Preparation and characterization of a dissociated 14-S form from 30-S dynein of Tetrahymena cilia

Bioehimiea et Biophysica Acta, 351 (1974) 142-154 © Elsevier Scientific Publishing Company,~Amsterdam - Printed in The Netherlands BBA 36702 P R E P A R A T I O N A N D C H A R A C T E R I Z A T I O N OF A DISSOCIATED 14-S F O R M F R O M 30-S D Y N E I N OF T E T R A H Y M E I V A CILIA

MINORU HOSHINO Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyoku, Tokyo (Japan) (Received December 3rd, 1973)

SUMMARY Dissociation of 30-S dynein of Tetrahymena cilia was investigated by the use of sodium dodecylsulfate and urea. By treatment with 0.01 ~ sodium dodecylsulfate, some of 30-S dynein could be dissociated into the 14-S form. 0.02-0.03 ~ sodium dodecylsulfate treatment showed only one peak of 14-S ATPase. Sucrose density gradient centrifugation of 30-S dynein in 2 M urea newly revealed 18-S and 23-S ATPases. When 30-S dynein was treated with 4 M urea for 10 min, the 14-S form of dynein (dissociated dynein) possessing comparably high ATPase activity was obtained. ATPase activity of the dissociated dynein was enhanced by Mg 2+ with an optimum at 2 mM, and was sharply activated by Ca e+ at 2 mM and almost completely inhibited at higher concentrations. The C a 2+ :Mg 2+ ratio at 1 mM was 1.5. KC1 inhibited Ca 2+ATPase activity, but affected Mg2+-ATPase activity to a lesser extent. The pH optimum of Mg2+-ATPase was 7.5 with shoulders near 6.5 and 9.0. The pH optimum of Ca2+-ATPase was 8.0 with a shoulder around 7.0. The Km for ATP was 14-15 #M. The dissociated dynein had two components in common with 30-S and 14-S dyneins on sodium dodecylsulfate-polyacrylamide gel electrophoresis.

INTRODUCTION Ciliary and flagellar axonemes have an ATPase named dynein, which is a structural protein forming the projections ("arms") bound to the A tubules of the outer doublets [1, 2]. Since ATP is the energy source for ciliary and flagellar motility [3, 4], dynein presumably plays an important functional role in the process which underlies motility [5]. Cilia of Tetrahymena pyriformis always yield two forms of dynein sedimenting at 14-S and 30-S [1]. Both dyneins differ in several enzymatic properties [6]. The presence of 30-S dynein is required for the sensitivity of the light-scattering properties of axonemes to ATP [7], and 30-S dynein preferentially recombines with the outer fibers [2]. According to these data, the 30-S form of dynein has been considered to be physiologically more active [6]. Electron microscopic observations have revealed that 30-S dynein is a linear connection of several globular particles, which coincide in size with globular 14-S

143 dynein [1]. So it has been thought that 30-S dynein is an oligomeric form of 14-S dynein "monomers" [1]. This paper shows that 30-S dynein can be dissociated to the 14-S form by treatment with sodium dodecylsulfate or urea. The enzymatic properties of the dissociated dynein are presented and compared with 30-S and 14-S dyneins. The relationship between 14-S and 30-S dyneins is also discussed. MATERIALS AND METHODS

Preparation of dynein Cilia were isolated from Tetrahymena pyriform&, strain W, by the calciumethanol method [2]. The cells were grown until the late logarithmic phase in a medium containing 2 ~ (w/v) polypeptone, 0.5 ~ (w/v) yeast extract and 0.87 ~ (w/v) glucose under continuous aeration. The isolated cilia were once washed with 30 m M Tris-HC1 (pH 8.2) containing 3 m M MgC12 and 0.1 m M dithiothreitol, and the axonemes were prepared by suspending the isolated cilia in 1 ~ (w/v) Triton X-100 plus Tris-HC1-M,C12-dithiothreitol [8]. The resulting axonemes, after being washed twice with Tris-HCI-MgC12-dithiothreitol, were suspended in a small volume of 1 mM Tris-HC1 (pH 8.2) containing 0.1 mM E D T A and 0.1 mM dithiothreitol and dialyzed overnight against Tris-HC1EDTA-dithiothreitol [2, 6]. The dialysate was centrifuged at 45 000 × g for 20 min. The supernatant fraction (Fraction I by Gibbons [2]) consisted of a mixture of 14-S and 3O-S dyneins together with some non-ATPase proteins having a sedimentation coefficient of about 4-S [1, 2, 6]. In some experiments, this crude dynein fraction was used without further purification. For preparing 30-S and lzI-S dyneins, the crude dynein fraction was centrifuged through a sucrose density gradient. In some preparations dithiothreitol was omitted, but no significant difference in the results of the experiments was found between preparations with and without dithiothreitol. During the procedures the temperature was kept at 0-4 °C. Treatment with sodium dodecylsulfate To a crude dynein solution or 30-S dynein fraction an adequate volume of 1 sodium dodecylsulfate in an ice bath was added so that a desirable concentration of sodium dodecylsulfate was obtained. The enzyme fraction containing sodium dodecylsulfate was centrifuged through a sucrose density gradient without sodium dodecylsulfate. In most of experiments enzyme fractions were used for zonal centrifugation shortly after the addition of the detergent. In some experiments the enzyme fractions containing sodium dodecyl sulfate were left standing overnight before zonal centrifugation. The time of the treatment made no effect on the centrifuge patterns. Treatment with urea The crude dynein fraction was dialyzed overnight against 10 mM Tris-HC1 (pH 8.2) containing 0.1 mM E D T A and a varying concentration of urea, and then layered on top of a 5-20 ~ sucrose density gradient in the same solution. A short treatment was performed as follows. The crude dynein fraction was mixed in a dialyzing tube with an equal volume of 8 M urea, which was freshly

144 prepared just before use, and left standing for 10 min in the cold. The mixture was then dialyzed against 10 m M Tris-HC1 buffer (pH 8.2) containing 0.1 m M EDTA and 0.1 m M dithiothreitol. The sample was used for zonal centrifugation. The isolated 30-S dynein fraction was also treated in the same manner.

ATPase assay The standard assay medium consisted of 25 mM Tris-HC1 (pH 8.2), 1 mM MgC12 and 1 mM ATP(disodium), and incubation was performed at 25 °C. The reactions were initiated by the addition of ATP and stopped by the addition of 5 ~ trichloroacetic acid. The precipitates, if any, were removed by brief centrifugation. Inorganic phosphate in the supernatant fluid was determined by the method of Fiske and SubbaRow [9]. In the experiments with Ca ~+ activation, MgCI 2 in the standard assay medium was replaced by CaCI2. For estimation of the Michaelis constant, [7-32P]ATP was used and HCIO4 was substituted for trichloroacetic acid. Nucleotides were removed by adsorption to Norit [10]. Radioactivities of the liberated inorganic phosphate were counted by a liquid scintillation system (Beckman model LS-200B). All experiments for the ATPase assay used preparations of dynein which were less than 2 weeks old.

Polyacrylamide gel electrophoresis Samples were run on sodium dodecylsulfate-polyacrylamide gel (6.0 ~o acrylamide-0.16 ~ methyler~ebisacrylamide) as described by Weber and Osborn [11] with 25 mM Tris-glycine buffer (pH 8.5) containing 0.1 ~ sodium dodecylsulfate. The gels were stained with Coomassie brilliant blue in 4 0 ~ (v/v) m e t h a n o l - 1 0 ~ (v/v) acetic acid and destained by diffusion in 7 ~ acetic acid. Proteins were reduced by dialyzing against 0.1 M fl-mercaptoethanol containing 8 M urea, 0.1 ~ sodium dodecylsulfate, 5 mM EDTA and 20 m M Tris-HC1 (pH 8.2) at room temperature. The proteins were applied without alkylation.

Protein determination Protein concentrations were determined by the method of Lowry et al. [12] with bovine serum albumin as a standard.

Sucrose density gradient centrifugation The enzyme fraction was layered on top of a 5-20 ~ (w/v) sucrose density gradient containing 10 mM Tris-HC1 (pH 8.2), 0.1 mM EDTA and 0.1 mM dithiothreitol, and centrifuged in either an RPS 25-1 or RPS 25-2 rotor of an Hitachi model 65P centrifuge at 2413130 rev./min for 21 h. The contents of the centrifuge tubes were fractionated by using a syphon in samples of either 25 drops for RPS 25-1 or of 50 drops for RPS 25-2 each. Each sample was assayed for protein and for ATPase activity. A mixture of horse apoferritin (17.6 S, ref. 13) and bovine ~,-globulin (7.3 S, ref. 14) was centrifuged simultaneously in a separate centrifuge tube for estimation of the sedimentation coefficients [15]. For the centrifugation in a solution containing urea, the sucrose density gradient was made in a solution containing 10 mM Tris-HCl (pH 8.2), 0.1 mM EDTA, 0.1 mM dithiothreitol and varying concentrations of urea. The enzyme fraction was

145 dialyzed against the same solution without sucrose, and layered on top of the gradient. Centrifugation was performed under the same conditions as described above. RESULTS

Treatment of crude dynein fraction with sodium dodecylsulfate As sodium dodecylsulfate is known as a reagent which dissociates various protein molecules into their subunits, the dissociation of 30-S dynein into its subunits, probably 14-S dynein, was examined. A typical centrifuge pattern of the crude dynein fraction without sodium dodecylsulfate treatment is presented in Fig. 1. Most of the activities of dynein appeared as the 30-S form and the 14-S form was a minor component of ATPase. 2o 4S

0.3

./-\

3os

o

E

0.2

o2

0.1

0.1

, . ../ ," i k.. o o

I

"-J

" /

o o OT o o"

10

~ a. o

E

or9

20

30

o

40

5

No.

Tube

Fig. 1. Sucrose density gradient centrifugation of the crude dynein fraction. 0.2 m! of each fraction was used for the ATPase assay and was incubated with 1 ml of the standard assay medium. The reactions were performed at 25 °C for 20 min. H , absorbance at 280 nm; G---O, ATPase activity (/~moles Pi/min per ml of the fraction).

After treatment with 0.01% (w/v) sodium dodecylsulfate, the peak of protein (absorbance at 280 nm) corresponding to 30-S dynein fell in height and the peak corresponding to 14-S dynein rose (Fig. 2). The pattern of the ATPase activity was parallel to the protein profile. Specific activity at the peaks was almost as high as that of the £. a

8L 4S

0.10

E

oo,

,

v

0.05 ~-I,.,.M~I o

•

e

..o"

0.02

i •

\

~ a. o

E

....... I()

20 Tube

30

40

No.

Fig. 2. Sucrose density gradient centrifugation of the crude dynein fraction treated with 0.01% sodium dodecylsulfate. 0.5 ml of each fraction was used for the ATPase assay. The reactions were performed at 25 °C for 40 min. 0 - - - 0 , absorbance at 280 nm; G---O, ATPase activity (~moles Pj/min per ml of fraction).

146 untreated enzymes. Some of the 3O-S dynein must have been dissociated into the 14-S form. There were no ATPase peaks other than those of the 14-S and 30-S dyneins. When the crude dynein fraction was treated with sodium dodecylsulfate of a higher concentration (0.02~0.03 ~ (w/v)), the 30-S dynein almost disappeared with respect to both the protein profile and ATPase activity, and only one peak of ATPase activity sedimenting at 14-S remained, although the activity of the sodium dodecylsulfate-treated ATPase was lowered to about one-fifth of the untreated 30-S dynein (Fig. 3). However, the protein peak shifted from 14 to 11 S. It can, therefore, be concluded that by the treatment with sodium dodecylsulfate 30-S dynein was dissociated into subunits, which suffered a slight distortion by sodium dodecylsulfate to present the sedimentation coefficient of 11 S. Some resistant subunits still had activities at 14 S.

4S

Q 46

0.15 11 S

./\

oo2

/~ V

010

~E

•

/ ? ~o 0.05

b

0.01

a.

,

% ....... .-"

,/

\

.o.-O'" 0

~

I0

1

n-

.o"

I

20

*~ I

30

o.

.n

4'0

0

Tube No

Fig. 3. Sucrose density gradient centrifugation of the crude dynein fraction treated with 0.03 ~ sodium dodecylsulfate. 0.5 ml of each fraction was used for the ATPase assay. The reactions were performed at 25 °C for 1 h. @--O, absorbance at 280 nm; (3---(3, ATPase activity Cumoles Pl/min per ml of fraction).

In the presence of 0.02-0.03 ~ sodium dodecylsulfate, the original 14-S dynein lost its ATPase activity almost completely. Treatment of 30-S dynein with 0.03 sodium dodecylsufate, on the other hand, retained the ATPase activity by one-fifth of its original level, and only one protein peak was found at 11 S. This indicated that 30-S dynein could be a homo-oligomer. Distortion of the dynein also occurred when a much higher concentration of sodium dodecylsulfate (0.1 ~ ) was used in the treatment. 0.1 ~ sodium dodecylsulfate caused all proteins included in the crude dynein fraction to change into a single peak at about 4 S. It was not the case that the 30-S and 14-S dyneins were dissociated into much smaller subunits, because the molecular weights of both dyneins were estimated to be several hundred thousand in sodium dodecylsulfate-polyacrylamide gel electrophoresis with a buffer containing 0.1 O//o sodium dodecylsulfate.

Sucrose density gradient centrifugation in urea It was suggested that 30-S dynein could be dissociated into its subunits, probably 14-S dynein, in the presence of low concentrations of sodium dodecylsulfate. However, the subunits obtained had a remarkably low ATPase activity. For the

147 purpose of obtaining subunits still possessing high activity, urea replaced the sodium dodecylsulfate. First, an overnight treatment with urea was performed in a concentration of 2 or 4 M. A typical centrifuge pattern in 2 M urea is shown in Fig. 4. The peak of 30-S dynein disappeared in respect to both the protein profile and ATPase activity and 23- and 18-S peaks newly appeared with regard to both profiles of protein and ATPase activity. Furthermore, no activity could be detected at the 14-S protein peak. The activity of the 30-S dynein fraction assayed in 2 M urea was as high as that of the untreated enzyme, while 14-S dynein completely lost its activity in 2 M urea. 30-S dynein seemed to be dissociated into the 23-S and 18-S forms, and the protein peak at

4S

o.o6

"

23_s

,o,

/

\'. .."

-oo2

\.

0.04 _

o

o.o

0

'~°"°

10

~

i ,o 210

/°"°"°-n

30

I

40

!

Tube No.

Fig. 4. Sucrose density gradient centrifugation of the crude dynein fraction in 2 M urea. 0.5 ml of each fraction was assayed at 25 °C for 1 h for ATPase activity. 0 - - 0 , absorbance at 280 nm; O---O, ATPase activity (Ftmoles Pi/min per ml of fraction).

14-S must have been mostly derived from denatured 14-S dynein itself. The 18-S form of dynein had slightly higher ATPase activity than the 23-S form. Two possibilities were brought out about the dissociation process of 30-S dynein in 2 M urea. Firstly, 30-S dynein was dissociated into the 18-S form through the step of the 23-S form. Secondly, 30-S dynein consisted of an equal number of moles of 23-S and 18-S ATPases and 2 M urea dissociated the 30-S dynein into these two forms. The second possibility could be denied, because the amounts of 18-S and 23-S ATPases were not equal judging from the protein profile. It was remarkable that the specific activity of the dissociated 23-S and 18-S ATPases are almost comparable to that of intact 30-S dynein. When the crude dynein fraction was treated with an increasing urea concentration of 4 M, 30-S dynein was no longer found with respect to the protein profile and ATPase activity. The time course of losing ATPase activity in 4 M urea clearly indicated that 30-S dynein lost its activity quickly within 60 min. The sucrose density gradient centrifugation, therefore, revealed only 4-S and 14-S protein peaks and no ATPase activity was detected in any fraction.

148 S h o r t t r e a t m e n t with urea

2 M urea caused partial dissociation of the 30-S dynein, that is, to the 23-S and 18-S forms of dynein but not to 14-S dynein. 4 M urea dissociated 30-S dynein into 14-S dynein, but the ATPase activity was lost at the same time. It was found that dialysis of 4 M urea-treated 30-S dynein largely resumes its activity if the time of the treatment is 10 rain. A successful dissociation of 30-S dynein into an active 14-S form was performed as follows. The 30-S dynein fraction was treated with 4 M urea for 10 rain in the cold and dialyzed quickly against l0 m M Tris-HC1 (pH 8.2), 0.1 m M E D T A and 0.1 m M dithiothreitol. The ATPase profile in sucrose density gradient centrifugation showed one major peak corresponding to 14 S and a minor peak of 22 S (Fig. 5). The profile of Ca2+-activated ATPase was almost parallel in a rather higher level to that of Mg2+-activated ATPase. Four protein peaks were detected at c

g

0.3

E c:

Q2

23S

•~

;

,t',X

,,

o~o ~

*,,/,o,,

~

,,,,"

"~

16S

./~

.,,

\

22S 0.1

0.15

14 S

~ o

,=,

0.05

,,t..~, ,,~ ',

,,~ m

",,.,¢ , o

...........

1~3

210

i-o ....

30

".-,

40

0

E ::t

Tube No.

Fig. 5. Sucrose density gradient centrifugation of the 30-S dynein fraction treated with 4 M urea for 10 rain. 0.1 ml of each fraction was used for the ATPase assay. The reactions were performed at 25 °C for 40 rain. O - - O , absorbance at 280 nm; O---©, ATPase activity (#moles P~/min per ml of fraction).

16 S, 23 S, 8 S and 4 S. The former two (16 S and 23 S) probably corresponds to the ATPase peaks of 14 S and 22 S, respectively. Though the heights of those peaks varied with the preparation, the 16-S protein peak was usually higher than the peak of 22 S in most preparations. Table I summarizes the manner of preparation and specific activities of all forms of dynein. Further purification of the 30-S dynein could be done by applying the crude dynein fraction to a hydroxyapatite column before density centrifugation (Kaji, K., personal communication). When this purified 30-S dynein was treated with 4 M urea in a similar manner and subjected to sucrose density gradient centrifugation, the sedimentation pattern was principally similar to the result mentioned above except that the 8-S and 4-S protein peaks could not be detected. Therefore, the 8-S and 4-S peaks were attributed to contaminants: Differences in the sedimentation coefficients between the ATPase peaks (14 S and 22 S) and the protein peaks (16 S and 23 S) were probably due to a slight change in the conformation of the enzyme molecules by urea. The 22-S form of ATPase might be an incompletely dissociated form, through which the 14-S form was formed. It was hardly conceivable that the 22-S form was formed from the 14-S form of dynein in a backward process. This 22-S ATPase could be identical with the 23-S ATPase found in centrifugation in 2 M urea. The 18-S ATPase detected in centrifugation in 2 M urea, however, was not found in the case of the short treatment with 4 M urea.

149 TABLE I COMPARISON OF SPECIFIC ACTIVITIES OF DYNEINS OBTAINED BY VARIOUS PROCEDURES The activity was measured under standard assay condition (see Materials and Methods). Values in parentheses indicate Ca2+-ATPase activity. Sample

Treatment

Form of ATPase

Specific activity (/~moles Pt/mg protein per min)

Crude dynein fraction

None

30 S 14 S

0.514 5- 0.134 (0.462 5-4-0.110) 0.906 5- 0.035 0.537 ± 0.165 (0.344 5- 0.076) 0.659 ± 0.061

Crude dynein fraction

0.01 ~ sodium dodecylsulfate (1 h-1 day)

30 S 14 S

0.384 5- 0.076 0.462 5- 0.111

Crude dynein fraction

0.02q3.03 sodium dodecyl sulfate 14 S (1 h-1 day)

Ca 2÷ : Mg2+ ratio

0.0934 5- 0.0144

Crude dynein fraction

centrifugation in 2 M urea

23 S 18 S 14S

0.327 0.412 0

Crude dynein fraction

centrifugation in 4 M urea

14S

0

30-S dynein

4 M urea for 10 min

22 S 14 S

0.145 5- 0.027 (0.161 ± 0.008) 1.29 ± 0.02 0.364 ± 0.023 (0.561 ± 0.037) 1.54

This enzymatic p r o p e r t i e s o f the dissociated 14-S f o r m were studied especially in c o m p a r i s o n with the 30-S and 14-S dyneins. In the following this 14-S f o r m will be n a m e d dissociated dynein.

Characterization of the dissociated dynein F o r the c h a r a c t e r i z a t i o n o f the dissociated dynein, the fraction at the p e a k o f the d i s s o c i a t e d d y n e i n was used, b u t fractions next to the p e a k were also used in some e x p e r i m e n t s . As it t o o k a few d a y s to p r e p a r e the dissociated dynein, experiments for the c h a r a c t e r i z a t i o n were p e r f o r m e d within a week at the earliest a n d at the latest within 2 weeks after i s o l a t i o n o f the cilia. The specific activity o f the dissociated d y n e i n m e a s u r e d in the s t a n d a r d assay m e d i u m was a b o u t a h a l f as high as that o f 30-S dynein. The dissociated dynein could be activated by either M g z+ o r Ca 2+ at a low c o n c e n t r a t i o n similar to the 30-S a n d 14-S dyneins [6]. The C a 2+ : M g 2+ r a t i o at 1 m M d i v a l e n t cations was consistently a b o u t 1.5 t h r o u g h o u t every fraction o f the p e a k in a sucrose g r a d i e n t centrifugation. This value was rather closer to that o f 30-S dynein (0.95) t h a n that o f 14-S dyr~ein (0.39) [6]. The specific activity o f the dissociated dynein is shown as a function o f the d i v a l e n t c a t i o n c o n c e n t r a t i o n in Fig. 6a. The dissociated dynein was activated b y M g 2+ with an o p t i m u m a r o u n d 2 m M a n d was slightly inhibited with an increasing M g 2+ c o n c e n t r a t i o n b e y o n d 10 m M . This b e h a v i o r o f the dissociated dynein to M g 2+

151 I 0.6,

"'o. "'o.

£ '~"~'~"o~ ~ o,~%o~

/

~o xo

~x xo

0.2 £ :::L

00

012

014

O.n6

018

110

KCI (M)

Fig. 7. Effects of KCI on the dissociated dynein. ATPase activity of the dissociated dynein was assayed in 1 ml of a medium containing 25 mM Tris-HCl (pH 8.2), 1 mM ATP, 1 mM MgC12 or CaCI~, varying concentrations of KCI as indicated, and 12.4/~g protein of the enzyme fraction. The reactions were performed at 25 °C for 1 h. @--@, Mg2+-ATPase activity; C)---O, Ca2+-ATPase activity. stant (1 raM). The Lirteweaver-Burk plot gave values o f 1 4 - 1 5 / ~ M for ATP, which was close to the Km o f the 30-S dynein ( 1 1 - 1 3 / ~ M ) [6]. The Km o f the I4-S dynein, obtained by Gibbons [6], was 33-35/~M. The dissociated dynein had a low value for Km in spite o f a drastic treatment with urea. Furthermore, the activity o f the dissociated dynein was comparably stable, while the 14-S dynein was unstable. After aging for

"2 0.8

f.

>_ ~ 0.6

~

0.2

0

I* 5.0

J 60

[ 7.0

1 8.0

T 9.0

1 10.0

pH

Fig. 8. Effect of pH on the ATPase activity of the dissociated dynein. The assay medium consisted of 1 m M MgCI2 or CaCI2, 1 m M ATP, and 25 m M Tris-ma]eate (pH 5.2-8.4) or borate-NaOH (pH 8.3-10.0). The Mg2+-ATPase assay used 16.4/~g of the protein in ] m! of the assay medium, and the Ca2+-ATPase assay used 18.5 # g of the protein. For the CaZ+-ATPase assay, a fraction next to the peak of the dissociated dynein was used. O-~@, MgCI2 and Tris-maleate; A - - A , MgCI2 and borateNaOH; © - - © , CaC12 and Tris-maleate; A - - / k , CaCI2 and borate-NaOH.

152 1 day, the loss of activity was 24, 2 and 3 ~o for the 14-S dynein, 30-S dynein and the dissociated dynein, respectively. 30-S dynein showed two adjacent bands on sodium dodecylsulfate-polyacrylamide gel electrophoresis (Fig. 9). 14-S dynein also showed two adjacent banas

Fig. 9. Electrophoresis of the dissociated dynein, 30-S dynein and 14-S dynein on sodium dodecylsulfate-polyacrylamide gels. From left to right: the dissociated dynein, 30-S dynein and 14-S dynein. Arrows indicate dyneins. corresponding to those of the 30-S dynein together with many other bands of contaminants which ran faster. The dissociated dynein showed a similar pattern to that of the 30-S dynein. Therefore, it was evident that the dissociated dynein still consisted of the two components of the 30-S dynein. DISCUSSION The dissociation of the 30-S dynein into the 14-S form can be obtained by treatment with urea and sodium dodecylsulfate. With the urea treatment new forms of dynein sedimenting at 18 S and 22 S (or 23 S) appear, which cannot be found in the routine extraction procedure of dynein from cilia. 30-S dynein is successively dissociated into 22-S, 18-S and 14-S forms by urea. 22-S and 18-S ATPases may be the tetramer and dimer of 14-S dynein, respectively. This speculation needs evidence other than that of the sedimentation coefficients. In contrast, sodium dodecylsulfate at a concentration below 0.03 ~ dissociates most of the 30-S dynein into the 14-S form in one step. As sodium dodecylsulfate is a detergent which attacks hydrophobic bonds

153 and urea is a reagent which destroys hydrogen bonds, associating forces between the subunits of 30-S dynein presumably arise from hydrogen bonds and hydrophobic bonds, but not from covalent bonds. Shifts of the protein peaks from the ATPase peaks could be the results of conformational changes caused by the reagents. The enzymatic properties of the dissociated dynein are generally different from both the 14-S and 30-S dynein. The activation by Ca ~+ is striking, which cannot be seen in the case of 14-S and 30-S dyneins. Variations of activity as a function of the varying concentrations of Ca ~+, Mg 2+ and KCI and the varying pH do not resemble those of either 14-S or 30-S dynein [6]. Although the Michaelis constant of the dissociated dynein and the stability of the ATPase activity are close to those of 30-S dynein [6], the properties of the dissociated dynein might be changed to various extents during the dissociation process by urea treatment. Preliminary experiments showed that the dissociated dynein could recombine only partly to the outer fibers. It is not clear whether the dissociated dynein has lost the recombination ability or the subunits of 30-S dynein no longer recombine with the outer fibers like the intact 14-S dynein. Properties of the dissociated dynein do not agree with those of the 14-S dynein with only one exception (the recombination ability to outer fibers). At present the dissociated dynein which has ATPase activity can be obtained only by short treatment with urea, which might be drastic enough to distort the protein molecules, although the affinity of the dissociated dynein to ATP does not change. Urea contains a small amount of HCN, which strongly binds to protein molecules. It must affect the properties of the dissociated dynein. Connecting forces between subunits of the 30-S dynein are very strong and the 30-S dynein cannot be easily dissociated. High salt concentrations (4 M KC1 or NaCI) can also dissociate 30-S dynein into the 14-S form, which no longer has ATPase activity and its activity cannot be restored even after removal of the salt. Milder conditions must be sought for further studies on dissociation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the dissociated dynein shows two adjacent bands corresponding to those of the 30-S and 14-S dyneins. Therefore, the dissociated dynein, 30-S dynein and 14-S dynein contain common components. However, it is obscure as to whether the three forms of dynein are exactly the same protein. Recently, the outer and inner arms have been extracted separately from sperm flagella of the sea urchin [16] and two types of dynein of different molecular weight have been reported [17]. In addition, Allen [I 8] has shown the structural asymmetry of the two arms of Tetrahyrnena cilia. The two bands of Tetrahymena dynein on polyacrylamide gel might correspond to the outer arms and inner arms, respectively. The occurrence of dyneins of the 30-S type in respect to the sedimentation coefficients has been reported with some biological sources other than Tetrahymena. Chlamidomonas flagella have both the 30-S and 14-S forms of dynein [19]. When extracted with 0.6 M KC1, sea urchin sperm flagella contains two forms [20]. In the author's preliminary experiments, the 22-S, 17-S and 12-S forms of dynein corresponding to those of Tetrahymena were detected. The 22-S and 12-S ATPases of sea urchin flagella differ in some of their properties. Interconversion between these two forms has not yet completely succeeded. It has been considered that 30-S dynein is physiologically more active than 14-S dynein in view of the experiments of light scattering [7] and recombination to the

154 outer fibers [2]. It is n o t unlikely that 14-S dynein splits off from 30-S d y n e i n a n d suffers some d e n a t u r a t i o n during extraction. There is still a n o t h e r possibility that localization of the 14-S a n d 30-S dyneins is different in the axonemes a n d they act separate functions or that 14-S a n d 30-S dyneins play cooperatively i m p o r t a n t roles on ciliary motility. Success in complete interconversion between 30-S a n d 14-S dynein or in the selective extraction of 30-S and 14-S dyneins will be one of the requirements for further studies on ciliary motion. ACKNOWLEDGEMENTS The a u t h o r wishes to express his t h a n k s to D r H. Sakai for his suggestions a n d discussions. The author is greatly obliged to D r K. Kaji for his kind suggestions t h r o u g h o u t this work. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Gibbons, I. R. and Rowe, A. J. (1965) Science 149, 424~426 Gibbons, I. R. (1965) Arch. Biol. 76, 317-352 Brokaw, C. J. and Benedict, B. (1968) Arch. Biochem. Biophys. 125, 770-778 Gibbons, B. H. and Gibbons, I. R. (1972) J. Cell Biol. 54, 75-97 Summers, K. E. and Gibbons, 1. R. (1971) Proc. Natl. Acad. Sci. U.S. 68, 3092-3096 Gibbons, I. R. (1966) J. Biol. Chem. 241, 5590-5596 Gibbons, I. R. (1965) J. Cell Biol. 26, 707-712 Stephens, R. E. and Levine, E. E. (1970) J. Cell Biol. 46, 416-421 Fiske, C. H. and SubbaRow, Y. (1925) J. Biol. Chem. 66, 375-400 Tsuboi, K. K. and Price, T. D. (1959) Arch. Biochem. Biophys. 81, 223-237 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406--4412 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265275 Rothen, A. (1944) J. Biol. Chem. 152, 679-693 Koenig, V. L. and Pedersen, K. O. (1950) Arch. Biochem. Biophys. 25, 97-108 Martin, R. G. and Ames, B. N. (1961) J. Biol. Chem. 236, 1372-1379 Gibbons, I. R. and Fronk, E. (1972) J. Cell Biol. 54, 365-381 Gibbons, I. R. (1973) FEBS on The Functional Anatomy of the Spermatozoon, 1973, Stockholm, Allen, R. D. (1968) J. Cell Biol. 37, 825-831 Watanabe, T. and Flavin, M. (1973) Biochem. Biophys. Res. Commun. 52, 195-201 Brokaw, C. J. and Benedict, B. (1971) Arch. Biochem. Biophys. 142, 91-100