Kinetic analysis of axoneme and dynein ATPase from sea urchin sperm

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Kinetic Analysis

165, 288-296

(1974)

of Axoneme

and Dynein ATPase from

Sea Urchin Sperm MASAO Institute

of Molecular

Biology,

Faculty

HAYASHI

of Science, Received

Nagoya January

University,

Chikusa-ku,

Nagoya,

Japan

464

8, 1974

The behavior of the ATPase of axoneme (detergent-treated flagellum) and dynein from sea urchin sperm was investigated. The activation of the ATPase by divalent cations was attributed to formation of a complex of ATP and the divalent cation; the metal-ATP complex is an effective substrate. However, free ATP is a modifier of the ATPase. Free ATP markedly changes the affinity of the metal-ATP complex to the enzymes. Calciumactivated ATPase activity of axoneme decreased at high concentration of CaCl,, but that of dynein did not decrease.-

There are two hypotheses for the molecular mechanism of flagellar or ciliary movement (1). One is the local contraction hypothesis in which one outer doublet actively contracts. The other is the sliding filament hypothesis in which active sliding between outer doublets is the motive force for flagellar or ciliary bending. Satir (2) supported the sliding mechanism in ciliary bending with serial sectioning. Recently, under an optical microscope active sliding was found to take place between outer doublets in the axoneme briefly digested by trypsin (3). It has been shown that flagellar movement of glycerinated spermatozoa is coupled with the hydrolysis of ATP (4-8). Recently, Gibbons and Gibbons (9) reported that spermatozoa treated with 0.04% Triton-X 100 could be reactivated by ATP. A ciliary model of Purumecium was also reported by Naitoh and Kaneko (10) using Triton X-100. These models indicated that the movement of flagella or cilia always required the presence of ATP and magnesium. Gibbons and Gibbons (9) and Douglas and Holwill (11) also suggested that the substrate for flagellar movement was a metal-ATP complex. Dynein, an ATPase protein from flagella or cilia, was identified to be the arm projecting from the outer 288 Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserved.

doublets (12). The nucleotide specificity and divalent cation requirement for flagellar or ciliary motility are similar for enzymatic activity of axoneme and dynein (9, 13, 14). It was previously reported by Hayashi and Higashi-Fujime (15) that the magnesium-activated ATPase of axoneme was inhibited by high concentration of ATP. This report describes the results of further investigation on the effect of added magnesium on the ATPase of axoneme and dynein. The Mg-ATP complex is an effective substrate and free ATP is a modifier of the Mg-ATPase. MATERIALS

AND

METHODS

Preparation of axoneme. The procedure for preparation of axoneme from sea urchin Pseudocentrotus depressus was that reported previously (15). Flagella detached from heads and midpieces by homogenization were collected and washed by 30 mM KC1 and 20 rn~ Tris-HCl buffer (pH 8.0). The purified flagella were treated with 1% Triton X-100 in a solution containing 30 mM KCl, 3 mM MgCl,, and 20 mM Tris-HCl buffer (pH 8.0) for 30 min in an ice bath with gentle stirring. Then the solubilized membranes and other dissolved materials were washed out by 30 mM KC1 and 20 mM Tris-HCI buffer (pH 8.0) with repeated centrifugation at 8,000 rpm for 30 min. The structure of purified axoneme was confirmed by electron microscopically.

AXONEME

AND

Purification of dynein. Dynein was purified by bydroxylapatite column chromatography according to Ogawa and Mohri (16) with slight modifications. The axoneme suspension was dialyzed against 1 mM Tris-HCl buffer (pH 8.0) and 0.1 mM EDTA overnight and was centrifuged at 105,OOOg for 60 min. The supernatant (Tris-EDTA extract) was applied to a hydroxylapatite column (2.1 x 65 cm) equilibrated with 0.1 M potassium phosphate buffer (pH 7.3) containing 1 mM MgC1, and 0.1% 2-mercaptoethanol. The column was then washed with 250 ml of the equilibrating buffer. A linear gradient of 0.1-0.7 M potassium phosphate buffer (pH 7.3) containing 1 mM MgCl, and 0.1% 2-mercaptoethanol was applied for the elution of dynein. Fractions (15 ml) were collected at flow rate of 1.5 ml/cm2/min. In contrast to the results of Ogawa and Mohri (16), the elution pattern (Fig. 1) always showed a single peak of dynein. Fractions showing ATPase activity (fractions 45-53) were pooled and applied to a Sephadex G-25 column previously equilibrated with 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0). The protein fractions were pooled and centrifuged at 105,OOOg for 60 min. The supernatant showed a specific activity of 4.00 pmole P,/mg/min at 30°C and was used as purified dynein. This dynein preparation examined by Na dodecyl sulfate-polyacrylamide gel electrophoresis contained some impurities, but did not contain microtubules or tubulin (28). The purification processes and a result of a typical preparation are summarized in Table I. ATPase assay. The initial rate of inorganic phos-

I

I

0

0 0

10

20

30 Froctlon

40

50

60

70

NO

FIG. 1. Hydroxylapatite column chromatography of Tris-EDTA extract of flagellar ATPase. TrisEDTA extract (322 ml) was applied to a hydroxylapatite column (2.1 x 65 cm) equilibrated with 0.1 M potassium phosphate buffer (pH 7.3) containing 1 mM MgCl, and 0.1% 2-mercaptoethanol. The column was then washed with 250 ml of the equilibrating buffer. A linear gradient of 0.1-0.7 M potassium phosphate buffer (pH 7.3) was applied. Fractions (15 ml) were collected at flow rate of 1.5 ml/cm2/min. (O---O) Absorbance at 280 nm; (-) potassium phosphate buffer concentration (M).

DYNEIN

289

ATPase TABLE

I

SUMMARY OF PURIFICATIONOF DYNEI~ Step

Total protein (mg)

Total activity (@mole cl/

2% activity (rmole

Yield (%)

mm) Pi;f/

I. Axoneme II. Tris-EDTA extract III. Hydroxylapatite chromatography IV. Centrifugation

516 197 40.0 15.2

165 157

0.320 0.797

100 95.1

109

2.72

66.0

4.00

36.7

60.6

a The purification procedures were as described in Methods. About 80 ml of dry sperm was used. ATPase activity was measured at 2 mM MgCl,, 1 mM ATP, 30 mM KCl, and 20 mM Tris-HCl buffer (pH 8.0) at 30°C.

phate liberation was determined from four points. 0.3 ml of enzyme solution in 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) and 4.8 ml of 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) containing appropriate concentrations of ATP and divalent cation were separately incubated at 30°C. After a few minutes, 0.2 ml of enzyme solution was added to the latter substrate solution. A l-ml portion of the reaction mixture was withdrawn after an appropriate time interval and pipetted into a tube containing 2 ml of distilled water and 0.5 ml of reagent of Murphy and Riley (17). After incubation at 30°C for 10 min, the absorbance was measured at 882 nm. The absorbance increased linearly with the amount of inorganic phosphate up to 0.03 @mole/ml. When reaction mixture contained more than 1.5 mM ATP, it was appropriately diluted by distilled water after enzyme reaction. The final nucleotide concentration was always reduced to less than 0.5 mM in the color-developing solution, since higher concentrations of ATP inhibit color developments. Protein concentration. Protein concentration was determined by the microbiuret method of Goa (18) using recrystallized bovine serum albumin as a standard. Association constants between ATP and diualent cations. There have been many reports on the association constants between ATP and divalent cations under various conditions. Those reported under the condition close to those of our experiments, 30 mM KC1 and 20 mM Tris-HCI buffer (pH 8.0) at 3O”C, were as follows: 11,500 M-’ for Ca-ATP at the ionic strength of 0.10, neutral pH, 37°C (19); 16,000 M-’ for Mg-ATP and 9,300 Mm’ for Ca-ATP at the ionic strength of 0.10, neutral pH, 25°C (ZO), and 20,000

290

MASAO

HAYASHI

Mm’ for Mg-ATP at the ionic strength of 0.13, pH 7.9 (21). Accordingly, we used 20,000 M-’ for the Mg-ATP complex and 10,000 Mm’ for the Ca-ATP complex. Reagents. ATP was a disodium salt from Boehringer Mannheim Chem. Co. with no contaminating divalent cations. Hydroxylapatite was prepared according to Siegelman et ~2. (22). Triton X-100 was purchased from Wako Chem. Co. and other chemicals were analytical grade reagents from Katayama Chem. co.

RESULTS

The Effect

of Magnesium ATPase

on Axonemal

The addition of 1 mM M&l, stimulates axonemal ATPase activity about 15-fold compared with the activity measured in the absence of divalent cations in the presence of 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) at 30°C. As previously reported (15), the magnesium-activated ATPase of axoneme was inhibited by high concentration of ATP. In order to investigate systematically the effect of magnesium on axonemal ATPase, we measured the velocity surface both when the total ATP concentration was fixed and the total magnesium concentration was varied, and when the total magnesium concentration was fixed and the total ATP concentration was varied. The former results are shown in Fig. 2 and the latter in Fig. 3. In Fig. 2, the activity was measured at various fixed ATP concentrations (0.4, 1, and 2 mM) with increasing MgCl, (0, 0.05, 0.2, 1, 2, 3, and 10 mM). This figure shows that the activity increased monotonically with MgCl, concentration. At lower concentrations of MgC12, the activity was higher at lower ATP concentrations than at higher ATP concentrations. Inversely, at higher concentrations of MgC12, the activity was lower at lower ATP concentrations than at higher ATP concentrations. This suggests that magnesium does not simply activate the ATPase and ATP does not react as a simple substrate. In Fig. 3, we show the velocity surface measured at various fixed magnesium concentrations (0, 0.05, 0.2, 1, 3, and 10 mM) with increasing ATP (0.2, 0.4,0.6,0.8, 1, 2, 3, and 5 XnM). This shows that a small amount of added MgCl, was effective in

MgCl2

( mM1

FIG. 2. The effect of MgCl, on axonemal ATPase. The concentrations of total ATP were constant: 0.4 mM (a), 1 mM (a), and 2 mM (0). The activities were measured at various concentrations of added MgCl,: 0, 0.05, 0.2, 1, 2, 3, and 10 mM. Reactions were carried out in a medium containing 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) at 30°C.

ATP

(mhi)

3. Behavior of magnesium-activated ATPase of axoneme. Concentrations of added MgCI, were 0 rnM (m), 0.05 rnM (O), 0.2 rnM (O), 1 rnM (O), 3 rnM (O)rmand 10 mM (0). Concentrations of added ATP were 0.1, 0.2, 0.4, 0.6,0.8, 1, 2, 3, and 5 mM. Reactions were carried out in a medium containing 30 mM KCI and 20 mM Tris-HCl buffer (pH 8.0) at 30°C. FIG.

producing an increased activity. For example, in the presence of 0.05 mM MgCl,, a maximum activity appeared at 0.2 mM ATP, which is 7 times as large as that in the absence of MgCl,. At high concentration of ATP, however, the activity de-

AXONEME

AND

DYNEIN

291

ATPase

creased. With increasing concentration of Mg-ATP complex on the abscissa are the higher activities were shown in Fig. 4. Four straight lines were added MgCl,, obtained at all ATP concentrations examobtained at each concentration of free ATP ined, and the concentration of ATP needed within the experimental error. Limiting activities obtained by extrapolation to infito reach the maximum activity became of free ATP were also on higher. When 10 mM MgCl, was added, a nite concentration Michaelis-Menten type relation was ultia straight line as shown by the broken line mately obtained. These results suggest in Fig. 4. The method to obtain limiting that the complex of magnesium and ATP is activities is described later. In the presence of 10 mM MgCl,, the free ATP concentraan effective substrate for axonemal ATPtion and, hence, inhibition by free ATP ase. In order to confirm that the Mg-ATP was assumed to be negligible. In Fig. 4, the complex was actually a substrate and free four straight lines intersected in one point ATP was an inhibitor, the Mg-ATPase was on the ordinate. Therefore, the inhibition measured at various fixed concentrations by free ATP is competitive. The value of of free ATP. The concentrations of the Michaelis constant depends on the concentration of free ATP; it is 2.6 x 1O-5 Mg-ATP and free ATP were determined with certain concentrations of total ATP M in the absence and 3.7 x low4 M at and total magnesium as shown in Table II infinite concentration of free ATP. Some results, however, show that free (a), (b). Lineweaver-Burk plots with the reciprocal of the concentration of the ATP is a partial inhibitor (modifier). In TABLE FREE ATP

AND

Mg-ATP

CONCENTRATIONS

Total ATP (mM) 0.0500 0.0667 0.100 0.200

0.100 0.117 0.150 0.250

(b) Total ATP b-4 0.0200 0.0250 0.0333 0.0500 0.100 0.200 0.300 0.500 1.00 4.00

I

r”~~g2+ 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0

Free ATP

i

Total

FROM ADDED TOTAL ATP

Cc)

(a) 0.050 mM free ATP

II

CALCULATED

2;

Total ATP hM)

0.100 0.133 0.200 0.400

0.150 0.167 0.200 0.300

Free ATP

(mh4) 0.0001 0.0001 0.0002 0.0002 0.0005 0.0010 0.0016 0.0026 0.0055 0.0329

D The free ATP and Mg-ATP concentrations and ATP was 20,000-’ (see Methods).

0.0249 0.0331 0.0498 0.0995 0.199 0.298 0.497 0.994 3.97

Free ATP

r

0.20 rnM Mg-ATP

rotal ATP

Total Mgz+

(rnM)

0.400 0.600 0.800 1.20 2.20 3.20 4.20 5.20

l.OrnM Mg-ATP

TOTAL Mg2+ 4.0 rnM Mg-ATP

-

L

CrnM)

rota! t rrota1 tig2’ ATP ( mM1 I (mM

rotat ngz+ mM)

0.250 0.225 0.217 0.210 0.205 0.203 0.203 0.202

1.20 1.40 1.60 2.00 3.00 4.00 5.00 6.00

1.25 1.12 1.08 1.05 1.03 1.02 1.01 1.01

4.20 4.40 4.60 5.00 6.00 7.00 8.00 9.00

5.00 4.50 4.33 4.20 4.10 4.07 4.05 4.04

/ 0.30 mM Mg-ATP

1 1 ) 0.50 mM Mg-ATP

0.750 0.833 1.00 1.50 2.50 assuming

that

a

7

rota1 ATP :mM)

0.250 0.333 0.500 1.00 2.00

were calculated

T

AND

the association

constant

0.600 0.575 0.550 0.525 0.513 of Mg*+

292

MASAO

Fig. 5, the Mg-ATP concentration was kept constant and free ATP concentration was varied as shown in Table II (c), (d). The values at 0 mM free ATP were approximately obtained by the addition of 10 mM MgCl, as shown in Table II (b) . At a fixed concentration of the Mg-ATP complex, the activity decreased with increasing concentration of free ATP. It should be noted that the limiting activities at infinite concentration of free ATP did not tend to zero. When free ATP is a simple competitive inhibitor, these limiting activities must tend to zero. The following analysis was used to interpret the data of Fig. 5. The degree of inhibition (Uinh) was defined to be the difference between activities in the absence of free ATP (u,) and those at the individual concentrations of free ATP (u):

The double reciprocal plots of &,h against the concentration of free ATP showed good linearity (Fig. 6). The limiting activities at infinite concentration of free ATP depending on the concentration of Mg-ATP were obtained from the intercepts of straight lins on the ordinate in Fig. 6. These values are indicated by open squares in Fig. 4. This result gives the very important information that the effect of free ATP is

HAYASHI

0.L

_ Tc E ;”

0.3

~

Oo

I

2

3

[ATPfl

1 l/M)

FIG. 4. Lineweaver-Burk plots of axonemal ATPase assuming that Mg-ATP is the substrate. The calculated concentrations of free ATP were 0 mM (O), 0.05 mM (a), 0.1 mM (0). and limit to the infinite concentration (Cl) (see in the text). Added concentrations of total ATP and total magnesium are presented in Table II (a), (b). Reactions were carried out in a medium containing 30 mM KC1 and 20 mM Tris-HCI buffer (pH 8.0) at 30°C.

5 1

FIG. 5. Inhibitory effect of free ATP on the Mg-ATPase of axoneme. Each line was obtained by keeping the concentrations of Mg-ATP constant: 0.2 mM (0),0.3 mM (0). 0.5 mM (al, 1 mM (a), and 4 mM (0). Added concentrations of total ATP and total magnesium are presented in Table II (b), (c), (d). Reactions were carried out in a medium containing 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) at 30°C.

1i3/

I I IMg-ATPI

L (mM

[ATP‘I

(M-‘1

FIG. 6. Partial inhibition of Mg-ATPase of axoneme by free ATP. l/u,,, vs l/[ATP,] was plotted at various constant [Mg-ATP], using the data in Fig. 5. [Mg-ATP]: 0.2 mM (01, 0.3 mM (01, 0.5 mM (a), 1 mM (a), and 4 mM (0). Reactions were carried out in a medium containing 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) at 30°C.

partial inhibition (modification) for the Mg-ATP complex. Axonemal ATPase requires the Mg-ATP complex as a substrate and is modified by free ATP. The above analysis was made without consideration of the effect of free Mg2+. Actually it was

AXONEME

AND

found that free Mg 2+ had no influence the activity of the enzyme.

Kinetic

on

Analysis

London and Steck (26) reported on the kinetics of an enzyme reaction in which magnesium and ATP combined with the enzyme and with each other. They treated three kinetic models. Model I assumes that the magnesium-ATP complex combines with the enzyme to form the final complex. In model II, magnesium essentially activates the enzyme whose substrate is the magnesium-ATP complex. Model III assumes modification of the enzyme by a modifier and that the magnesium-ATP complex reacts with the enzyme to form final complexes. There are two paths ot product formation only in model III. Plots of activity against the total magnesium concentration at fixed total ATP concentrations were able to distinguish between both models I and II, and model III. In models I and II, the activity has a maximum value, then decreases, and tends to zero with increasing of total magnesium. On the other hand, in model III, the activity increases and reaches a saturated value with increasing total magnesium. Figure 2 shows that the activity of axonema1 ATPase increases monotonically with total magnesium at fixed concentrations of total ATP. This suggests that the ATPase reaction of axoneme can be analyzed with model III reported in the paper of London and Steck (26). The results from Figs. 4-6 also showed that axonemal ATPase could be explained by the reaction mechanism of model III, in which Mg-ATP was a substrate and free ATP was a modifier. Therefore, the reaction scheme is described as follows (23, 26).

DYNEIN

(free ATP), and products (inorganic phosphate and ADP), respectively. Two or three letters combined with hyphen indicate a complex. The concentration of each component is represented by brackets. K with a subscript is a dissociation constant, v is the reaction velocity. We assume that each enzyme species is in the steady state, and furthermore that the rates of transitions from one equal the rates of the reverse transitions. Under this assumption, the final results of the calculation are as follows:

l/v = K&L

E-S E-M

1hh

+ +

M

l/KEs

-

1/K,M

S +

E-M

V is the maximum velocity. At a given value of w 1, l/v linearly relates to l/[S]. Figure 4 shows the relation between l/v and l/[S], with @I] as a parameter. Vand K, are obtained from intercepts on the ordinate and abscissa, respectively, at [M] = 0, KEM from intercepts on the abscissa at WI = m. K,, is calculated from the following relation, using the value of the intercept on the abscissa at an appropriate value of [M 1:

--

Km+ PII (III) K,.& + K En,‘N I ’

KM is calculated from the following relation: K ix, = KsK,slK,,.

(IV)

The next result of the calculation follows:

is as

lluinh

(KS + [Sl)W&,s + K,,[S])

l,[Ml

VW Eiv - Ks)[Sl

(I)

E-S-M J+ ’ E-S-M

+ Km, IN I l/[S] + -+. (11)

V(K,, + [Ml)

= EfM

293

ATPase

E-M

+

P

I

E, S, M and P denote the enzyme (axoneme), substrate (Mg-ATP), modifier

+ (KS + [Sl)W,, + [s]) . (V) VW EZI - K,) [S ] This relation shows that l/vinh linearly relates to l! [M ] at a given value of [S 1. The linearity shown in Fig. 6 is in agreement

294

MASAO

HAYASHI

with equation (V). Then K,, can be also calculated from the intercept on the abscissa in Fig. 6, according to the following relation:

&Km

+ K&i

.

From the analyses of the data in Figs. 4 and 6 by means of the method mentioned above using Eqs. (II)and (VI), the following results were obtained. V KS KEM K,, K,

= = = = =

0.34 hmole PJmglmin

2.6

x 1o-5M

3.7 x 1O-4 5.4 x 1O-4 3.8 x lo-‘M.

M M

All the lines obtained experimentally in Figs. 4-6 could be superimposed on the lines calculated from Eq. (II) and (V) in good agreement, using the above constants. This good agreement also strongly supports that our kinetic analyses are consistent with axonemal ATPase activity. The Effect of Calcium Axonemal ATPase Axonemal

ATPase

on

was activated

about

‘0

1

I

2 3 ATPimM)

L

FIG. 7. Behavior of calcium-activated axoneme. Concentrations of added calcium (W), 0.05 mM (Cl), 0.2 mM (O), 1 mM CO), (CD). Reactions were carried out in a medium ing 30 mM KC1 and 20 mM Tris-HCl buffer 30°C.

5

ATPase of were 0 mM and 3 mM contain(pH 8.0) at

3

L Cd12

5 (mb.4)

6

7

FIG. 8. The effect of CaCl, on axonemal ATPase. The concentrations of total ATP were constant: 0.1 mM (O), 1 mM (O), and 5 mM (0). The activities were measured at various concentrations of added CaCl,: 0, 0.2, 0.5, 0.8, 1, 2, 3, 4, 5, 6, and 7 mM. Reactions were carried out in a medium containing 30 mM KC1 and 20 mM Tris-HCl buffer (pH 8.0) at 30°C.

fivefold by the addition of 1 mM CaCl,. This calcium-activated ATPase was also systematically examined as shown in Fig. 7. The activation by added CaCI, and the inhibition by high concentration of ATP were similar to those in Fig. 3. At a constant concentration of CaCl,, with increasing concentrations of total ATP, the activity markedly increased, and after reaching a peak, a gradual decrease was ‘bbserved. The position of this peak shifted to higher concentrations of total ATP when the concentration of added CaCl, was increased. Below 1 mM total ATP, the activity at 3 mM CaCl, was lower than that at 1 mM CaCl,. This decrease of the activity is interpreted as the effect of CaCl, from the result shown in Fig. 8. When total ATP concentration was fixed, at first the activity increased with increasing concentration of CaCl, and then decreased, tending to zero. The Effect

Oo

2

of Magnesium or Calcium on Dynein ATPase

Dynein, discovered by Gibbons (12, 241, is an ATPase protein of flagella or cilia which has a very large molecular weight (25). Axoneme is mainly constituted from dynein and outer doublets. The ATPase of dynein is also activated by divalent cations and is influenced by binding of outer doublets (15, 29). Therefore, it was important

AXONEME

AND

to elucidate whether the characteristics of the axonemal ATPase are due to dynein itself or the interaction between dynein and outer doublets. The change of the activity with the concentration of total ATP was measured at various concentrations of added MgCl,. As shown in Fig. 9, three characteristics were observed. The activity decreased at high concentrations of ATP except with 10 mM and no MgCl,. With increasing concentration of added MgCl,, higher activities were obtained and the concentrations of total ATP producing maximum activity became higher. These characteristics are the same as those for the ATPase of axoneme. Therefore, it is concluded that the Mg-ATP complex is also an effective substrate for dynein ATPase and free ATP modified the magnesiumactivated ATPase of dynein. The effect of calcium on dynein ATPase was different from that on axonemal ATPase. Figure 10 shows that dynein ATPase is not inhibited by CaCl, even at higher concentration. With increasing concentrations of CaCl,, the activity showed satura-

DYNEIN

295

ATPase

L ‘0

I I

2

3 CaCl2

L 5 1 mM )

6

FIG. 10. The effect of CaCI, on dynein ATPase. The concentrations of total ATP were constant: 0.3 mM (0) and 1 mM (0). The activities were measured at various concentrations of added CaCl,: 0, 0.2, 0.5, 0.8, 1.2, 2, 3, 4, 5, and 6 mM. Reactions were carried out in a medium containing 30 mM KC1 and 20 mM Tris-HC1 buffer (pH 8.0) at 30°C.

tion. When the total calcium concentration was fixed and the total ATP concentration increased, the profiles of activity were similar to those in F’ig. 9 except ror tower specific activities (data not shown). This leads to the conclusion that, in the presence of calcium, Ca-ATP is also a substrate and free ATP is a modifier. DISCUSSION

-0

1

2 ATP

3 (mt-4)

4

5

FIG. 9. Behavior of magnesium-activated of dynein. Concentrations of added MgCI, (m), 0.05 mM (0). 0.2 mM (a), 1 mM (0), and 10 rnM (0). Reactions were carried medium containing 30 mM KC1 and 20 mM buffer (pH 8.0 at 3O’C.

ATPase were 0 mM 3 mM (a), out in a Tris-HCl

It is well-known that the ATPase activity of cilia or flagella is activated by magnesium or calcium. From our kinetic analysis of the ATPase using axoneme and dynein, this activation was found to be due to the formation of the complex between the divalent cation and ATP. Mg-ATP is a very effective substrate. Free ATP inhibits the Mg-ATPase by decreasing the affinity of the substrate to the enzyme. Axoneme and dynein hydrolyzed ATP even in the absence of divalent cations. This activity was neglected in our kinetic analysis. The consideration of this activity, however, does not influence our kinetic analysis except for slight corrections of the maximum velocity and dissociation constants. When the total calcium concentration was fixed and the total ATP concentration increased, the activity of axonemal ATPase was similar to that of Mg-ATPase (Figs. 3 and 7). The activity of dynein

296

MASAO

ATPase in the presence of calcium was also similar. Therefore, although precise kinetic analysis was not done of calcium-activated ATPase, it may be concluded that Ca-ATP is a substrate and free ATP a modifier. A large amount of CaCl, inhibited axonemal ATPase. Such a phenomenon was not observed for dynein ATPase. Wilson et al. (27) reported that microtubule protein precipitated with calcium. Therefore, the inhibition of axonemal ATPase by CaCl, might be due to the aggregation between microtubules (outer doublets) induced by calcium. We did not succeed in showing whether Mg-ATP and Ca-ATP bound to the same site or not. But we think that Mg-ATP and Ca-ATP compete for the same site, since several profiles of activation of the ATPase by calcium were similar to those by magnesium. ACKNOWLEDGMENTS The author would like to express his thanks to Dr. S. Higashi-Fujime, Professors F. Oosawa and S. Hatano for their helpful suggestions and critical reading of this manuscript. He is also very grateful to Mrs. K. Kunda-Hayashi for her technical assistance, and to Dr. K. Ishihara, Dr. Y. Tonegawa (Saitama Univ.), Misaki Biological Station, and Sugashima Marine Biological Laboratory for supplying sea urchins. REFERENCES 1. BROKOW, C. J. (1968) in Aspects of Cell Motility Cambridge (Miller, P. L., ed.) pp. 101-116, Univ. Press, London. 2. SATIR, P. (1968) J. Cell Biol. 39, 77-94. 3. SUMMERS, K. E., AND GIBBONS, I. R. (1971) hoc.

Nat. Acad. Sci. USA 68, 3092-3096. 4. HOFFMANN-BERLING, H. (1955) Biochim. Biophys. Acta 16, 146-154. 5. BISHOP, D. W., AND HOFFMANN-BERLING, H. (1959)

J. Cell. Comp. Physiol. 53, 445-466. S. (1958) J. Fat. Sci. Univ. Tokyo

6. KINOSHITA,

HAYASHI Sect. 4 8, 219-228. S. (1959) J. Fat. Sci. Uniu. 7. KINOSHITA, Sect. 4 8, 427-437. 8. BROKOW, C. J. (1967) Science 156, 76-78. 9. GIBBONS, B. H., AND GIBBONS, I. R. (1972)

Tokyo J. Cell

Biol. 54, 75-97. 10. NAITOH, Y., AND KANEKO, H. (1972) Science 176, 523-524. 11. DOUGLAS, G. J., AND HOLWILL, M. E. J. (1972) J. Mechanochem. Cell Motility 1, 213-223. 12. GIBBONS, I. R. (1963) Proc. Nat. Acad. Sci. USA 50, 1002-1010. 13. GIBBONS, I. R. (1966) J. Biol. Chem. 241, 5590-5596. 14. STEPHENS, R. E., AND LEVINE, E. E. (1970) J. Cell Biol. 46, 416-421. 15 HAYASHI, M., AND HIGASHI-FUJIME, S. (1972) Biochemistry 11, 2977-2982. 16. OGAWA, K., AND MOHRI, H. (1972) Biochim. Bio-

phys. Acta 256,142-155. 17. MURPHY,

J., AND RILEY,

J. P. (1962)

Anal. Chin.

Acta 27, 31-36. 18. GOA, J. (1953) Stand. J. 218-222. 19. DISTEFANO, V., AND NEUMAN,

Clin. Lab. Inuest. 5, W. F. (1953)

J. Biol.

Chem. 200, 759-763. 20. KHAN,

M.

M.

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