Partial resolution of the enzymes catalyzing photophosphorylation. XI. Magnesium-adenosine triphosphatase properties of heat-activated coupling factor I from chloroplasts.

Abstract Conditions are reported under which purified coupling factor 1 from spinach chloroplasts, suitably activated, exhibited Mg++-dependent ATPase activity at rates of about 15 µmoles of Pi released per mg of protein per min. The presence of a carboxylic acid and proper MgCl2 concentrations were the main factors determining the rate of hydrolysis. At pH 6, with 8 mm MgCl2, sodium maleate accelerated the Mg++ATPase activity of coupling factor 1 up to 30-fold. At pH 8, 2 mm MgCl2 was optimal and bicarbonate was more effective than maleate as an activator. Higher concentrations of MgCl2 inhibited the reaction. A monospecific antibody against coupling factor 1 inhibited both the Mg++- and the Ca++-dependent ATPase activities. The specificity for triphosphonucleotides was similar for the two reactions. The Km of the Mg++ATPase for ATP was 0.1 mm at pH 6 with 2-(N-morpholino)ethane sulfonic acid buffer and 1.1 mm with maleate buffer. It is concluded that carboxylic acids increase the Vmax by removing inhibitory products of the reaction. From the mode of inhibition by ADP, existence of at least two active sites acting cooperatively, is proposed.

Unlike the mitochondrial coupling factor 1 (Fi),' the purified coupling factor from chloroplasts (CFJ has no ATPase activity unless properly activated. Treatment by trypsin (I), heat (1, 2), or DTT (3) induced a rapid BTPase activity which was dependent on the presence of Ca+f.
Other divalent ions such as Ni+f, Mg++, Mn+f, Co++, and Sr++ at 10 mM were less than 3y0 as effective as Ca++. The Ca++ATPase activity of the Since the photophosphorylation and light-induced ATPase activities of chloroplasts (4)(5)(6) are dependent on Mg+f, the Ca++ATPase activity of CFI is not directly applicable to the study of the phosphorylating mechanism of the chloroplast. It is the purpose of this paper to describe conditions under which CFI can be induced to catalyze a rapid hydrolysis of ATP dependent on the presence of Mg++ without loss of coupling activity.
The cooperative properties of this Mg++ATPase were studied and the regulatory featured of the enzyme are discussed.

Materials
Tricine, ATP, ADP, CTP, GTP, UTP, ITP, digitonin, and bovine serum albumin were obtained from Sigma. TPCKtrypsin and soybeau trypsin inhibitor were from Worthington. [y-32P]ATP was obtained by photophosphorylation of ADP in the presence of 32Pi (7). The resulting [s2P]ATP was purified on a Dowex l-Cl column according to the procedure of Cohn and Carter (8). Tricine buffers and Tricine-maleate buffers were prepared by adjusting the pH with NaOH.

Analytical Methods
ATPase activity was measured by release of 32Pi from ylabeled ATP as follows.
In a final volume of 1 ml, approximately 4 mM ["ZPJATP (50,000 cpm) and 8 InM CaCh or MgClz were incubated at 37" and the reaction was started by addition of 2 to 10 pg of activated CF1. After 10 min the reaction was terminated by addition of 0.1 ml of 30% trichloroacetic acid and 4 ml of 1.2% ammonium molybdate in 1 N HCl. The liberated 32Pi was extracted with 7 ml of isobutyl alcoholbenzene-acetone (9). The extract, 1 ml, was dried and counted in a Nuclear Chicago gas flow counter.
The water phase of a zero time sample was counted to give total counts added.
Protein concentration was determined according to the method of Lowry et al. (10).

Preparations
Heat Activation of CF1-Spinach chloroplast CFr was prepared as previously described by Lien and Racker (11). Only those fractions w&h a fluorescence ratio at Aaoa:Aa6" of more than I were used. Samples of about 2 mg of CF1 stored at 4" in 50r0 saturated ammonium sulfate were centrifuged at 10,000 x g for 10 min. The precipitate was dissolved in 1 ml of a solution containing 10 mM Tricine, pH 8, and 1 mM EDTA. The enzyme (1 to 2 mg per ml) was heated either directly, 01 after desalting on a &hades G-50 coarse column equilibrated with the same solution.

Preparation of Stable EDTA-treated
Chloroplasts--EDTAt.reated chloroplasts have been reported to be unstable (13). We found now that in the presence of high concentrations of bovine serum albumin, t,hese particles can be preserved at, -70". Spinach chloroplasts were prepared as previously described (14) except t,hat 1 mg of bovine serum albumin per ml was used and swelling of the chloroplasts was performed with 10 InM Tricine (~1-1 8) at 4". The suspension was centrifuged at 20,000 X g for 10 min and the resulting pellet was suspended at a chlorophyll concentration of 2 to 3 mg per ml in a medium containing 0.4 M sucrose, 0.01 M NaCl, 0.01 M Tricine (pH 8), and 1 7. bovine serum albumin.
These chloroplasts were diluted t,o a concentration of 0.1 mg per ml with 0.75 m&I EDTA (pH 7.2). After 10 min at 4", the chloroplasts were centrifuged at 35,000 X g for 15 min and suspended in the sa,me medium described above at, a chlorophyll concentration of about 2 mg per ml. The EDTX particles were divided to 0.5~1x11 portions frozen immediately and thawed just before use. These preparations were stable at -70" for months and gave rise to photophosphorylat,ion in the absence of CF, of 2 to 4 pmoles of ATP per mg of chlorophyll per hour, which was increased to 60 t,o 120 pmoles on addition of CFI.

Actzvation of Mg++-dependent
ATPase Activity of CF1-When CFI was heat-activated and tested at pH 8.0 (2), as described previously, little Mg++-dependent ATPase activity could be detected (about 3% of the ratd with Ca+f).
However, when t.he enzyme was tested at pH 6.0 in the presence of maleate buffer, considerable Mg++ATPase activity was observed. An exploration of this observation revealed that the activity in the presence of 8 m&I MgClp was dependent both on the presence of male&e and the lower pH. As shown ia Fig. 1, increasing concentrations of maleate-stimulated Mg++ATPase activity up to 15-fold whereas Ca++ATPasc activity was inhibited so that above 60 nl M maleate the activity with T\lg++ was actually higher than with Ca++.
With 8 mM MgCl, in the reaction mixture, 6 was the optimal pH for Mg+f-4TPase activity of heat-activated CF1 (Fig. 2  sodiurn maleate increased the V,,,,, to 14 pmoles of Pi per mg of protein per min and the K, for ATP to 1.1 mM (Fig. 5). This shows clearly that the acceleration of the lZT1'ase activity by maleate is not caused by a greater affinity of A'l'l' for CF1. .1t pH 8, with sodium maleate in the reaction mixture the K, for AT1 was 1.8 mM. It was previously observed (1) that XDI' strongly inhibited the Ca++ATPasc activity of CF1. As can be seen from Fig. 6 ADP also inhibited the hlg++-ATPase activity.
In the presence of 4 mM ATP 50y0 inhibition was obtained at the following concent.rations of ADP: 0.7 IIIM for Ca++hTPase; 0.4 MM for Mg++ATl'ase at pH 8, and 0.3 KIM for Mg++ATPasc at pH 6. Hammes and Hilborn (16) reported that in the case of the mitochondrial ATPase, ADP is a competitive inhibitor of ATP. As can be seen from Fig. 7, IDP inhibition of the M&f-ATPase activity of CF1 did not follow the same pattern; both the affinity toward ATP and the V,,,, were modified.
Moreover, ADP changed the saturation curve of ATP from a hyperbolic to a sigmoid shape. The apparent reaction order changes from 1.0 in the absence of ADP to 2.3 in t,he presence of ADP (Fig. 8). The same phenomenon was observed for Ca++hTPase and Mg++-Al'Pase activities at pH 6. In agreement with the findings of Hammes and Hilborn (16), we found that the Vlllax of the mitochondrial ATPase was not modified by ADP, but the reaction order as determined by Hill plots changed from 0.9 to 1.4.

T.\ISLE III
EJect of Die-9 on CCL++-and Mg++ATPase ac/ivities of CF, The experiments were performed ns described in Table II

6509
Coupling Activity of ,Mg++ATPase-As shown in Table IV, CFI with Mg++ATPase activated by heat in the presence of digitonin retained its coupling activity.
The mode of action of digitonin is now under active investigation.

DISCUsSION
Previous studies of the mechanism of action of CF1 in its relation to photophosphorylation were complicated by the fact that after activation of the AT'Pasc activity by either trypsin or Rxperimental conditions were as described in Fig. 7 (1,2). similarities between the two effects. The availability of anti-Even mild activation by dithiothreitol yielded a Cn++-dr-bodies against each of the five subunits of CF,2 (15) may permit pendrnt ~ZTPase activity (3) while membrane-bouud CF1 (3, 6) a niorc direct al)l)roacli to this problem. catalyzed in the light, a ?\Ig ++-dependent ;\'l'l'ase activity (4,5) It is very likely that the inhibition of the AYYI'ase activities of as well as l)llotol)tlosphorgl~~tiol~.
A 3-fold increase thcrcfore, to d&ermine which of the subunits int,eract with X)1' in 1Ig+.\Tl':~se activity after treatment of DT'r-activated at the active site and which at, the cooperative site. The -YI'Pasc with TlY'K-trypsin was reported by Lien and Kacker isolation of individual CF1 subunits should make a direct (18), which was further iucreased about 3-fold by addit,ion of approach to this problem feasible. C'F1-deplctctl subchloroplast, particles.
In all these studies, the There is a remarkable similarity between the properties of highest sllccific activity of M g++-dependcxt Al'l'ase was less mitochondrial F1 and chloroplast CFI in amino acid composition, than 1 ,~nlole of ;\Tl' cleaved per mg of CF1 protein per min, subunit st,ructure, cold lability, and iuhibition by X111'. Xn while the soluble preparations described in this paper had apparent diference in their properties has disappeared mit,h the specific actiritics exceeding 15 without loss of coupling activit,y, preparation of an active 1\Ig++.1TPase from chloroplasts with provided digitonin was present during heat activat,ion.
Sexual aspects of this activation are of interest.
Heat trentmerit in the presence of digitonin results in an alteration of l)rotcill st ructurc which is under investigation.
The ot,her main Factors contributing to high YIg++llTPase activity are the presence of an organic acid or bicarbonate, and the choice of a suitable 1Ig++ and H+ concentration during assay. The kinetic data suggest that the acceleration of the Mg++ATPase b\ mnleatc is due to acceleration of the turnover of the enzyme, b> increasing t,he rate of either the catalytic step or the product relcasc step, whichever is rate-limiting.
Mthough the major effect of the organic acids was bcliercd to bc to supply an internal buffer capacity to the chloroplasts, it might be that they also play a role in the activation of the CF1 on the chloroplast membrane.
Wang et al. (20) reccnt,ly dcmolxtrated that maleate or succinate accelerated -YI'I-' formation in the dark after a brief illumination of chloroplasts.
In thesr experiments succinate, which has better buffering capacit,y in the range of experimental condit,ions employed, should have been more effective than maleate.
The fact that it was not suggests that the stimulation might be partly due to an activation of CFI.
13atra and Jagendorf (21) studied the effect of bicarbonate on I)llotol)llos,lior~lation and observed acceleration of photophosphorylation whiln tile yield of the high energy intermediat,e (X,) decreased.
Thus the acceleration of ATPax activity and of :Yl'I' formation by bicarbonate may be related.
The effect of LII)l' 011 the activity of Mg++hTl'ase was at least partially cooperative in nature as indicated by the kinetic behavior of the system. Since CF1 contains five diffcrcnt subunits (22) conformational changes may result in different interactions between t.he subuuits.