THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 18, Issue of June 25, pp. 88364843,1988 Printed in U.S.A.

Radiation Inactivation Analysis of Chloroplast CFo-CFI ATPase* (Received for publication, October 22, 1987)

May Yun Wang, Lee Feng Chien, and Rong Long Pan4 From the Institute of Radiation Biology, College of Nuclear Sciences, Hsin Chu 30043, Taiwan, Republic of China

Radiation inactivation technique was employed to measure the functional size of adenosine triphospha- tase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 460 f 24 kilo- daltons; for phenazine methosulfate-mediated ATP synthesis, 613 f 33 kilodaltons; and for methanol- activated ATP hydrolysis, 280 f 14 kilodaltons. The difference (170 f 67 kilodaltons) between 460 f 24 and 280 f 14 kilodaltons is explained to be the molec- ular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, 11, and I11 of coupling factor 0 is 1:2:16. Ca2'- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as S0s2- and COS2- increased the molecular mass for both ATPase's (except trypsin-activated MSa'-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation.

The energy-transducing adenosine triphosphatase (CFo- CF, ATPase)' of chloroplasts is a complex system responsible for the synthesis of ATP during photophosphorylation (5). The enzyme complex is composed of a water-soluble compo- nent (CF1) and a membrane sector (CFo). CFl carries catalytic sites for ATP synthesis and hydrolysis and contains five nonidentical subunits designated as a, p, y, 6, and t in order of decreasing molecular masses of 59,56,37, 18, and 13 kDa, respectively. The stoichiometry of these five subunits has been recently determined as a&y6c with total molecular mass of 420 kDa (18, 19). CFo is responsible for proton transport across membrane and consists of three different kinds of subunits (I, 11, and 111) with decreasing molecular masses of 17, 11, and 9 kDa, respectively (17, 23). The stoichiometry of these three subunits is 0.3:0.7 to 0.9:4 to 6 per CFI (17, 23). CFo-CF1 ATPase has been the subject of intensive studies during last decades (17). However, the molecular mechanisms and functional units involved in ATP synthesis and hydrolysis remains to be elucidated.

Thylakoid membrane-bound CFo-CF1 ATPase can catalyze

* This work was supported by Grant NSC75-0201-B007-03 from National Science Council, Republic of China (to R. L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom all correspondence should be addressed. ' The abbreviations used are: CFo, the hydrophobic part of chloro-

plast ATP synthase-ATPase complex; CF1, chloroplast coupling fac- tor 1; PMS, phenazine methosulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Hepes, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid; Bicine, N,N-bis(2-hydroxy- ethy1)glycine; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl] glycine.

ATP synthesis but not ATP hydrolysis. Furthermore, isolated CFl from thylakoid membrane either by low ionic strength or by chloroform extraction shows little or no ATPase activity. A number of treatments for the activation of latent CF,- ATPase (either soluble or membrane-bound) such as heat, proteolytic digestion, thiol reduction, or organic solvent have been developed (10). The mechanisms and change in func- tional architecture during activation are only partly under- stood.

Recently, the technique of radiation inactivation has been used effectively in the studies on functional size of many membrane-bound components such as enzymes, transporters, and receptors (9, 11, 20). This method involves irradiating membrane preparation with ionizing radiation such as elec- tron beams, x-rays, or y-rays and analyzing the dose response of inactivation. The functional size can then be estimated by applying target theory analysis. This technique offers several advantages not always available with more conventional bio- chemical methods. In particular, the procedure can be per- formed on the proteins in situ, the measurements are inde- pendent of the degree of purity and the molecular mass obtained is the minimum functional size.

In this work, we report the functional size of spinach CFo- CF1 ATPase for either ATP synthesis and hydrolysis from radiation inactivation measurements. In addition, we have examined the effects of several activation procedures and anions on the functional unit of ATPase.

EXPERIMENTAL PROCEDURES

Preparation of Thylakoid Membranes and Soluble CFl-Thylakoid membrane were isolated from commercial spinach (Spirwcia olerock L.) as described elsewhere (20) and finally resuspended in a storage medium containing 25 mM Hepes (pH 7.5), 10% glycerol, 10 mM NaCl, and 2 mg/ml bovine serum albumin. The chloroplast prepara- tions were then stored at -70 'C until irradiation. The irradiated chloroplast preparations were either ready for immediate activity assay or stored again at -70 "C until used. The chlorophyll concen- tration of the isolated chloroplasts was measured according to the method of Arnon (1). Soluble CF1 was isolated from spinach as described elsewhere (4). The protein concentrations were determined by a modified Lowry assay with bovine serum albumin as a standard (13).

Irradiation Procedure-Irradiation was performed with a @'Co ir- radiator (1000 Ci) at our institute. The dose rate was 1.10 f 0.06 megarads/h which was determined by the method of Hart and Fricke (8) using Fe2+/Fe3+ or Ce'+/Ce3+ couple. Glucose-6-phosphate dehy- drogenase was used as internal standard as described previously (20). The molecular mass of glucose-6-phosphate dehydrogenase is 104 kDa. Both chloroplasts (1 mg chlorophyll/ml) and soluble CFl(1 mg/ ml) were irradiated at -25 'C maintained by a cryothermostat. The control samples were run concurrently under the same condition but without irradiation.

Activity Assay-The activation treatment for ATPase of soluble and membrane-bound CFl were reported elsewhere (cf. Table I of Ref, 10). Assay conditions for ATPase activity were based on those described by Anthon and Jagendorf (2). The standard assay medium contained 25 mM Bicine (pH 8.8), 20 mM NaC1, 2.5 mM M&12 (or CaC12), 5 mM ATP, and 33.3% methanol (if present). The concentra-

8838

Radiation Inactivation of CFo-CFl ATPase 8839

tions of SO3’- and C03*- were 20 and 10 mM, respectively, if added. Reactions were started by addition of the thylakoid or CF1 and incubated at 37 “C for 2 min. Assays were stopped and the released Pi was determined colorimetrically by the method of LeBel et al. (15).

Acid-base-induced ATP synthesis was carried out using the method similar to that of Uribe and Jagendorf (24). PMS-mediated cyclic photophosphorylation was performed by illumination of thylakoid membrane for 2 min. Assay mixture contained 50 mM Tricine-NaOH (pH 8.0), 100 mM sucrose, 5 mM MgC12, 10 mM NaC1,0.25 mM ADP, 5 mM Pi, 25 p~ PMS, and 25 pg of chlorophyll/ml chloroplasts. ATP formed in samples was determined with luciferin-luciferase using Lumac Biocounter (Lumac/3M Biocounter M2010, Ref. 14). The luciferase signal was calibrated for each sample with a standard amount of ATP.

The activity of glucose-6-phosphate dehydrogenase was determined spectrophotometrically as the rate of decrease at AM in a medium containing 50 mM Tris-C1 (pH 7.81, 3 mM MgC12, 3 mM NAD, 3 mM glucose-6-phosphate, and 1 pg/ml of glucose-6-phosphate dehydro- genase (20).

SDS-PAGE Electrophoresis-Irradiated soluble CF1 was analyzed by SDS-PAGE as described by Laemmli (12). The slab gel was stained by Coomassie Blue R-250 (Merck) and subjected to scan by densitom- eter (E-C Apparatus C o p , St. Petersburg, FL). The areas on recorder paper under peaks of each subunit at various radiation exposure were cut and weighed. The survival fraction of each subunit was then calculated.

Calculation of Target Size-Molecular weights (target size) were calculated according to the equation of Beauregard and Potier (3):

log m = 5.89 - log D37,f - 0.0028 t

where m is the functional size in daltons, t is the temperature (“C) during irradiation, D3, is the dose of radiation in megarads required to reduce the activity to 37% of that found in unexposed control at temperature t (“C).

Materials-Glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroids, EC 1.1.1.49), luciferase-luciferin, PMS, and dithiothre- it01 were purchased from Sigma. All chemicals were of reagent grade and used without further purification.

RESULTS

Radiation Inactivation of Chloroplust ATP Synthesis- Light-driven ATP synthesis (photophosphorylation) involves two enzyme systems: photosynthetic electron transport chain and its associated CFo-CF1 ATP synthase. Irradiation of thy- lakoid membrane at megarad dosage caused a marked decrease in photosynthetic electron transport rate (20). For simplicity, acid-base-induced ATP synthesis (24) was employed here to avoid the complicated involvement of electron transfer. When thylakoid preparations were exposed to high energy y-ray irradiation, the acid-base-induced ATP synthesis was reduced with increasing radiation doses. The activity decays as a simple exponential function of dosage, allowing straightfor- ward application of target theory for determination of func- tional mass involved in ATP synthesis (Fig. 1). The line in Fig. 1 was determined from least square fits of averages from 12 assays ( r = 0.98). The result yields a D37 value of 2.00 +: 0.11 megarads, which corresponds to a radiation-sensitive mass of 450 f 24 kDa according to the equation of Beauregard and Potier (3). This molecular mass may represent the func- tional size for H+-translocation across CFo-CFl channel plus machinery for ATP synthesis at CF1. The dose response of enzyme marker, glucose-6-phosphate dehydrogenase, is also shown in Fig. 1 to demonstrate the feasibility of this meas- urement (for details, see Ref. 20). The D3, of 7.7 f 0.4 megarads implies the molecular mass of 116 f 6 kDa for glucose-6-phosphate dehydrogenase compared to 104 kDa de- termined by conventional biochemical methods.

To make further radiation inactivation analysis of cyclic photophosphorylation, PMS was included as electron media- tor. The inactivation of PMS-mediated cyclic photophos- phorylation by increasing dose of radiation is shown in Fig. 1

2 6 8 10 1 .oo 0.80 0.60 ‘ 0 . 4 0 ..

> + .- > 0 . 2 0 .- + a u

Q, 0.1 0

0.0 1 1 I

1 2 3 4 5

D o s e , Mrads FIG. 1. Radiation inactivation of acid-base-induced and

PMS-mediated ATP synthesis of chloroplasts. Reaction condi- tions were described under “Experimental Procedures.” 0, acid-base- induced ATP synthesis; 0, PMS-mediated photophosphorylation. All data points are means of 12 assays with lines fitted by regression analysis ( r > 0.98). The control activities were 400 and 1950 pmol of ATP formed/mg of chlorophyll/h for acid-base-induced ATP synthe- sis and PMS-mediated photophosphorylation respectively. The inter- sects on lines a t 37% activities of control give the D37 dose values. The functional size was calculated using Beauregard and Potier equation (3). The survival curve of glucose-6-phosphate dehydrogen- ase ( m = 104 kDa) is also shown (A-A) to demonstrate the feasibility of radiation inactivation technique (for details, see Ref. 20). D37 value for glucose-6-phosphate dehydrogenase is 7.7 f 0.4 megarads (Mrads) which yields a functional size of 116 f 6 kDa.

as well. The survival curve indicates also a simple exponential loss of the activity of PMS-cyclic photophosphorylation. The line with best fits from 12 assays ( r = 0.99) yields a D37 value of 1.47 f 0.08 megarads which corresponds to a functional size of 613 f 33 kDa. Since PMS-cyclic photophosphorylation involves two protein systems ( i e . cyclic electron transport around photosystem I and CFo-CF1 ATPase), and the func- tional mass for latter is 450 f 24 kDa as determined above, the difference between these two molecular masses, about 163 f 57 [=(613 f 33) - (450 k 24)] kDa, represents the molecular mass of cyclic electron transport chain around PSI. As we measured a molecular mass of 73 f 4 kDa for photosystem I core complex (from N,N,N’,N’-tetramethyl-p-phenylenedi- amine to methylviologen, Ref. 20). We thus obtained a differ- ence of about 90 kDa for the rest of proteins besides photo- system I core complex involved in PMS-mediated cyclic elec- tron transport (Table I). The identification of polypeptides accountable for this molecular mass of about 90 kDa requires further studies. The functional size for cyclic photophosphor- ylation mediated by other electron carriers such as menadione and ferredoxin were larger than 1000 kDa (data not shown). The interpretation and assignment of polypeptides involved are difficult and beyond the scope of this study.

Functional Size of CFl-ATPase Activated by Various Treat- ments-The molecular mass of membrane-bound CFI-AT-

8840 Radiation Inactivation of CFo-CFl ATPase TABLE I

Functional size of CFO-CF, ATP synthuse-ATPase complex of spinach chloroplasts The reaction conditions and the determination of D37 and functional size were as described under “Experimental

Procedures.” The M, of possible components were obtained either from the comparison of results in this work or from previous work (20). PSI, photosystem I; ETC, electron transport chain.

No. Systems DI? Functional size Possible components Remarks

megarads kDa 1 PMS-cyclic photophosphorylation 1.47 f 0.08 613 f 33 ETC + CFO-CFl ATPase

450 f 24 CFo-CFl ATPase No. 2 73 f 4 PSI Ref. 20 90 Others = [613 - (450 + 70)] kDa

2 Acid-base-induced ATP synthase 2.00 f 0.10 450 f 4 CFo-CF1 ATPase 2 8 0 f 14 CF1 170 +. 38 CFo = [(450 f 24) - (280 f 14)] kDa

3 Membrane-bound ATPase 3.13 f 0.17 280 f 14 CFI Methanol-activated

4 Soluble CF1-ATPase 2.00 f 0.10 450 f 24 CF1 Me-ATPase Methanol-activated M2+-ATPase

Pase activated by 33.3% methanol was also measured by radiation inactivation. The loss of the activity of ATP hy- drolysis by thylakoid membrane-bound CFl was dependent on the exposure dosage. The survival fraction of activity was a simple exponential function of radiation (Fig. 2). A D37 value of 3.21 f 0.17 megarads was calculated from the line deter- mined from least square fits of averages from 21 assays ( r = 0.99). According to the equation of Beauregard and Potier (3), a molecular mass of 280 f 14 kDa is thus obtained for methanol-activated membrane-bound CFl-ATPase.

We also carried out radiation inactivation analysis of CFl- ATPase activated by other treatments. Table I1 lists the D37

1 2 3 4 5 ,009 .80 - .60 -

20 -

, l o -

01 - 2 4 6 8 10

Dose , Mrads

bound CFI-ATPase activated by methanol. Reaction conditions FIG. 2. Radiation inactivation of soluble and membrane-

were as described under “Experimental Procedures.” 0, soluble CF1; 0, membrane-bound CF1. All data points are means of 21 (0) and 10 (0) assays, respectively, with lines best fitted by regression analysis ( r > 0.98). The control activities were 18 pmol of Pi released/mg of protein/h and 380 pmol of Pi released/mg of chlorophyll/h for soluble and membrane-bound CF1, respectively. The calculation of functional size is the same as in Fig. 1. Mrads, megarads.

values and corresponding functional mass for our investiga- tion. The target size of CFl-ATPase activated by methanol, heat, and trypsin digestion are in the range of 265 & 13 to 288 k 14 kDa. It is obvious that the functional mass of CFl- ATPase is very close in spite of the different activation treatments. Table I1 also shows that M P - and CaZ+-ATPase have the similar functional size, although Anthon and Jagen- dorf (2) showed that both ATPase’s possessed different rate- limiting steps. The functional mass of CaZ+-ATPase activated by methanol was not done in our studies due to its low activity (data not shown).

Many studies demonstrated that anions such as s03’- and c03*- might stimulate the activity of CF1-ATPase (2). The mechanism of anion stimulation is still ambiguous. We there- fore explored whether the target size of CF1-ATPase is differ- ent in the presence of these anions. It is interesting that the addition of anions increased the functional mass of ATPase under several conditions (ie. in the presence of different cations and activation treatment) except for M$+-ATPase activated by trypsin digestion (Table 11). These results imply that the anion effects on ATPase activity might be due to the conformational change yielding in reorganization of subunits involved in ATP hydrolysis rather than mere charge screening (2).

Target Analysis of Soluble CFl--Radiation inactivation of soluble CF1 extracted by ethylenediaminetetraacetic acid from chloroplasts was investigated. Soluble CFl was a well known cold-labile protein complex. It might decrease its activity to about 50% after being stored 3 days at -25 to -70 “C and then remained constant for at least 2 other weeks (data not shown). We therefore irradiated soluble CFl on the 3rd day after it was frozen and then measured its activity immediately following exposure. The latent soluble CF1 ATPase was acti- vated by 33.3% methanol, and the activity was assayed follow- ing increasing radiation dosage. The semilogarithm survival fraction was obtained with best fits ( r = 0.98) of averages from 10 assays (Fig. 2). The application of target analysis yields a functional size of 450 f 24 kDa which is larger than that of membrane-bound CFl-ATPase (cf. 280 k 14 kDa) but close to total molecular mass of a3fi3y6e complex (420 kDa). There are two possible explanations for the difference in target size of soluble and membrane-bound CF1 ATPase. 1) Cold-warm cycle caused the dissociation and reassociation of CF1 subunits (27) which in turn yielded larger functional size. 2) The extraction of CF1 from chloroplasts made it more susceptible to radiation resulting in the illusion of a larger functional unit.

Radiation Inactivation of CFo-CFl ATPase 8841 TABLE I1

Functional size of membrane-bound CFl under various conditions The activation treatments and reaction conditions were described as under “Experimental Procedures.” All D3,

values were obtained from survival curves determined from best fits of averages from at least 12 assays ( r > 0.98). The values in parentheses are the percentage increase of functional size in the presence of anions to their absence for both Ca2+- and Md+-ATPase. chl. chloroDhvk N.D.. not determined.

Activation treatments

Methanol Heat Trypsin ATPase Additions

Activity Functional D37 Activity size D37

Functional size Activity Da7

Functional size

pmol PJ mg chlf megarads

h Me-ATPase Control 373.5 3.21 f 0.17

+S03’- 410.0 2.48 f 0.13

+C03’- 1010.0 2.69 f 0.14

Ca2+-ATPase Control N.D. N.D.

+SO2- N.D. N.D.

+c03’- N.D. N.D.

kDa

280 f 15 (1.00)

363 f 19 (1.30)

335 f 18 (1.20) N.D.

N.D.

N.D.

am1 Pd mg chlf megarads

h 877.0 3.40 f 0.18

1160.0 3.04 f 0.16

1350.0 3.04 f 0.16

630.0 3.30 f 0.18

1975.0 2.55 f 0.14

1924.2 2.55 f 0.14

kDa

265 f 15 (1.00)

296 f 16 (1.12)

305 f 16 (1.15)

273 f 15 (1.00)

353 f 19 (1.30)

353 f 19 (1.30)

P m l Pi/ mg c w

h 120.8

199.5

150.5

70.5

76.0

90.0

megarads kDa

3.20 f 0.17 281 f 15

3.20 f 0.17 281 f 14

3.33 f 0.18 270 f 13

3.24 f 0.17 278 f 15

2.83 f 0.15 318 f 17

2.80 f 0.15 321 f 17

(1.00)

(1.00)

(0.96)

(1 .OO)

(1.14)

(1.15)

Target analysis of soluble CF1 was also observed using SDS- PAGE (Fig. 3A). The degree of staining by Coomassie Blue for each subunit on gel decreased as the exposure dosage increased. The radiation sensitivities of each subunit on stained gel were clearly visualized by densitometer and plotted as function of radiation dosage (Fig. 3B). Table I11 lists D37 values and apparent target size of each subunit. Surprisingly the apparent target size for each subunit is larger by 1.15- 8.93-fold than their molecular weights determined by their relative mobility on SDS-PAGE gel. We speculate that cold treatment of soluble CF1 might cause small subunits more sensitive to radiation either through energy transfer or other mechanism. We therefore felt that such an analysis for cold- labile soluble CF1 was trivial and beyond the scope of this paper.

DISCUSSION

In the current investigation, the functional unit of the CFo- CFl synthase-ATPase complex of spinach chloroplasts in native environment has been determined by radiation inacti- vation. The functional size of acid-base-induced ATP synthe- sis appeared to be 450 f 24 kDa whereas that for membrane- bound CF1 ATPase was 280 & 14 kDa. Therefore the differ- ence of about 170 & 39 [=(450 & 24) - (280 f 14)] kDa (Table I) represents the functional mass for H+-translocation chan- nel, CFo (Table I). Although the polypeptide composition and overall topology of CFo have not yet been well established, it is believed that CFo consists of three proteins designated as I, 11, and I11 in order of decreasing molecular weights (17, 23). Subunit I11 has been characterized as dicyclohexylcarbodi- imide-binding protein. The function of other subunits is still in dispute. Nevertheless, the stoichiometry of subunits I, 11, and I11 was found to be 0.3:0.7 to 0.9:4 to 6 copies per CF1 (17, 23). The molecular mass for CFO complex is thus about 156-230 kDa accordingly. We suggest that the stoichiometry of these subunits be 1:2:15 which corresponds to molecular mass of about 170 kDa as obtained by radiation inactivation.

The smaller functional size of membrane-bound ATPase (280 & 14 kDa) than that of acid-base-induced ATP synthesis (420 & 23 kDa) indicates that CFO is not involved in ATP hydrolysis. In other words, thylakoid ATPase is not coupled

to proton pumping under our conditions. It is generally ac- cepted that CFI consists of a3/33y6t with total molecular mass of 420 kDa (18, 19). Catalytic sites are believed to be on the subunits or at a-/3 subunit interface (22). Reconstitution studies showed that ATP activity is expressed by a&y; no single pure subunit had ATPase activity (7). The molecular mass of this smallest active complex is 382 kDa, which is larger than that 280 f 14 kDa obtained by radiation inacti- vation. Our data show obviously that the collaboration be- tween all the subunits in a3P3y moiety is not needed for ATP hydrolysis. Only 80% (280/382 kDa) of the molecular mass of this complex may be involved in the functional ATPase. The rest of mass may play only the structural role.

A body of evidence supports the model developed by Boyer and co-workers (6,21) that CF1-ATPase has multiple catalytic sites with identical /3 subunits, which exist in alternating conformations and function in the catalytic cycle during steady state hydrolysis. On the other hand, hypothesis of CFl- ATPase with nonequivalent /3 subunits has also been proposed (16, 25-27). It is assumed that the steady state hydrolysis of ATP takes place at one specialized /3 (p ’ ) subunit with other two /3 subunits (/?”) playing a regulatory role (25, 26). With this evidence, we interpret our finding from radiation inacti- vation, 280 & 14 kDa for functioning membrane-bound AT- Pase, in two possibilities. 1) Three /? subunits, which make up functional mass of about 177 kDa, are involved in ATP hydrolysis leaving about 103 (=[280 - 1771) kDa for only two out of three subunits (about 112 = 2 x 56 kDa) accommodat- ing in functioning complex as proposed by alternating site model (6, 21). The subunit y plays only the structural role in this complex. 2) One /? subunit (59 kDa) may be involved in ATP hydrolysis while about 221 (=[280 - 591) kDa functional mass are shared by the rest of remaining subunits 8, a, and y (nonequivalent model, Refs. 25 and 26). Radiation inactiva- tion analysis can not distinguish these two possibilities. The reconstitution studies are carried out currently in this labo- ratory to further justify these two models.

The requirement and inhibition of free divalent cations for CF1-ATPase have been scrutinized by many workers showing specificity dependence for different activation treatments (2). However, the radiation inactivation analysis gives a very

8842 Radiation Inactivation of CFO-CF, ATPase

A n TABLE I11

:: 0 - UPJ g)(*)+eJao- g 2 o m V-r-NUrnL-rqq The determination of D37 and "apparent target size" for subunits 0 x 0. 0. -* -* d m ' 4 m*n'c9ao of soluble CFI was described as in Fig. 3. The values in parentheses

DS7 values for subunits of soluble CF, determined on SDS-PAGE

are the ratios of apparent target size to molecular mass determined W - (v m u m a F"D" by their relative mobility on SDS-PAGE gel.

I I I I I I I l l 1

1 d

Subunits Molecular mass

kDa megarads kDa

Ds7 Apparent target size

a 58 13.4 f 1.8 67.3 f 8.7 (1.16) B 56 13.9 f 2.4 64.5 k 10.9 (1.15) Y 34 8.5 f 1.4 102.0 f 16.3 (3.00) I 3 24 6.8 k 0.9 132.0 f 18.5 (5.50) c 18 5.6 f 1.9 160.7 f 56.3 (8.93)

Y -

6 -

t -

2 4 6 8 10

Dose , Mrads FIG. 3. Relationship between dose and the degree of Coo-

massie Blue staining for subunits of soluble CFI on SDS-PAGE gel. After electrophoresis of soluble CF1 irradiated at dosage as indicated, the gel was stained by Coomassie Blue and subjected to scan using densitometer as described under "Experimental Proce- dures." The survival curves in B were obtained by comparing on the recorder paper the areas under peaks of every subunit a t each dosage to that of control samples. 0, a; A, p; 0, y; A, 6; 0, e. Mruds, megarads.

similar functional size for both Ca2'- and Mg2+-ATPase acti- vated by organic solvent, heat, and trypsin digestion. This result indicates that under proper conditions the functional moiety for ATP hydrolysis is likely the same regardless the cation species employed.

A wide variety of inorganic anions are reported to stimulate the ATPase activity of either soluble or membrane-bound CFl. Of these anions, S032- and C03'- have been shown to be the most effective (2). The anion stimulation was presumably interpreted as the reduction in the true free cation concentra- tion which in turn relieved the extent of cation inhibition. Since the presence of anions increased the Ki of free cation but with no effect on the K,,, for cation-ATPase, it is more likely that anion may also interact directly with the moiety other than catalytic sites at CFl to prevent the inhibitory cation binding (2). Our results further show the presence of anions increased the functional size of both Ca'+- and M$+- ATPase by 12-30% (Table 11). We believe the primary anion effect is to cause the conformational changes resulting in larger functional size. The elucidation of the structural reor- ganization at the presence of anions requires further investi- gation.

Acknowledgments-We thank Dm. H. M. Huang, S. D. Yang, and Y. K. Lai for reading the manuscript and Dr. A. T. Jagendorf for his encouragement and valuable suggestions during the course of this work. The expert secretarial assistance of Y. S. Liu and J. H. Chow is acknowledged. We also thank F. C. Song and S. T. Sheu for irradiation of samples.

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