THE JOURNAI. OF BXX.OCICAL CHEMISTRY Vol. 2.54, No. 22, I.s.sue of November 25, pp. 11561-11565, 1979 Printed m U.S.A.

Ciliary Adenosinetriphosphatase from a Slow Swimming Mutant of Paramecium caudatum*

(Received for publication, May 18, 1979)

Masao Hayashi and Mihoko Takahashi

From the Institute of Biological Sciences, The University of Tsukuba, Zbaraki 300-31, Japan

The slow swimming mutant (strain 11C 104), isolated from Paramecium caudatum, which swims at a speed about 70% of the wild type, is not different from the wild type (strain Kyky-1) in cell shape, density of cilia over the cell surface, and length of cilia. Cross-sec- tioned structures of the somatic cilia also do not give any difference between the mutant and the wild type.

ATPase activities of ciliary axonemes and Tris/ EDTA extract from the mutant are found to be about 70% of those from the wild type. Sucrose density gra- dient centrifugation of the Tris/EDTA extract from the wild type suggests the presence of two ATPase enzymes in 14 S and 30 S regions each. The mutant shows considerably lower ATPase activity in the 14 S fraction, while the 30 S fraction shows similar activity to that from the wild type. The extent of reduction of the ATPase activity by the 14 S fraction coincides well with the decrease in the swimming speed of the mutant.

In relatively high molecular weight polypeptide chains analyzed by SDS-polyacrylamide gel electro- phoresis, the 30 S fraction from the mutant gives the same band pattern as that from the wild type showing the molecular weights of 388,000 and 380,000. The 14 S fraction from the mutant represents identical polypep- tides of 380,000, 370,000, 360,000, and 340,000 with the wild type, and gives one differently migrated poly- peptide whose molecular weight is 268,000 lower than the 280,000 present in the wild type.

Ciliary motility has been shown to be generated by sliding between doublet microtubules (1,2). Arms projecting from A- submicrotubules contain an enzyme with ATPase activity, dynein, and in response to ATP interact with B-submicrotu- bules of the adjacent doublet microtubules to produce me- chanochemical force (3-9).

A low ionic strength extract from Tetrahymena cilia is separated into two differently sedimenting dyneins of 14 S and 30 S in a sucrose density gradient centrifugation (3,4, lo- 12), while that from sea urchin sperm flagella sediments as only one peak at 10 S to 14 S (4, 13, 14). Gibbons (3, 4) has succeeded in rebinding 30 S dynein to outer doublet micro- tubules at pH 8.3 and has found that 30 S dynein localizes as arms. Under his conditions, however, 14 S dynein failed to rebind to doublet microtubules. Shimizu (15) has reported that 14 S dynein would bind at pH 6.0 but precise localization in the cilia is still obscure.

Behavioral mutants are powerful tools for investigation of * This work was supported in part by grants to M. H. (No. 234045)

and to M. T. (No. 411802) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

the molecular mechanism of ciliary motility (16-18). Recently, Takahashi and Naitoh (19) have isolated some behavioral mutants from Paramecium caudatum. One of them, strain llC, swims slowly at a speed 62% of the wild type. This behavioral characteristic is retained in Mg2+-ATP-reactivated Triton models, the speed being 73% of the wild type. This indicates that the mutant would have some defect in the motile system of cilia. This report describes results which show that low ATPase activity of 14 S dynein causes slow swimming speed in strain 11C.

MATERIALS AND METHODS

Preparation of Axoneme, Tris/EDTA Extract, and Dynein- Strains of P. caudatum used were the slow swimmer mutant, 11C 104, which was a F2 segregant from the original mutant IlC, and the wild type, Kyky-1, which was used for producing F2 segregants. They were grown at 27’C in fresh lettuce medium inoculated with Klebsiella aerogenes (20). Ten to 32 flask cultures, each of which contained 1.6 liters of culture medium, were prepared. Final cell densities were about 1,200 to 1,5OO/ml. The cells were harvested in a continuous flow centrifuge (Tomy Seiki Co., Ltd., Tokyo) at 2,300 rpm at room temperature. In this centrifugation, the cells were not formed into a packed sediment, since it was difficult to detach cilia from tightly sedimented cells in the following preparation. Immediately after the cells in suspension (about 500 ml) were sedimented and washed rapidly with 1 liter of 34 mM NaCl, 1 mM KCl, and 1 mrvf CaC12 at 800 x g for 2 min with conical centrifuge tubes at room temperature, the cells were suspended in 300 ml of 12% ethanol, 20 mM CaC12, 4 mM

MgC12, and 20 mM Tris-HCl buffer (pH 8.3), and chilled for 3 to 5 min for the detachment of the cilia (21). From this step on, all preparations were carried out at 0 to 4°C. The deciliated cells were removed at 1,800 x g for 3 min. The detached cilia were collected and washed two times with TME solution (30 mM Tris-HCl buffer (pH 8.3), 3 mM

MgCls, and 0.1 mM EDTA) at 8,000 x g for 8 min. The cilia were then demembranated by 1% (w/v) Triton X-100 for 30 min in an ice bath and washed three times with TME solution to obtain axonemes. A 32-liter culture of the wild type yielded about 12 mg of axonemal protein and a 48-liter culture of the slow swimmer yielded about 15 mg. After dialysis against Tris/EDTA solution (1 mM Tris-HCl buffer (pH 8.3) and 0.1 mM EDTA) for 21 h, axonemes were centrifuged at 105,000 x g for 30 min. The sediments were washed with Tris/EDTA solution and finally suspended in TME solution. This preparation is outer fiber. The 105,000 x g supernatant (Tris/EDTA extract) was layered on top of a 5 to 30% (w/v) sucrose density gradient in Tris/ EDTA solution, and centrifuged at 41,ooO rpm for 12 h at 4°C in the SW 41-Ti rotor of a Beckman model L5-65 centrifuge. The bottom of the centrifuge tube was punctured and the contents were collected from the top in samples of 5 drops (0.3 ml) each with an ISCO fractionator, model 185. Each sample was assayed for protein and for ATPase activity, and sometimes used for SDS’-polyacrylamide gel electrophoresis. The assay of ciliary ATPase was always completed within 48 h after the end of preparation of cilia.

Since Gibbons (3) first discovered ciliary dynein using Tetrahy- mena, it has been a widely used source of ciliary dynein. Therefore, besides the wild type (strain Kyky-1) of Paramecium, Tetrahymena was used as another control. Tetrahymena pyriformis, strain W, was grown axenically in the medium containing 0.5% yeast extract, 1%

I The abbreviation used is: SDS, sodium dodecyl sulfate.

11561

by guest on January 26, 2020http://w

ww

.jbc.org/D

ownloaded from

11562 Ciliary ATPase from a Mutant of Paramecium

proteose peptone, and 0.87% glucose at 27’C to the late log phase (22). Cilia, axonemes, Tris/EDTA extract, and dynein were prepared by the same procedure as described in those of Paramecium, except that stronger centrifugal force was used for the collection of cells and cilia.

In some preparations, 0.1 mM dithiothreitol was added to both Tris/EDTA solution and TME solution in order to protect enzyme activity. However, it did not give any significant effect.

ATPase Assay-Enzymes were reacted at 20°C in 0.5 ml of TME solution containing 0.5 mM ATP neutralized previously by NaOH. The ATP from Boehringer Mannheim Corp. was used because it contains no inhibitory impurity as described by Gibbons et al. (23). Immediately after the enzyme had been added, a 100~1.11 portion of the solution was withdrawn and pipetted into a tube containing 100 4 of Murphy-Riley acid molybdate reagent (24, 25) and 300 ~1 of water in an ice bath to terminate enzyme reaction. Further portions were withdrawn and pipetted after l%, 3, and 4Yz min. Each tube was incubated at 37°C for 10 min for the color development, and the color was measured at 750 nm. The amount of phosphate liberated was proportional to time within 10 min. In the case of a prolonged incubation of purified dynein, not axonemes, the rate of inorganic phosphate liberation was decreased with time due to inactivation of the enzyme (IO). A blank was run under the same conditions. Mg2+ was used in excess of ATP, because the substrate for dynein ATPase is MgATP complex (26,27) and free ATP inhibits the ATPase activity (27, 28). The presence of EDTA in the assay mixture was to obtain higher and more reproducible values of specific activity as reported by Gibbons (10).

For analysis of sucrose density gradients, the routine reaction was begun by adding lo-y1 portions of the gradients to 290 ~1 of TME solution containing 0.5 mM ATP in tubes at 20°C. After 4 min, the reaction was terminated by adding 100 al of the acid-molybdate reagent and 306 pl of water.

SDS-Polyacrylamide Gel Electrophoresis-For good separation in the region of high molecular weight polypeptides, the system of Laemmli (29) was used in the concentrations of 3% (w/v) polyacryl- amide, 0.08% (w/v) N,W-methylenebisacrylamide, 0.05% (v/v) N,iV,iV’,N’-tetramethylethylenediamide, 0.1% (w/v) SDS, and 0.375 M Tris-HCl buffer (pH 8.8). The stacking gels were omitted. The gel was polymerized by adding 0.1% (w/v) (final) ammonium persulfate in a pipe (0.7 x 10 cm) of acrylic resin, which made the gel extrude easily. The gels were run at 4 ma/tube for about 70 min. After the gels were stained with 0.25% Coomassie blue R in 9% acetic acid and 45% methanol overnight, they were diffusion-destained extensively by repeated washing in 7.5% acetic acid and 5% methanol.

The following proteins were used for drawing the calibration curve (molecular weight in parentheses): rabbit muscle myosin (200,000), its dimer (400,000) prepared by the method of Davies and Stark (30), and bovine serum albumin cross-linked by 0.05% (w/v) glutaraldehyde at 25°C for 24 h (31). The bovine serum albumin represented mono- (68,000), di- (136,000), tri- (204,000), tetra- (272,000), and pentamer (340,000). The pentamer was stained very weakly.

Samples were dialyzed against excess amount of water for the removal of sucrose and Tris/EDTA solution in the gradients, dried in vacuum, and then dissolved in a small amount of the sample buffer. After standing at 25°C overnight, 50-pl portions were layered on the top of the gel. In order to compare the migrated bands exactly, two or three samples were run on the same gel if necessary.

Electron Microscopy-Samples were fixed by 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). After 1 h, they were centrifuged to a pellet, and washed with the same phosphate buffer. The pellet was then postfixed for 1 h with 1% osmium tetroxide in the same phosphate buffer, dehydrated through a graded series of ethanol solution, and was embedded in Araldite epoxy resin. Thin sections were stained with 2% uranyl acetate in 95% ethanol for 10 min and then with 0.2% lead citrate for 5 min. Electron micrographs were taken with a JEM 1OOC at 80 kV.

Protein Assay-Protein concentrations were determined by the method of Lowry et al. (32) using bovine serum albumin as standards.

RESULTS

No significant differences in cell shape, density of cilia over the cell surface, and length of cilia were observed between the wild type and the slow swimmer under the phase-contrast optical microscope. Cross-sectioned structures of somatic cilia of the slow swimmer were the same as those of the wild type as shown in Fig. 1. They showed fine 9 + 2 arrays of micro-

tubules with normal inner and outer arms and radial spokes. We tried to detect the difference in ATPase activity of the cilia.

The phenotypic difference is rather subtle; the swimming speed of the mutant is about 70% of the wild type (19). Besides, dynein ATPase is known to be very labile (10, 33). Therefore, in order to compare ciliary ATPase activity, we must pay special attention to doing experiments in parallel and preparing enzymes freshly. We always assayed ATPase activity within 48 h after the end of the preparation of cilia.

The slow swimmer had about 70% specific activities of ATPase in both axonemes and Tris/EDTA extract compared to the wild type as shown in Table I. Specific activity of the enzymes from Tetrahymena was the same level as that of the wild type. Protein and ATPase activity extracted in Tris/ EDTA extract from axonemes averaged 23% (range 19 to 27%) and 80% (range 72 to 99%), respectively. These values did not differ greatly between the wild type and the slow swimmer. Tetrahymena gave 32% (range 31 to 33%) in protein and 98% (range 87 to 102%) in ATPase activity. Sometimes, however, extracted ATPase activity was as low as 55 to 60%, when axonemes were dialyzed for 30 h or more. In that case, however, there were no remarkable differences in the following results, whereas outer fiber had slight high specific activity. That the ratio of the specific activities was in good agreement with the ratio of the swimming speed indicates that the slow swimming is due to low specific activity of ciliary ATPase.

Ciliary dyneins are known to be separated into differently sedimenting dyneins of 14 S and 30 S (3, 4). One of them should be charged with the low specific activity of Tris/EDTA extract, because the mutant character of the slow swimmer is controlled by a single recessive gene (19). Tris/EDTA extract from Paramecium yielded a similar sedimentation pattern of protein peaks to that of Tris/EDTA extract from Tetrahy- menu when centrifuged in paired gradients as shown in Fig. 2.

Kykv-1 IIC 104

0.1 pm FIG. 1. Cross-sectioned structure of somatic cilia of the wild

type (left) and the slow swimmer (right). ~120,000

TABLE I

Specific activity of axonemes and Tris/EDTA extract The ATPase activity was measured in 30 mM Tris-HCl buffer (pH

8.3), 3 mM M&l*, 0.1 mu EDTA. and 0.5 mM ATP at 20°C. The unit is expressed as prnol of P,/mg/min.

Exneriment 1 2 3 4

Axonemes Kyky-1 11c 104 DC 104/Kyky-1

0.300 0.312 0.282 0.320 0.237 0.226 0.201 0.241

79% 72% 71% 75%

Tris/EDTA extract Kyky-1 11c 104 1lC 104/Kyky-1

0.714 0.964 0.622 0.710 0.592 0.511 0.500 0.630

83% 53% 80% 89%

by guest on January 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Ciliary ATPase from a Mutant of Paramecium 11563

Although both 4 S and 30 S peaks sedimented slightly faster than those from Tetrahymena, the peaks present in Tris/ EDTA extract from Paramecium will be designated as 4 S, 14 S, and 30 S after Gibbons (4) for convenience of comparison. Fig. 2 shows that the ATPase activity in the 14 S region is very much lower in the slow swimmer than in the wild type.

Details in profiles of the amount of protein and ATPase activity from the wild type showed that protein peaks did not coincide with that of the ATPase activity in both 14 S and 30 S regions (Fig. 2). In the 14 S region, the activity gave a somewhat skewed peak with a prominent trailing shoulder in addition to a small leading shoulder. After heat inactivation at 37°C for 15 min, activities in both leading and trailing shoulders almost disappeared. The activity in the leading shoulder was more labile to heat inactivation than in the trailing shoulder. These results imply that the 14 S region was composed of two differently sedimenting and differently heat- sensitive ATPase enzymes. We designated them as 14 SL (L means ‘light’) and 14 SH (H means ‘heavy’) dyneins.

In the 30 S region, the activity also gave a skewed peak with a prominent leading shoulder as easily recognized in the profile of the specific activities. The position of the activity peak changed somewhat toward the trailing region after heat inactivation at 37°C for 15 min. This suggests that the 30 S region was also composed of two differently sedimenting ATP- ase enzymes. We called them 30 SL and 30 SH dyneins in the same designation as for 14 S dyneins.

In the slow swimmer, Tris/EDTA extracts gave very low ATPase activity in the 14 S region but not in the 30 S region compared with the wild type. The position of the activity remained in the 14 S region corresponded to that of 14 SH dynein. Thus, the mutation diminished the activity of 14 SL dynein tremendously but not completely, because the activity appeared at the position of 14 SL dynein after the heat inactivation. The activity of 14 SL dynein might be too low to

Ky ky-1 11c 104

Fraction Number

FIG. 2. Sucrose density gradient centrifugation of Tris/ EDTA extract from the wild type (left) and the slow swimmer (tight). Tris/EDTA extract was applied on 11.5 ml of a 5 to 30% (w/ v) sucrose gradient containing 1 mM Tris-HCl buffer (pH 8.3) and 0.1 rnM EDTA, and centrifuged at 41,000 rpm for 12 h at 4°C in the SW 41-Ti rotor of a Beckman model L5-65 centrifuge. The contents were collected from the top of the centrifuge tube in samples of five drops (0.3 ml) each. Upper column, protein concentrations (0); middle column, ATPase activity. The ATPase activity was measured in 0.3 ml of 30 mu Tris-HCl buffer (pH 8.3), 3 mM MgCls, 0.1 mM EDTA, and 0.5 mM ATP at 20°C for 4 min before (0) and after (0, X) heat inactivation at 37°C for 15 min. The reaction was started after the addition of each lo-p1 portion of the fraction. X, the activities when 50 ~1 of the sample is used. Lower column, the specific activities before (0) and after (0) the heat inactivation; X, their difference. Vertical lines, the positions of protein peaks which appeared, when Tris/EDTA extract from Tetrahymena was centrifuged in paired gradients.

TABLE II The ratios of the amount ofprotein and ATPase activity

distributed in the 14 S region to those in the 30 5’ region after the sucrose density gradient centrifugation

Exueriment

Protein Kyky-1 11c 104

ATPase activity Kyky-l 11c 104

1 2 3

0.88 0.71 0.84 0.82 0.67 0.78

0.91 0.73 0.82 0.24 0.15 0.24

0 2040

1 O-3/AT P ( M-’ ) FIG. 3. Lineweaver-Burk plot of dynein ATPase activity.

The activity was measured under the same conditions as described in the legend to Fig. 2, except for the ATP concentrations which were varied as indicated. A, the slow swimmer 14 S dyneins; 0, the wild type 14 S dyneins; a, the slow swimmer 30 S dyneins; 0, the wild type 30 S dyneins.

be detected behind the activity of 14 SH dynein before the heat inactivation. In the 30 S region, the activity showed similar behavior as the wild type.

Since the specific activity of the 30 S region of the slow swimmer was the same as that of the wild type, total amount of ATPase activity in the 14 S region can be compared as a ratio of that of the 30 S region. The same presentation was taken for the total amount of protein. Table II shows that the 14 S region in the slow swimmer sustained one-fourth of the ATPase activity and almost the same amount of protein. This suggests that the low activity of ciliary ATPase in the slow swimmer can be attributed fully to the low activity in 14 S region, while 30 S dyneins were normal.

Three fractions, a peak fraction of protein and the two neighboring fractions, were pooled for 14 S dyneins; three fractions, a peak fraction of protein and the two leading shoulder fractions, were pooled for 30 S dyneins. After dialysis against TME solution at 4°C for 8 h, the ATPase activity was assayed. Unfortunately, however, 14 S dyneins lost their ac- tivity considerably, while 30 S dyneins did not. Therefore, the specific activity of the 14 S dyneins became considerably lower

by guest on January 26, 2020http://w

ww

.jbc.org/D

ownloaded from

11564 Ciliary A TPase from a Mutant of Paramecium

14s 30s 14s+3os T K C TKC TK

FIG. 4. SDS-polyacrylamide gel electrophoresis of dyneins. Z’, K, and C indicate samples from Z’etrahymena, the wild type of Paramecium, and the slow swimmer of Paramecium, respectively. The arrow indicates a differently migrated band in the slow swimmer.

than the expected values from Fig. 2. Lineweaver-Burk plot of both dyneins gave further confirmation that 30 S dyneins were unchanged but 14 S dyneins were changed in the slow swimmer (Fig. 3). The 30 S dyneins gave a K, of 2.4 x lo-” (M) and V milx of 1.2 ,umol of P,/mg/min in both the slow swimmer and the wild type. However, the 14 S dyneins showed 5.5 X 1O-5 (M) as K, and 0.06 pmol of P,/mg/min as V,,, in the slow swimmer, and 1.9 X lo-” (M) and 0.23 pmol of P,/mg/ min in the wild type. Thus, we conclude that the mutational lesion in the slow swimmer relates closely to an active site in 14 SL dynein of cilia.

Both the 14 S and 30 S dyneins were dialyzed against an excess amount of water, dried in vacuum, solubilized with a small amount of water, and applied on a gel electrophoresis after treatment with SDS. Fig. 4 shows the result. The 14 S dyneins from the wild type represented strongly stained poly- peptides showing the molecular weights 380,000 and 340,000, and weakly stained polypeptides showing 370,000, 360,000, and 280,000 as relatively high molecular weight polypeptides. The slow swimmer gave only one difference. A band at 280,000 disappeared and a band at 268,000 appeared. On the other hand, 30 S dyneins from the slow swimmer gave an identical band pattern with the wild type. They represented polypep- tides showing the molecular weights 388,000 and 380,000. Tetrahymena dyneins gave polypeptides showing the molec- ular weights 375,000 and 358,000 in 14 S dyneins and 392,000 in 30 S dyneins. Thus, the band pattern of dyneins from Paramecium was different in both 14 S and 30 S dyneins from that from Tetrahymena.

DISCUSSION

Our results have clearly shown that the slow swimming speed of a slow swimming mutant from P. caudatum is caused by considerably low ATPase activity of 14 SL dynein in the cilia. Tris/EDTA extract distributed the ATPase activity 45% to the 14 S region and thus reduction of about 30% in the ATPase activity of Tris/EDTA extract corresponded to the result of one-fourth the amount of activity in the 14 S region. The swimming speed is reduced by about 30% in the mutant. The extent of the reduction of the swimming speed corre- sponded to that of ciliary ATPase. 14 SL dynein seems to be the only defect in the mutant, because the mutational defect is controlled by a single gene (19). This suggests that 14 SL dynein plays a role in generating ciliary beating in proportion to its ATPase activity.

Recombination of 14 S dyneins to outer doublet microtu-

bules was morphologically obscure, while 30 S dyneins formed arms on A-submicrotubules (3,4, 15). The reconstituted arms were bound to B-submicrotubules in the absence of ATP and then were detached by the addition of ATP (9). Although 14 S dyneins had been reported to be a subunit constituting 30 S dyneins (ll), Mabuchi and Shimizu (31) distinguished 14 S dyneins from 30 S dyneins in their polypeptides by SDS- polyacrylamide gel electrophoresis. Warner and Satir (34) proposed that radial spokes were a part of force generation in ciliary beating from cytochemical observations of the locali- zation of Mg”+-activated ATPase activity in the region of the radial spokes (35, 36). Witman et al. (18) found that Chlam- ydomonas mutants lacking radial spokes and central micro- tubules, whose axonemes underwent ATP-induced disintegra- tion, could not bend their flagella. Therefore, the 14 S dyneins are possibly localized in radial spokes and may generate ciliary bending. To elucidate localization and function of 14 S dyneins in cilia, further study will be required.

Tris/EDTA extract from Paramecium cilia showed similar distribution of ATPase activity and protein to that from Tetrahymena cilia in a sucrose density gradient centrifuga- tion, but not identical in the following respects. In the fist place, the protein peaks of 4 S and 30 S shifted toward heavier position in comparison with those from Tetrahymena. Sec- ondly, Paramecium gave two differently sedimenting ATPase activities in the 14 S region but Tetrahymena did not. Two ATPase activities were also present in the 30 S region from Paramecium. Recently, however, we found two differently sedimenting ATPase enzymes in the 30 S region from Tetra- hymena (to be published elsewhere). This suggests that the two ciliates are common in that the 30 S region contains two ATPase enzymes.

The polypeptide chains of dynein are known to show very high molecular weight ranging from 320,000 to 560,000 when analyzed by SDS-polyacrylamide gel electrophoresis (31, 37- 39). Mabuchi and Shimizu (31) have reported that the molec- ular weight of the polypeptide chains was 520,000 in 14 S dynein and 560,000 in 30 S dynein from Tetrahymena cilia. Gibbons et al. (6) discovered four closely migrated bands (A, B, C, and D) from sea urchin sperm flagella. Two (A and D) of them were found to show ATPase activity. The A band migrated at a position with slightly higher molecular weight than the D band whose molecular weight was reported to be 325,000 f 40,000 (5). In contradiction to the results of Mabuchi and Shimizu (31), our results from Tetrahymena showed two bands of the molecular weight of 375,000 and 358,000 in 14 S dyneins and one band of 392,000 in 30 S dyneins. Although it can be hardly recognized from Fig. 4, repeated experiments make us consider that the band of 392,000 in the 30 S dyneins was composed of three closely migrated bands within the difference of +5,000 in their molecular weight. It is also the case that the band of 340,000 in Paramecium 14 S dyneins would be composed of two closely migrated bands.

The slow swimmer from Paramecium gave only one differ- ent band from the wild type in 14 S dyneins not in 30 S dyneins. The band of 280,000 disappeared and the band of 268,000 appeared. The molecular weight reported hitherto as dynein polypeptides is higher than 280,000. We do not have any evidence that the 280,000 polypeptide has ATPase activ- ity. The 280,000 polypeptide, however, might be an active ATPase enzyme polypeptide and change to an inactive poly- peptide of 268,000 by the mutation.

We had been worried that the slightly low extraction yield (80%) of the activity in Tris/EDTA extract from axonemes might not strictly reflect ciliary ATPase. In a comparative study, however, it does not seem critical, because the extrac- tion yield did not differ between the wild type and the mutant,

by guest on January 26, 2020http://w

ww

.jbc.org/D

ownloaded from

Ciliary ATPase from a Mutant of Paramecium 11565

and the ratio of the mutant to the wild type in specific activity almost retained in Tris/EDTA extract from axonemes. Tet- rahymena axonemes gave 98% extraction yield in parallel experiments. Electron microscopy using Paramecium re- vealed that no detectable amounts of arms, central microtu- bules, and radial spokes remained but a small amount of membrane had remained. However, the membrane remaining would not be enough to account for the residual ATPase activity. These residual ATPase also had been noticed in sea urchin sperm flagella (14) and emphasized as a still unidenti- fied ATPase enzyme in Tetrahymena cilia by Blum (33).

Watanabe and Flavin (40) have found 3 S Ca2+-activated ATPase in Tris/EDTA extract from Chlamydomonas flagella. We could not detect such Ca”+-activated ATPase activity in the region around 4 S from both Paramecium and Tetrahy- menu.

The fact that low ATPase activity in 14 SL dynein causes slow swimming speed in the slow swimmer of Paramecium makes the mutant useful material to investigate the function of 14 SL dynein in cihary motility and the molecular mecha- nism involved. Paramecium will become very useful material for biochemistry if additional similar behavioral mutants can be isolated.

Achnowlec&nents-We thank Yutaka Naitoh for his suggestion in initiating this work and Yoshio Watanabe for his critical reading of this manuscript. Thanks also are due to Kazuo Oonishi for electron microscopy, to Shunichi Hamazaki for the technical assistance in phase-contrast optical microscopy, and to Kazuko Hayashi for gen- erous assistance throughout this work.

1. 2.

3.

4. 5.

6.

7.

REFERENCES 32. Satir, P. (1968) J. Cell Biol. 39, 77-94 Summers, K. E., and Gibbons, I. R. (1971) Proc. N&l. Acad. Sci.

U. S. A. 68, 3092-3096 Gibbons, I. R. (1963) Proc. Natl. Acad. Sci. U. S. A. 50, 1002-

1010

33. 34. 35.

Gibbons, I. R. (1965) Arch. Biol. 76, 317-352 36. Ogawa, K., and Gibbons, I. R. (1976) J. Biol. Chem. 251, 5793- 37.

5801 38. Gibbons, I. R., Fronk, E., Gibbons, B. H., and Ogawa, K. (1976) in

Cell Motility (Goldman, R., Pollard, T., and Rosenbaum, J., eds) pp. 915-932, Cold Spring Harbor Laboratory, N. Y.

Sale, W. S., and Satir, P. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,2045-2049

39.

40.

10. 11. 12. 13.

14.

Gibbons, I. R. (1966) J. Biol. Chem. 241, 5590-5596 Gibbons. I. R.. and Rowe. A. J. (1965) Science 149.424-426 Raff, E. C., and Blum, J. J. (1969) J. Biol. Chem. 244,366-376 Mohri, H., Hasegawa, S., Yamamoto, M., and Murakami, S. (1969)

Sci. PapColl. Gen. Educ. Univ. (Biol. Part) 19, 195-217 Hayashi, M., and Higashi-Fujime, S. (1972) Biochemistry 11,

2977-2982 15. 16.

17.

18.

Shimizu, T. (1975) J. Biochem. (Toh.yo) 78,41-49 Kung, C., Chang, S., Satow, Y., van Houten, J., and Hansma, H.

(1975) Science 188.898-904 Piperno, G., Huang, B., and Luck, J. L. (1977) Proc. Natl. Acad.

Sci. U. S. A. 74, 1600-1604 Witman, G. B., Plummer, J., and Sander, G. (1978) J. Cell Biol.

76,729-747 19. Takahashi, M., and Naitoh, Y. (1978) Nature (Land.) 271, 656-

659 20. 21.

Hiwatashi, K. (1968) Genetics 58, 373-386 Watson, M. R., and Hopkins, J. M. (1962) Exp. Cell Res. 28, 280-

295 22. 23

Watanabe, Y. (1963) Jpn. J. Med. Sci. Biol. 16, 107-124 Gibbons, I. R., Cosson,-M. P., Evans, J. A., Gibbons, B. H., Houck,

B.. Martinson. K. H.. Sale. W. S.. and Tang. W. H. (1978) Proc.

24. 25 26 27 28 29 30.

31.

9. Takahashi. M.. and Tonomura. Y. (1978) J. Biochem. (Z’ohvo) 84. 1339-1355

8. Warner, F. D., and Mitchell, D. R. (1978) J. Cell Biol. 76, 261- 277

Natl. Acad. Sci. U. S. A. 75, 2220-2224 - Murphy, J., and Riley, J. P. (1962) Anal. Chim. Acta 27, 31-36 Hayashi, M. (1976) Anal. Biochem. 76,9-15 Gibbons, B. H., and Gibbons, I. R. (1972) J. Cell Biol. 54, 75-97 Hayashi, M. (1974) Arch. Biochem. Biophys. 165, 288-296 Hayashi, M. (1976) Biochim. Biophys. Acta 422, 225-230 Laemmli, U. K. (1970) Nature (Land.) 227,680-685 Davies, G. E., and Stark, G. R. (1970) Proc. Natl. Acad. Sci. U.

S. A. 66,651-656 Mabuchi, I., and Shimizu, T. (1974) J. Biochem. (Tokyo) 76,991-

999 Lowry, 0. H., Rosebrough, N. J. Farr, A. L., and Randall, R. J.

(1951) J. Biol. Chem. 193,265-275 Blum, J. J. (1973) Arch. Biochem. Biophys. 156, 310-320 Warner, F. D., and Satir, P. (1974) J. Cell Biol. 63, 35-63 Anderson, W. A., Personne, P., and Andre, J. (1968) J. Microsc.

(Paris) 7,367-390 Burton, P. R. (1973) J. Morphol. 140, 185-196 Linck, R. W. (1973) J. Cell Sci. 12, 951-981 Kincaid. H. L.. Gibbons. B. H.. and Gibbons. I. R. (1973) J.

Supramol. Struct. 1,481-470 Borisy, G. G., Marcum, J. M., Olmsted, J. B., Murphy, D. A., and

Johnson, K. A. (1975) Ann. N. Y. Acad. Sci. 253, 107-132 Watanabe, T., and Flavin, M. (1973) Biochem. Biophys. Res.

Commun. 52, 195-201

by guest on January 26, 2020http://w

ww

.jbc.org/D

ownloaded from

M Hayashi and M Takahashicaudatum.

Ciliary adenosinetriphosphatase from a slow swimming mutant of Paramecium

1979, 254:11561-11565.J. Biol. Chem. 

  http://www.jbc.org/content/254/22/11561.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/254/22/11561.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on January 26, 2020http://w

ww

.jbc.org/D

ownloaded from