Inhibition of axoneme and dynein ATPase from sea urchin sperm by free ATP

225

Biochimica et Biophysica Acta, 422 (1976) 225--230 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 67692 INHIBITION OF AXONEME AND DYNEIN ATPase FROM SEA URCHIN SPERM BY FREE ATP

MASAO HAYASHI Institute of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464 (Japan) (Received August 1st, 1975)

Summary Sea urchin sperm flagellar ATPase (EC 3.6.1.3) has magnesium-ATP as an effective substrate and is inhibited by free ATP. The inhibition is prevented by high concentration of KC1 or NaC1. 0.4 M KC1 extracts 4 8 % o f ATPase activity from axoneme. The 0.4 M KC1 extract and 0.4 M KCl-treated axoneme are also inhibited by free ATP and this inhibition is reversed by KC1. Dynein purified twice by sucrose density gradient centrifugation is also inhibited by free ATP; this inhibition is also reversed by KC1.

Introduction

The ATPase dynein (EC 3.6.1.3), identified as " a r m " in the 9 + 2 structure [1,2], can be obtained in two enzymatically active forms. Cilia from Tetrahymena yield both a 14 S form and a 30 S form, while sea urchin flagella yield only a 10--11 S form [3]. Dynein ATPase is known to be stimulated by Mg2~ or Ca 2÷ [4], and is influenced by binding of microtubules in its KC1 dependency and pH dependency [5,6]. With a glycerine-model or Tritonmodel, the movement of flagella or cilia was found to couple with hydrolysis of ATP [7--9]. From the divalent cation requirement for the movement, Gibbons and Gibbons [9] and Douglas and Holwill [10] suggested that a magnesiumATP complex was an effective substrate. Recently, our kinetic analysis of axoneme and dynein ATPase showed that the effective substrate of the ATPase was a magnesium-ATP complex and free ATP inhibited ATPase [11]. This report describes the reversal by KCI of the inhibition by free ATP. Materials and Methods Sperm were collected from sea urchin Pseudocentrotus depressus. Axoneme was prepared as previously reported [11]. Axoneme was treated with

226

0.4 M KCI, 20 mM Tris • HC1 buffer (pH 8.0) and 0.1 mM dithiothreitol for 5 min at 0°C, and was then centrifuged at 8000 rev./min for 7 min with a Sorvall SS-34 rotor. The supernatant and precipitate were referred to as KCl-extract and KCl-treated axoneme, respectively. For the purification of dynein, Tris-EDTA extract [11] was fractionated by 5--20% (w/v) sucrose density gradient centrifugation with a SW 27 rotor at 74 900 X g for 43 h at 4°C. Fractions showing ATPase activity (fraction number 9--14 in Fig. 1, a) were collected and again centrifuged in the same sucrose density gradient with a SW 25.2 rotor at 75 500 X g for 35 h at 4°C. Fractions showing ATPase activity (fraction number 14--18 in Fig. 1, b) were collected and used as a purified dynein. ATPase activity was measured at 30°C as previously reported [11]. Inorganic phosphate liberated was measured by the m e t h o d of Murphy and Riley [12]. One unit of the enzyme activity was defined as the a m o u n t of enzyme which catalyzed the liberation of 1 pmol Pi per min. Protein concentration was determined according to the method of Lowry et al. [13]. Results Our previous report shows that axonemal ATPase requires magnesiumATP as an effective substrate and is inhibited by free ATP [11]. These effects were analyzed from the ATPase activities in 30 mM KC1 and 20 mM Tris • HC1 buffer (pH 8.0), when the magnesium-ATP concentration was fixed and free ATP concentration was varied. In the presence of 0.4 M KC1, however, axone-

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F i g . 1. ( a ) F i r s t s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f T r i s • E D T A e x t r a c t . T w o m l o f T r i s • E D T A extract of 6.24 mg/ml were applied on 5--20% sucrose containing 1 mM Tris • HCI buffer (pH 8,0), 0.1 m M E D T A a n d 0.1 m M d i t h i o t h r e i t o l , a n d c e n t r i f u g e d a t 7 4 9 0 0 X g f o r 4 3 h a t 4 ° C . E a c h 3 4 d r o p s w e r e collected from the bottom of a tube. (b) Second sucrose density gradient centrifugation. A sample was collected, fractions showing ATPase activity in Fig, la (fraction number 9--14) and applied on 5--20% sucrose containing 1 mM Tris • HCI buffer (pH 8.0), 0.1 mM EDTA and 0.1 mM dithiothreitol. Centrifugation was performed at 75 500 X g for 35 h at 4°C. o o, a b s o r b a n e e a t 2 8 0 n m ; • e, ATPase activity (arbitrary unit).

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Fig. 2. ( a ) A x o n e m a l A T P a s e i n h i b i t e d b y h i g h c o n c e n t r a t i o n s o f A T P . A T P a s e t h e p r e s e n c e o f 2 0 m M T r i s • HC1 b u f f e r ( p H 8 . 0 ) , A T P a n d 0 . 0 5 m M M g S O 4 ( o ) 3 0 ° C. ( b ) I n h i b i t i o n r e v e r s a l o f a x o n e m a l A T P a s e b y KC1. A T P a s e a c t i v i t y w a s o f 0 . 4 M KC1, 2 0 m M T r i s • HC1 b u f f e r ( p H 8 . 0 ) , A T P a n d 0 . 0 5 m M M g S O 4 ( o ) 3 0 ° C.

activity was measured in o r 0 . 5 m M M g S O 4 ( o ) at m e a s u r e d in t h e p r e s e n c e o r 0 . 5 m M M g S O 4 ( e ) at

mal ATPase showed a typical Michaelis-Menten type relationship and was not inhibited by high concentrations of ATP (Fig. 2b) as compared with that in the absence of KC1 (Fig. 2a). ATPase activity was always higher in the presence of 0.4 M KCI than in the absence of KC1 at all range of ATP concentrations used. This suggests that 0.4 M KC1 protected axonemal ATPase from free ATP. Fig. 3 shows that axonemal ATPase was stimulated by KCI more strongly in the presence of 1.5 mM ATP and 0.05 mM MgSO4 than that in the presence of 0.2 mM ATP and 0.05 mM MgSO4. These two activity curves crossed at 0.2 M KC1. Axonemal ATPase started to decrease in the presence of KC1 concentrations greater than 0.2 M. The difference between both activities changed gradu-

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Fig. 3. I n h i b i t i o n a n d r e v e r s a l o f i n h i b i t i o n o f a x o n e m a l A T P a s e . A T P a s e a c t i v i t y w i t h rising c o n c e n t r a t i o n o f K C I w a s m e a s u r e d i n t h e p r e s e n c e o f 0 . 0 5 m M M g S O 4, 2 0 m M T r i s • HC1 b u f f e r ( p H 8 . 0 ) a n d 0 . 2 m M A T P ( e ) o r 1 . 5 m M A T P (©) at 3 0 ° C . Fig. 4. K C I stimulation of dynein ATPase. A T P a s e activity was measured in the presence of 20 m M Tris • H C I buffer ( p H 8.0), 1 m M A T P , and 0.1 m M M g S O 4 (A), 1 m M M g S O 4 (o) or 20 m M M g S O 4 (e) with different concentrations of K C I at 30°C.

228 TABLE I INHIBITION AND REVERSAL FIED DYNEIN

OF INHIBITION AT AXONEME, TRIS • EDTA EXTRACT AND PURI-

ATPase activity was m e a s u r e d in the presence HCI b u f f e r ( p H 8.0) at 3 0 ° C . Conditions

0.2 1.5 0.2 1.5

mM mM mM mM

ATP ATP A T P a n d 0.4 m KC1 A T P a n d 0 . 4 KC1

o f e n z y m e , A T P , KC1, 0 . 0 5 m M M g S O 4 a n d 20 m M T r i s •

ATPase activity (units/mg) Axon~me

Tris • E D T A extract

Dynein

0.125 0.0575 0.110 0.150

0.248 0.186 0.244 0.398

0.620 0.310 0.672 1.21

ally with rising c o n c e n t r a t i o n of KC1. This suggests t hat inhibition by free ATP was strongest in the absence of KC1 and was gradually reversed by KC1. The same profile was observed in the case of NaC1 substituted for KC1. F r o m Fig. 3, a rough idea of inhibition and inhibition reversal of the ATPase could be deduced after comparison with four levels of activity. The results o f the measurement of inhibition of axonemal ATPase in the presence of 1.5 mM and 0.2 mM ATP and the reversal of the inhibition p r o d u c e d by addition o f 0.4 M KC1 are shown in Table I. T r e a t m e n t with 0.4 M KC1 at 0°C for 5 min extracted 48% of ATPase activity and 14% of protein from axoneme, and it was shown that bot h KC1 e xtr act and KCl-treated a x o n e m e were inhibited by free ATP and that this inhibition was reversed by KC1, although the former ATPase activity was about 4 times higher than the latter's (Table II). Usually, dynein was extracted f r om a x o n e m e by dialyzing against I mM Tris • HC1 buffer (pH 8.0) and 0.1 mM EDTA according to Gibbons [ 1 , 2 ] . 0.1 mM dithiothreitol was added in our system to prevent denat urat i on of dynein ATPase. Dithiothreitol did n o t influence the characteristics of inhibition or inhibition reversal of the ATPase. 0.4 M KC1 could n o t extract more than 60% of the ATPase from sea urchin sperm axoneme, while dialysis against 1 mM Tris • HC1 buffer (pH 8.0), 0.1 mM EDTA and 0.1 mM dithiothreitol extracted 90% or more ATPase. Tris • EDTA-extracted ATPase showed the same inhibition

T A B L E II INHIBITION AND REVERSAL

OF INHIBITION OF KC1-EXTRACT ABD KC1-TREATED AXONEME

ATPase activity was m e a s u r e d in the presence HC1 b u f f e r ( p H 8.0) at 3 0 ° C . Conditions

0.2 1.5 0.2 1.5

mM mM mM mM

ATP ATP A T P a n d 0 . 4 M KCI A T P a n d 0 . 4 M KCI

o f e n z y m e , A T P , KC1, 0 . 0 5 m M M g S O 4 a n d 20 m M T r i s •

ATPase activity (units/mg) KC1 e x t r a c t

KCl-treated

0.281 0.194 0.393 0.536

0.0864 0.0381 0.0662 0.0876

axoneme

229 and reversal of inhibition properties as axonemal ATPase (Table I). This suggests that the inhibition and inhibition reversal property of the enzyme was independent of microtubule binding. Dynein was purified from Tris. EDTA extract by 2 sucrose density gradient centrifugations (Fig. 1). The ATPase fractions from the first sucrose density gradient centrifugation were pooled. ATPase activity of this sample showed 3.00 units/mg in the presence of 30 mM KC1, 2 mM MgSO4, 1 mM ATP and 20 mM Tris • HC1 buffer (pH 8.0) at 30°C. This sample was again centrifuged in a sucrose density gradient (Fig. lb). From this figure double peaks of protein were observed; one near the bottom, the other in the middle. The protein concentration of the middle fractions was coincident with ATPase activity. Fractions 14--18 in Fig. l b were used as purified dynein. ATPase activity of the purified dynein was 4.02 units/mg in the presence of 30 mM KC1, 2 mM MgSO4, 1 mM ATP and 20 mM Tris-HC1 buffer (pH 8.0) at 30°C. The purified dynein also exhibited inhibition by free ATP and inhibition reversal by KC1 (Table I). In sea urchin sperm flagella, KC1 is known to stimulate axoneme and dynein ATPase [5]. Dynein ATPase at 0.1 mM and 1 mM MgSO4 in the presence of 1 mM ATP increased with rising concentration of KC1, while the ATPase at 20 mM MgSO4 in the presence of 1 mM ATP was independent of KC1 concentration (Fig. 4). Free ATP concentration decreases with rising concentration of MgSO4 and is negligible in the presence of 20 mM MgSO4. This suggests that KC1 stimulation of dynein ATPase can be interpreted as a result of a reversal of inhibition of the ATPase by KC1. Discussion Inhibition of flagellar ATPase by free ATP was reversed by KC1. With respect to the characteristics of the inhibition and inhibition reversal, there was no essential difference between axoneme and dynein. In P a r a m e c i u m demembraned by Triton X-100, calcium ions regulated the direction of ciliary beating at 10-6M [14,15]. Brokaw et al. [16] recently reported that the mode of flagellar beats of Triton X-100 treated spermatozoa was also regulated by low concentration of calcium ions. However, calcium ions did not influence the inhibition and inhibition reversal of axonemal ATPase at low concentration. Sea urchin sperm contained about 10 mM ATP [17]. But, there is no report about salt concentration and divalent cation concentration in sea urchin sperm flagella. Therefore, we cannot speculate on the biological function of the inhibition and inhibition reversal of dynein ATPase in vivo. Acknowledgements I would like to express my thanks to Professor Fumio Oosawa for his interest and valuable suggestions. I am also very grateful to Sugashima Marine Laboratory (Nagoya University} and Misaki Biological Station (Tokyo University} for supplying sea urchins.

230

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