE~XlOTO, S. KITAGAWA ANt) J. Gt~R(;~;LV, Bioche~z. Z., 345 (1966) 4 l°" 25 lx[. TAKAHASHr AND T. Y A s t ' I , .]. ]3iochc~. Tokyo, 62 (I967) I3~. 26 \V. H. F. M. MOSIS~AH~TS, J . Gc~z. Physiol., 36 (~948) 30I. 27 N. SHIBATA, N. TATSUIVII, Y. Moor, 1'2. TANAKA AND N. SENDA, in p r e p a r a t i o n . 28 J. RC, Ec~6, 1,2 STIIASSX'~ia axl3 R. H. SHII¢.~IER, Biochem. Z., 343 (1905) 7 TM 29 S. EgAsr~I, H . Iwai¢c~¢a, H. NAKAJI,~A, R. NA~A~IC'~A A,XD Y. Oot, Biochem. Z., 345 (1966) 2or 3 o F. l ) I c K v ; x s A.XD G. E. (;LocI~, 13iochim. Biophys. Hera, 7 (t95 I) 578. 31 ix[. ~'[ARUYAMA AND .~. \¥ATANABF2, .]. Biol. (7hem., 237 (~962) 3437. 32 M. BETTEX-(;AI.LAX~ .\XI) l'Z 1:. LUSCHEI~, Hiochim. l~iopkys. Acta, 49 (~961) 53 o. 33 S. HATA.XO a ~ ) M. TAZAWA, 14iockim. Biophys..4eta, ~54 (196~) 5o 7. 34 H. H O F F M A ~ - B m ¢ c l X G , 13iochim. Biophys. Acta, 14 (1954) ~72. 35 H. HOVFMA.X~x-BEI~LrXG, t3iochi~z. Biolgkys. ~4cta, it) (1956) 453. 36 S. HATANO, EXp. Cell Res., 6~ (197 ° ) 199. 37 N. SE~D:\, Na~g, 25 (1954) 7o7 . 3 S N. S~.'NDA, RVV. Hematol., 9 (1954) 41'~. 39 N. SE,XDA A.~D H. TA.~IF~A, Proc. 8lh l~l. Congr. Haematol., Tokyo, 296o, Vol. 2, P a n P a c i t i t P r e s s , T o k y o , t962, p. 820. 4o 1£. MEYE~ A,X'D H. H. \VEI3E~¢, Biochem. Z., 26(~ (I933) ~37.
Biochim. Biophys. Acta, 256 (1972) 5 6 5 - 5 7 ~
565
m~A 4~ 237 A CONTRACTILE PROTEIN
P O S S E S S I N G Ca 2+ S E N S I T I V I T Y ( N A T U R A L
ACTOMYOSIN) F R O M L E U C O C Y T E S ITS E X T R A C T I O N AND SOME OF ITS P R O P E R T I E S " N(~BUHII,~() SHIBATA, NORIYUKI TATSUMI, KAZUHIKO TANAKA, \-()SHIM[ OI,~AMURA AND NOBUYUKI SENDA Ccldcl'.fi~r Adult Diseases, Osaka (Japan)
(Received June 281h, 197f)
SUMMARY I. Methods have been developed for tile isolation of contractile protein from equine leucocytes resembling n a t u r a l actomyosin of the smooth nmscle. 2. This protein exhibited ATPase (EC 3.6.1.3) a c t i v i t y and u n d e r w e n t superprecipitation at low ionic strength in the presence of ATP, Mg 2+ a n d Ca"+, b u t revealed no superprecipitation in the absence of either Ca 2+ or Mg ~-+ even in the presence of ATP. Superprecipitation of this protein was enhanced b y an increase of Ca ~+ conc e n t r a t i o n in a m e d i u m c o n t a i n i n g Mg 2+ and its i n t e n s i t y was closely associated with e n h a n c e m e n t of the Mg~+-dependent ATPase (EC 3.6.1.3) activity of this protein by an increase of Ca 2+. The free Ca 2+ concentration at which the Mg~+-dependent ATPase a c t i v i t y of the protein was half activated was a b o u t I- io 4 M. Superprecipit a t i o n of this protein was e n h a n c e d b y an increase of Mg 2+ c o n c e n t r a t i o n in a m e d i u m c o n t a i n i n g Ca 2÷. I n this case, too, the superprecipitation was closely associated with e n h a n c e m e n t of the ATPase a c t i v i t y of this protein t33: increasing Mg 2+ with a c o n s t a n t Ca ~- concentration. The ATPase a c t i v i t y was half activated in the presence of a b o u t 3 mM Mg 2+. 3. Electron micrographs of the protein showed thick a n d thin filaments at low ionic strength a n d a relatively high A T P level conditions in which muscular actomyosin was dissociated into myosin aggregates a n d fibrous actin. At a relatively low A T P level, b o t h thin a n d thick filaments were aggregated with each other, as in actomvosin from the muscle, which seemed to be subject to superprecipitation.
INTR~ ) D U C T I O N
On the basis of the s t u d y of the motile form and function of leucocytes, we have suggested t h a t leucocvtes are most likely to possess a motile organ, a mechanochemical system coupled with ATP l, of which the essential substance m a y be a k i n d of actoAbbreviations: I';GTA, t,2dois-(2-dicarboxymethylarninoethoxy)ethane; KIU, trypsin l(allikrein inhibi~corvunit. * An outline of this paper was reported at the 8th Annual Meeting of the Japan Society of the l{cticuloendothelial System Research in ()saka, Japan, 197o. Biochim. l~i@hys. Acta, 256 (i972) 565 576
566
x. SlIIBAT.\ c! al.
myosin-like contractile p r o t e i n similar to t h a t of tile muscle". A contractile protein was first isolated b y us from equine leucocytes a in I967. The protein had various biophysical and biochemical properties characteristic of actomyosin from the muscle. However, it did not show a n y Ca 2+ s e n s i t i v i t y in superprecipitation, unlike n a t u r a l a c t o m y o s i n or nlyosin B from muscle. This finding posed the problem of whether " n a t i v e t r o p o n l y o s i n " , consisting of t r o p o m y o s i n and t r o p o n i n < s, in the presence of which Ca e+ effects the interaction of myosin and actin from the muscle, was subs t a n t i a l l y absent in leucocytes or i n a c t i v a t e d during the e x t r a c t i o n process. Thus, an a t t e m p t was m a d e to e x t r a c t a contractile protein from leucocytes possessing Ca e~- s e n s i t i v i t y silnilar to n a t u r a l actomyosin. EXP/~;RIMENTAL P R O C E D U R E
Isolation of leucocytes from whole blood Leucocytes were collected from fresh equine arterial blood. The isolation procedure of leucocytes from whole b l o o d was the same as t h a t e m p l o y e d previously except for some points 3. The modifications were the use of blood suspending solution containing d i t h i o t h r e i t o l and Trasylol< ~ of a strong inhibitor for protease. T h a t is, IO.O g of E D T A ( t e t r a s o d i u m salt), &5 g of NaC1, 3.6 g of cysteine, 2 m g of dithiothreitol a n d IO 5 K I U (I K I U corresponds to o.I 4/~g of Trasylol) of Trasylol dissolved in 1o ml of o.15 M saline solution were dissolved in I 1 of distilled w a t e r into which 9 1 of the blood was mixed. The w)tume of leucocyte buffy coat collected from a s t a r t i n g volmne of i~ t of b l o o d was 40-5 ° m l . As in the previous e x p e r i m e n t a, ttle leucocytes (I.O- IO4-i.3 • lO 4, m m a in whole blood) were c o n c e n t r a t e d to I.O. IoG-I.3 • Io6/mm a in the b u l l y coat at the final step of isolation. C o n t a m i n a t i o n with red cells in the huffy coat was less t h a n 5 °o, and t h a t of p l a t e l e t s was negligible. A b o u t 9o % of the collected leucocytes were usually counted to be neutrophiles on the smear p r e p a r a t i o n .
Extraction of contractile protein possessing Ca 2+ sensitivity from leucocytes All procedures were c o n d u c t e d at a b o u t 4 ' . The buffy coat of leucocytes was first m i x e d with ¼ vol. of T r a s y l o l in o.15 M NaCI (lO 5 K I U in IO mI), 2 mM dithiothreitol and 2.5 vo]. of tlle e x t r a c t i o n solution containing I mM A T P and 20 mM histidine buffer (pH 7.0). The final ionic s t r e n g t h of tllis m i x t u r e was about 0.o5. The leucocytes were homogenized in a Virtis homogenizer at half the maxinaum speed and, further, in a P o t t e r - E l v e h j e m homogenizer for 30 sec each. This homogenization procedure was v e r y i m p o r t a n t for o b t a i n i n g contractile protein possessing significant sensitivity for Ca 2÷ in s u p e r p r e c i p i t a t i o n , so it was carried out v e r y carefully. The contractile protein p r e p a r e d without the above care with regard to the homogenation d e m o n s t r a t e d only a p a r t i a l Ca `,+ sensitivity, although it was never devoid of Ca "~+ sensitivity. The resulting homogenate was not as viscous as tile leucocyte h o m o g e n a t e p r e p a r e d b y the previous procedure a. The fiomogenate was then centrifuged at 4oo00 7< g for I h to remove cell fragments and corpuscular material, leaving a pink s u p e r n a t a n t fluid. This s u p e r n a t a n t was dialysed for 2 d a y s against a b o u t 25 vol. of 5 mM histidine buffer (pH 7.o), containing 0.05 M KC1 and 2 mM ditlliothreitol in order to remove ATP. The resultant flocculent p r e c i p i t a t e was collected b y centrifugation at 3000 :>,~,~ for 20 rain. The sediment was dissolved in an tJMchbn
lTi,ptu,<~. At/a, 256 (T072) 505 570
C~)NTRACT1LE PROTEIN FROM LEUC()CYTES
5(i7
a p p r o p r i a t e volume of 2. 4 M KCI solution coutaining 2 mM d i t h i o t h r e i t o l whose ionic s t r e n g t h was finally a d j u s t e d to o.6 with water, The indissoluble m a t e r i a l was r e m o v e d b y centrifugation at 1oooo 7< g for I o rain. The s u p e r n a t a n t was again dialvsed for about I(~ h against a b o u t Ioo vol. of 5 mM histidine buffer (pH 7.o) containing o.24 M KC1 and 2 mM dithiothreitol. T h e r e s u l t a n t flocculent p r e c i p i t a t e was collected bv centrifugation at 3ooo z: g for I5 rain. The sediment was again dissolved in o.(i M KC1 (pH 7.o) containing 2 mM dithiothreitol. The h a r d l y dissolved m a t e r i a l was r e m o v e d b y centrifugation at 33ooo ~, g for 3 o rain. P r e c i p i t a t i o n and dissolution ~f the above s u p e r n a t a n t fluid was r e p e a t e d at least twice in KCI solution containing 2 mM d i t h i o t h r e i t o l at the final ionic strength of o.o6 and o.6, respectively. The p r o t e i n dissolved in o.0 M KC1 was k e p t at o ' a n d used for e x p e r i m e n t s within a d a y after the extraction. The protein e x t r a c t e d from 4 ° ml of the buffy coat of leucocytes u s u a l l y a m o u n t e d to a h o u t I6o mg. The a m o u n t of protein was d e t e r m i n e d b y the lnethod of LOWRY el al. 8 using bovine serum a l b u m i n as s t a n d a r d . .1 T P a s c a c t i v i ( v
The effect of Ca ~+ on the Mg'~+-dependent A T P a s e (EC 3.6.i.3) a c t i v i t y was d e t e r m i n e d in the coupled s y s t e m with p y r u v a t e kinase as an A T P generating s y s t e m b y m e a s u r i n g t h e time dependence of p y r u v a t e liberation according to the m e t h o d of RZYXA~I~ d alY. The reaction m e d i u m was composed of 0.4-0. 7 mg protein per ml, 0.6 #g p y r u v a t e kinase per ml, • m M p h o s p h o e n o l p y r u v a t e , I o mM T r i s - m a l e a t e (pH 7.o), I o mM MgC12, different a m o u n t s of Ca 2~ set u p bv C a - E G T A buffer (E(iTA, raM), 0.06 M KC1 and o.I mM ATP. The reaction was s t a r t e d b y an a d d i t i o n of A T P and was carried out at 3 7 . At a p p r o p r i a t e times, o.5 ml of the reaction m i x t u r e was t r a n s f e r r e d to a solution containing o.2 ml of 2.5 mM 2 , 4 - d i n i t r o p h e n y l h y d r a z i n in 3 M HCI to stop the reaction. This coupled system was not influenced b y the Mg ~+ c o n c e n t r a t i o n in the m e d i u m over the range from 0.5 to 2o raM. The effect of Mg 2+ on t h e A T P a s e (EC 3.6.I.3) a c t i v i t y u n d e r a constant Ca ~+ concentration was likewise determined. In the d e t e r m i n a t i o n of the A T P a s e a c t i v i t y of the reaction m e d i u m w i t h ~ mM A T P as s u b s t r a t e , the reaction was i n i t i a t e d b y adding A T P and was s t o p p e d b y a d d ing 5 %, trichloroacetie acid at a p p r o p r i a t e times. In this case, the coupling s y s t e m consisting of p h o s p h o e n o l p y r u v a t e a n d p y r u v a t e kinase was o m i t t e d . Then, the a m o u n t of Pi l i b e r a t e d was d e t e r m i n e d b y the m e t h o d of MARSIt 1°. ~uperprecipitation
The ionic s t r e n g t h of the reaction m i x t u r e used for observation of s u p e r p r e c i p i t a tion was brought to 0.06 b y m i x i n g the protein solution into an a p p r o p r i a t e reaction m e d i u m i n d i c a t e d in the text. The s u p e r p r e c i p i t a t i o n was followed with a H i t a c h i UV-VIS s p e c t o r o p h o t o m e t e r b y measuring the change in absorbanee at 660 nm in the reaction m i x t u r e on a d d i t i o n of A T P at room t e m p e r a t u r e following the m e t h o d developed by EBASH111. In some e x p e r i m e n t s the s u p e r p r e c i p i t a t i o n in the reaction m i x t u r e containing t h e protein was also macroscopically studied. I "i s c o s i m e t r i c d e t e r m i n a t i o n
This was p e r f o r m e d in an Ostwald viscosimeter with I ml c a p a c i t y at 25 :'. The r e l a t i v e viscosity was m e a s u r e d at p H 7.0 first in 0.6 M KC1 (~re0 and then after Biochim. Biopkys. Acta, _,5(i (1972) 565 ~576
568
x . S H I B A T A t'[ a[.
a d d i t i o n of o.oi nil of a solution containing 0.5 M MgC12 and o.~ M A T P in o.0 M KC1 (t]rel ATP). The s e n s i t i v i t y to A T P was calculated (in %) according to PoR'rzmm el al. 12 with the fornmla: logdb~, - l o g ~ q ~ A T P Iog~q~jATP
X IOO
( ~ltracentrif~tgc A H i t a c h i Model UCA-I a n a l y t i c a l ultracentrifuge was e m p l o y e d for the s e d i m e n t a t i o n - v e l o c i t y measurements.
Eleclronmicrophotographs Micrographs of the protein were t a k e n b y the n e g a t i v e - s t a i n i n g technique of HvxLI~v '3. One drop of an ice-cold p r e p a r a t i o n of the protein was placed on a microgrid coated with collodion carbon. The p r e p a r a t i o n was n e g a t i v e l y stained with 1 % u r a n y t acetate, and e x a m i n e d in a Nihon I)enshi Model J E M 6(; electronmicroscope with an acceleration voltage of 80 kV.
Chemicals The chenficals used were a n a l y t i c a l grade. Trasylot, was o b t a i n e d from B a y e r Leverkusen, G e r m a n y a n d E G T A of a Ca2+-chelating agent from Dojin I g a k u Institute, J a p a n . Various free Ca 2+ concentrations were set up b y means of C a - E G T A buffer p r e p a r e d in the m a n n e r described by P(mTZmtL el aI. '4. The w a t e r used in p r e p a r i n g the solution was redistilled in a glass vessel. RESULTS
Nuperprecipitation I t is a well-known characteristic of n a t u r a l a c t o m y o s i n , the contractile protein of the muscle, to undergo s u p e r p r e c i p i t a t i o n at low ionic strength in the presence of not only Mg 2+ and A T P b u t also Ca 2+ (ref. rx). As shown in Fig. ~, the e x t r a c t e d protein from leucocytes showed a m a r k e d s u p e r p r e c i p i t a t i o n on a d d i t i o n of o.x mM A T P in a reaction m i x t u r e with low ionic s t r e n g t h (0.06) of I o mM T r i s - m a l e a t e buffer (pH 7.o) containing i o mM MgC12 and x. xo-4 M Ca "2+ (Fig. I, right), b u t failed to d e m o n s t r a t e s u p e r p r e c i p i t a t i o n in the m e d i u m w i t h o u t free Ca 2+ (addition of x mM E G T A alone instead of I mM Ca-EGTA buffer) (Fig. I, middle). T h a t is, i m m e d i a t e l y after the a d d i t i o n of ATP, t u r b i d i t y of the reaction m i x t u r e r a p i d l y increased in tbe vessel containing I . i o -4 M Ca 2+ (Fig. i , u p p e r right), whereas it decreased to exhibit clearing when Ca 2+ was e l i m i n a t e d by the a d d i t i o n of o n l y E G T A i n s t e a d of C a - E G T A buffer (Fig. I., u p p e r middle). ~.o rain after the a d d i t i o n of ATP, the protein showed a m a r k e d s u p e r p r e c i p i t a t i o n (Fig. I, lower right), b u t it k e p t clearing in the m e d i u m not containing free Ca ~+ (Fig. i, lower middle) a n d no s u p e r p r e c i p i t a t i o n was observed in the control (left) containing no a d d e d ATP. I t was e v i d e n t t h a t the degree of s u p e r p r e c i p i t a t i o n which was m e a s u r e d b y an increase in absorbance at 660 nm (Fig. 2) was associated with the concentration of free Ca ~+ in the medium. T h a t is, a s u p e r p r e c i p i t a t i o n occurred significantly at conc e n t r a t i o n s a b o v e i . IO * M Ca "-~- and increased up to i . Io -4 M Ca z+, whereas the 13i(,cki,z. tHophys..qela. 25~ (t()72) 5~)5 57~
569
CONTRACTILE PROTEIN FI~,()3I LEUCOCYTES
l:ig. ~. S u p e r p r e c i p i t a t i o n of t h e n a t u r a l a c t o m y o s i n - l i k e c o n t r a c t i l e p r o t e i n e x t r a c t e d from leucocytes. T h e u p p e r series s h o w s c h a n g e s in t u r b i d i t y j u s t after an a d d i t i o n of ATP. T h e lower simws s e d i m e n t a t i o n of t h e p r o t e i n 2o rain after an a d d i t i o n of ATP. T h e left vessels in each series are controls for each c o n t a i n i n g no a d d e d ATP. T h e middle vessels in each series c o n t a i n i n151 lCGTA i n s t e a d of C a - E G T A buffer. The controls c o n t a i n I - lo -4 M Ca 2+. P r o t e i n c o n c e n t r a t i o n , 1.75 m g / m l ; ionic s t r e n g t h , o.o6; MgC1 z, io raM; Ca e~ c o n c e n t r a t i o n , i. IO - 4 M b y ~ m M Cat : ( ; T A buffer (pH 7 . o ) ; T r i s m a l e a t e buffer, io m M (pH 7.o). ATt ~ c o n c e n t r a t i o n used is o.l raM. Total vol., 1. 5 1111; t e m p . , zo:.
elimination
o f C a 2+ in a m e d i u m
containing
_~.o m M
EGTA
resulted
in a c o m p l e t e
inhibition of superprecipitation. Fig. 3 shows the effect on superprecipitation of t h e e x t r a c t e d p r o t e i n f o l l o w i n g t h e a d d i t i o n of MgC12 t o a r e a c t i o n m i x t u r e w i t h l o w i o n i c s t r e n g t h (o.o6) c o n t a i n i n g i o h i M T r i s - m a l e a t e b u f f e r ( p H 7.o), o . z m M A T P a n d z. I o -~ M C a ~+. U p t o 2o m M MgCI,, a d d e d , t h e s u p e r p r e c i p i t a t i o n was enhanced. In a medium containing 5 mM E D T A , o.~ m M A T P a n d T. - o -~ M C a 2+, b u t n o M g 2~-, t h e p r o t e i n s h o w e d n o s u p e r precipitation.
!~iochi~t. IHophyx. Acta, -'56 (I972) 565-576
57 °
X. SHIBATA £~
al.
From the above results, it was evident that the presently extracted protein required not only Mg ~+ and A T P but also Ca ~+ for superprecipitation.
Q40C
//f/~ /.,//
/
///
0.30C
/////
E
g 030C
0.200
0.I00
020C
- _z~-
/ / ,! ~ / / ,,~----,o
......
/// ---
~
. . . . . . .
o
.).100
o ......
o
............
Time (rain)
Time ( rain )
Fig. 2. Fffects of Ca 2÷ on s u p e r p r e c i p i t a t i o n a t 2o' o[ t h e e x t r a c t e d p r o t e i n . P r o t e i n c o n c e n t r a t i o n , o.65 m g / m l ; ionic s t r e n g t h , o.o6; T r i s - m a l e a t e buffer, To mM (pH 7.o); ATP, o.[ raM; p h o s p h o enolpyruvate, ~ raM; p y r u v a t e kinase, Eo p g ; MgCli, [o raM; t o t a l vol., i. 5 ml. The r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n of ATP. Free Ca ~ c o n c e n t r a t i o n w a s s e t u p b y C a - E G T A buffer, E G T A c o n c e n t r a t i o n b e i n g 2 raM. C o n c e n t r a t i o n s of Ca % in t h e r e a c t i o n m e d i u m were I • Io -4 M (X x ) , T-to SM ( O - - O ) , 5 " t o * M ( / ~ - - - ~ ) , t . ~ o 6M ( Q - - - Q ) , i - [ o ~3I (A ~k) a n d n e a r l y zero o b t a i n e d w i t h o n l y P;GTA w i t h o u t a d d e d CaC12 ( ~ - - - < ). Fig. 3. Effects of a d d e d Mg 2~ on t h e s u p e r p r e c i p i t a t i o n at 20 ° of t he e x t r a c t e d p r o t e i n . P r o t e i n c o n c e n t r a t i o n , o.45 m g / m l ; ionic s t r e n g t h , o.o6: T r i s - m a l e a t e buffer, To mM (pH 7.o); ATP, o.t raM; p h o s p h o e n o l p y r u v a t e , I raM; p y r u v a t e kinase, l o fig; Ca 2+ c o n c e n t r a t i o n , ~- i o -~ M (Ca-EGTA buffer); t o t a l vol., L 5 ml. The r e a c t i o n w a s s t a r t e d b y t h e a d d i t i o n of ATP. I q n a l concentration of a d d e d MgC12 were 20 mM (L" (~), ro nl3I ( x - - x ) , 5 mM (O -O), 2.5 h i m ( p j _ _ , A ) , [ nl3f ( ( -- (9), 0. 5 ii1~[ ( x - - x ) . ~ - - - ~ , w i t h [ mM ED TA .
.4 TPase activity F w m the above results, it is reasonable to expect that there m a y b e s o m e correlations between the activation of the Mg2+-dependent ATPase a c t i v i t y or the ATPase activity under a constant Ca 2+ concentration of the protein and the enhancement of superprecipitation due to an elevated concentration of Ca2. or Mg 2+ in the reaction medium. The Mg2+-dependent ATPase a c t i v i t y was activated b y an elevation of Ca"+ concentration in a m e d i u m containing the ATP-generating system consisting of phosphoenolpyruvate, pyruvate kinase and Mg 2+. It increased rapidly at i . i o * M reaching a m a x i m u m at I. i o 4 M (Fig. 4), showing correlation with the enhancement of the superprecipitation. That is, the ATPase activity was o.o2o 7 and o.o283 # m o l e pyruvate per mg protein per min w i t h o u t added Ca z+ and at I. Io 4 M Ca e+, respectively. The free Ca 2+ concentration at which the Mg"+-dependent ATPase activity of the protein was half activated was about ~ • i o ~ M. This value was almost the same as that of muscular actomyosin with Ca 2+ sensitivity 'a and actomvosin reconstituted
t3~ochhm Hiophy,< .4cla, 256 (t972)
565 576
571
CONTRACTILE PI{OTEIN FROM LEUCOCYTES
(ref. 16) from myosin and the actin/tropomyosin-troponin complex of the heart or skeletal muscle with regard to the above organs. 0.0350 c
~ OO300
L_ 0 . 0 3 0 0
8
c
c
~6 0 0 2 5 0 cx
bl E 0.02(30
/
E 0.0250
8 E ::z
/
D_ 010150
J
"6
~> 0.0100
0.0200
Y, O
110-8
110-7 110-6
110-8
16
110"4
IB
~b
C o n c n . of added M g C t 2 ( m M )
C o h e n of C a 2+ ( M )
Fig. 4" E f f e c t s of 171e+ c o n c e n t r a t i o n o n t h e M g ~ + - d e p e n d e n t A T P a s e a c t i v i t y of t h e e x t r a c t e d protein. I';xperimental c o n d i t i o n s are t h e s a m e as t h o s e in Fig. 2, except t h a t the t e m p e r a t u r e is 37 ° a n d t h e r e a c t i o n t i m e is 5 rain. Vig. 5. E f f e c t s of a d d e d Mg 2+ o n t h e A T P a s c a c t i v i t y u n d e r a c o n s t a n t Ca 2~ c o n c e n t r a t i o n ot t h e e x t r a c t e d p r o t e i n . E x p 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 t h o s e in ]rig. 3, e x c e p t t h a t the t e m p e r a t u r e is 37 ° a n d t h e r e a c t i o n t i m e is 5 rain.
Up to 20 mM added MgCI~, the ATPase activity of the protein was also activated by an increase of Mg ~+ concentration in a medium containing the ATP generating system and a constant amount of Ca2+ (Fig. 5). The ATPase activity was O.OLO7 and 0.0338 ffmole pyruvate per nag protein per rain at medium concentrations of 0.5 mM and 20 mM MgC12, respectively. This effect of Mg~+ on tim ATPase activity was similar to that seen with an arterial contractile protein of the smooth musclOL The extracted protein showed ATPase activity activated by Ca2+ and inhibited by Mg 2+ at high concentration of KC1. At low KC1 concentration, the ATPase activity of the protein was slightly activated by Ca~+ and Mg 2+ (Table I). The Ca"+-activated ATPase activity of actomyosin from striated muscle was to be greater at low ionic strength than at high ionic strength is, but in the extracted TABLE I ATPase ACTIVITY OF NATURAL ACTOMYOSIN L I K E CONTRACTILE PRO T E I N FROM LEUCOCYTJ~;S AT DIFFERENT
ATPase
KCI
activity
CONCENTRATIONS
was measured
under the following conditions:
IO m M T r i s - m a l e a t e
buffer
(pH 7.o) w i t h o r w i t h o u t i m*I MgC12 or Ca.C12, I mM ATP at 37°; p r o t e i n c o n c n . , i . I o m g / m l ; r e a c t i o n t i m e , 5 nlin.
A'Cl conch. (M)
0.06 0.60
Split P (ffmoles/mg protein per rain) No metal ions added
,$lgCl 2 added
CaCl 2 added
0.0201 0-0293
0.0205 0.0268
0.0268 0.0647
Biochim. Biophys. Acta, 256 (I972) 5 6 5 - 5 7 6
N. SHI/3AT.\ d[ ell.
572
1)rotein from leucocytes, the A T P a s e a c t i v i t y was higher at high ionic s t r e n g t h than at low. The same thing was true for actomyosin from the smooth muscle L%'°. I t seems reasonable to postulate that the contractile protein extracted from leucocytes e x h i b i t e d A T P a s e a c t i v i t y of the actomvosin t y p e which was a c t i v a t e d by Mg "~+a n d Ca 2+ at low ionic strength, a n d also A T P a s e a c t i v i t y of the myosin t y p e which was i n h i b i t e d b y Mg 2+ and a c t i v a t e d by ('a ")+ at high ionic strength, under conditions in wlfich actomvosin is dissociated into myosin a n d actin.
Change in viscosi O, by addition of A T P The relative viscosity of the protein was decreased b y the addition of A T P a n d 3Ig ~+ at high ionic strength. This was followed b y a g r a d u a l increase in viscosity towards the initial level, p r o b a b l y being b r o u g h t a b o u t by the hydrolysis of A T P (Fig. 6). The s e n s i t i v i t y t o w a r d s A T P c o m p u t e d according to the formula of PoIcrzl,: uJ et al? z was 19o %. This value was ahnost the same as t h a t o b t a i n e d for a c t o m v o s i n from s t r i a t e d musclO 2 or smooth musclO 7.
A nalvlical ultracentr@,gal pattern The ultracentrifugal p a t t e r n of the protein in I o mM T r i s - m a l e a t e buffer (pH 7.0) with ionic strength of 0.6 at 20 shows two peaks (Fig. 7) corresponding to 5.8 S a n d 37 S in the presence of A T P and Mg 2+. The former peak corresponded fairly well to t h a t of myosin from s t r i a t e d 2~ a n d smooth musclO 7,2° and the l a t t e r n e a r l y to t h a t of F - a c t i n from the smooth muscle 22 or from p l a s m o d i u m "3.
200 ATP
/*/
1.50
1.00 0 -20
0
30
60
90
120
150
180
210
rime (rain)
FiR.(i. Effect o f A T P o n t l l e ~ i s c o s i t y of the p r o t e i n from l e u c o c y t e s a t 25 . P rot e i n c o n c e n t r a t i o n , 4.o m g / m l ; ionic s t r e n g t h , o.0; T r i s - m a l e a t e buffer, 1o mM (pH 7.o). For d e t a i l s see t e x t . [:ig. 7" U l t r a c e n t r i f u g a l p a t t e r n of the e x t r a c t e d p r o t e i n . The e e n t r i f u g a t i o n was carri('(l out a t 542oo r e v . / m i n in io h i m T r i s - m a l e a t e buffer (pH 7.o) c o n t a i n i n g 6.5 m g p r o t e i n pe r ml, o.0 M KC1, IO mM 3IgCl 2, a n d 5 mM ATP. S e d i m e n t a t i o n proceeds from left to right. The p h o t o g r a p h was t a k e n at z5 nlin a f t e r t o p speed was reached.
Electronmicroscopic fi~zdit~gs The protein at low ionic s t r e n g t h was s t u d i e d b y electronmicroscopy I rain after an a d d i t i o n of the protein solution. The protein dissolved in o.6 M KC1 was a d d e d to IO mM T r i s - m a l e a t e buffer (pH 7.o) containing IO mM MgCI2 and either 5 mM A T P and I mM E G T A or o.I mM A T P and o.I mM CaC12. The final KCI concentration in these p r e p a r a t i o n s was o.o6 M. t3iochim. 13iophy,< Hera, 256 (1972) 565 .576
CONTRACTILE PROTEIN FROM I.EUCOCYTEg
57.3
Fig. 8. F.lectron micrograph of the extracted protein in clearing phase at low ionic strength. The p r e p a r a t i o n contained o. I5 mg of protein per ml, 0.06 M 1<(21, io mM MgC12, i mM EGTA, 5 mM ATP and :o mM Tris maleate buffer, p H 7.0.
In the preparation containing 5 mM ATP and I mM EGTA, the protein showed a clearing response. In the electron micrograph of this preparation, thick (1~I) and thin (A) filaments were seen about I5o A and 8o .~ in diameter, respectively (Fig. 8). It is suggested that the former corresponds to myosin aggregates and the latter to F-actin filaments. In the preparation containing o.I mM ATP and o.I mM CaC12, electron micrographs of negatively stained material showed the formation of m a n y large aggregates, apparently due to joining together both myosin aggregates and F-actin filaments, as observed in the superprecipitation of skeletal actomvosin ~4,2~ (Fig. 9)DISCUSSI()N
From the above experimental findings, it may be concluded that the protein under investigation corresponds to a so-called natural actomyosin-like contractile protein of leucocytes. The viscosity of the extracted protein was somewhat lower than that of actomyosin from striated muscle reported by MOSISrA~;i~TS2a. In this point, there arises an argument that the extracted protein should not be called natural actomvosin. Reconstitution of myosin- and actin-like protein obtained separately from the extracted protein of leucocytes demonstrated the superprecipitation characteristic of synthetic actomyosin2L This finding, together with biopl~ysical and biochemical features Biochim. Biophys. Acta, 256 (I97 z) 565-576
574
X. sH1z~;x'ra el a /
Fig. 9. Electron micrograph of the extracted protein superprecipitated at low ionic strength. The preparation contained o.15 mg of protein per ml, o.o6 M KC1, io mM Mg('l,,, o.~ mM Ca('l,,, o.I mM ATP and [o mM Tris maleate buffer (pH 7.o) of the protein described a l r e a d y in results, m a y indicate t h a t the e x t r a c t e d protein a c t u a l l y corresponds to n a t u r a l a c t o m y o s i n from muscle. In order to e x t r a c t the n a t u r a l actomyosin-like protein from leucocytes, leucocytes were homogenized in a solution with low ionic s t r e n g t h containing A T P and histidine buffer (pH 7.o) i n s t e a d of Weber-F2dsall's solution 40 with high ionic s t r e n g t h used for the e x t r a c t i o n of skeletal myosin B. This procedure was based on the finding t h a t the actomyosin-like contractile protein e x t r a c t e d previously bv us from leucocytes e x h i b i t e d biophysical and biochemical features similar to those of carotid myosin B, except for the absence of Ca `'.+s e n s i t i v i t y 3. S u p e r p r e c i p i t a t i o n of the carotid myosin B was accelerated b y increasing the concentration of a d d e d l~'Ig2+ (ref. I7). The contractile protein with Ca 2+ sensitivity from carotids was capable of being e x t r a c t e d with a low ionic s t r e n g t h solution containing A T P and histidine buffer (pH 7.o) ~,°s. F u r t h e r m o r e , the Ca'~÷-sensitizing protein contained in n a t u r a l actomyosin, a socalled troponin, is r e p o r t e d to be v e r y labile to proteinase digestion 2" and, since the ATPase of myosin is an S H e n z y m e 30, Trasylol, a n a t u r a l proteinase inhibitor, and dithiothreitol, an SH protector, were a d d e d to the above e x t r a c t i n g solution. The protein thus p r e p a r e d e x h i b i t e d the Ca 2+ s e n s i t i v i t y in s u p e r p r e c i p i t a t i o n (Figs. I and 2) and the Mg2+-dependent A T P a s e a c t i v i t y (Fig. 4), as well as biophysical a n d biochemical properties characteristic of myosin B or n a t u r a l actoinyosin from muscle. In n a t u r a l actomvosin or myosin B from s t r i a t e d muscle the o p t i m a l Mg -°+ Biochi~z. l~io[~h.vs. ,4cla, 256 (I97 z) 565 -576
COXTRACT1LE PROTEIN
FROM LEUCOCYTES
375
concentration necessary for superprecipitation is considerably lowat, whereas it was very high in the extracted protein from leucocytes (Fig. 3), just as observed in carotid smooth musclelL Furthermore, as described in 1~1~.svl:rs, the extracted protein demonstrated features resembling actomyosin from smooth muscle in the behaviour of the ATPase and analytical ultracentrifugal pattern. Thus, the presently extracted contractile protein from leucocytes seems to be similar to natural actomyosin from smooth muscle rather than that from striated muscle. In the analytical ultracentrifugal pattern (Fig. 7), the s°2o.w value of the retarding peak of the extracted protein was almost the same as that of the retarding peak of an actomyosin-like protein extracted previously from leucocytesa, but the value of the foregoing peak in Fig. 7 was somewhat greater than that of the foregoing peak of the protein prepared by the previous method a. This increase in s value of the foregoing component observed in the extracted protein having Ca '2+ sensitivity, may be explained by the assumption that the F-actin of the protein may be endowed with a native tropomyosin-like substance, The contractile protein has been extracted, so far, not only from muscle but also from free cells, for example from platelets 32, plasmodium as, fibroblasts a4, sarcoma cellsa~, and leucocytes3, dc.. These contractile proteins, however, showed requirement for Mg -°+, ATP and no Ca 2+ unlike natural actomyosin from muscle, which has a Ca"+ requirement for superprecipitation. Therefore, it has been posed that the question of whether or not the contractile protein of free cells other than muscle is different from that of muscle is decided on the basis of a need for a Ca`)+ concentration in the superprecipitation. From the nature of tire contractile protein presently extracted from leucocytes, it is suggested that even the contractile protein of free cells may be susceptible to Ca ')+ as observed in the contractile protein from the muscle. H.\T.xx~ 3" demonstrated that the movement of plasmodial fragments was subject to the specific effect of Ca ~'+ following caffeine treatment, although the contractile protein possessing ( ' a " ' sensitivity for the superprecipitation had not been isolated from plasmodium. From findings obtained in morphological, physiological and phylogenetic studies concerning the motility of leucocytes, we have reported that the mntile form and function of leucocvtes are coordinately controlled by ATP and that leucocx'tes po.~sess a motor organ consisting of an actomyosin-like contractile protein acting as a mechanochemical system coupled with ATP", a7 a~. In this paper, it has become evident that the contractile protein of leucocytes, as well as of the natural act.myosin of smooth muscle, exhibits the contraction and relaxation regulated by concentrations of free Ca2 ~ in the presence of ATP and Mg 2.. It is conceivable that the motor organ of leucocytes consists of natural actomyosin-like contractile protein similar to that of muscle and that the movement of leucocvtes is achieved by contraction and relaxation of the natural actomvosin-like contractile protein.
i 1(. IeUKUSIIIMA, N. SENDA, S. ISHIGAMr, J. ]~NDO, -'~f. ISHII, Y. MURAKAMI, l(. NISHIAN AND Y. UEDA, Alcd..[. Osaka Univ., 5 (I954) 23I. 2 N. ~ENDA AND ~{. TAMURA, A*z~Z~t. Rep. Center Adult Dis., Osaka, I (1961) I. 3 N. ~F. NDA, N. ~HIBATA, N. TATSUMI, I{. KONDO AND K. HAMADA, Biochim. Biophys. Ac/a, lSI (I969) 191. 4 "~- I'~BASHI AND 1~'. FBASH1, J. Biochem. Tokyo, 55 (1964) 604. 5 ~. tLm~sHi .~No A. KODA51A, J. Biochem. Tokyo, 60 (1966) 733.
Biochim. Biophys. Acla, 256 (1972) 5 6 5 - 5 7 6
5 7 ()
s . SHIBAT_\ v[ al
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Biochim. Biophys. Acta, 256 (1972) 5 6 5 - 5 7 ~