Partial Resolution of the Enzymes Catalyzing Photophosphorylation THE INHIBITION AND STIMULATION OF PHOTOPHOSPHORYLATION BY NJV-DICYCLOHEXYL-CARBODIIMIDE*

1. Exposure of chloroplasts to sonic oscillation in a medium of low osmolarity rapidly inactivated the Hill reaction, photophosphorylation, and photooxidation of ascorbate in the presence of dichlorophenyl-1 , 1 -dimethylurea. Concurrently with this inactivation plastocyanin was liberated from the chloroplasts. The addition of plastocyanin during sonic oscillation prevented net loss of plastocyanin from the chloroplasts and preserved the Hill reaction, cyclic and noncyclic photophosphorylation, and ascorbate photooxidation. These effects were not reversed by washing the particles. In contrast, the addition of plastocyanin to the assay mixtures after sonication did not enhance photophosphorylation even though it stimulated electron flow. This stimulation by external plastocyanin was abolished by washing. 2. A specific antibody against spinach plastocyanin had no effect on electron transport or photophosphorylation in chloroplasts and did not agglutinate chloroplasts. However, if chloroplasts were sonicated in the presence of the antibody, a more pronounced inhibition of the Hill reaction, of ascorbate photooxidation, and of photophosphorylation was observed than when sonic oscillation was performed in the presence of y-globulins from nonimmunized rabbits. 3. From these findings we conclude that plastocyanin functions in noncyclic electron flow between the two photosystems as well as in cyclic photophosphorylation. In contrast to the chloroplast coupling factor (CFt) and ferredoxinNADP+ reductase, plastocyanin in situ is not accessible to antibody added to the chloroplasts. Plastocyanin added to chloroplast particles can induce an artificial electron flow through Photosystem I. This process is not coupled to photophosphorylation but is sensitive to external antibody. The similarity between the function of plastocyanin in the

in beef heart submitochondrial particles. Lee, Azzone, and Ernster (1) and Racker and Monroy (2) demonstrated that the succinate-driven transhydrogenase reaction in partially resolved submitochondrial particles prepared by sonic oscillation in the presence of ammonia was greatly stimulated by oligomycin. Lee and Ernster (3) subsequently showed that phosphorylation in these particles was also stimulated by low concentrations of oligomycin.
These experiments led to the suggestion that coupling factors might stimulate phosphorylation by preventing the hydrolysis of a high energy intermediate rather than by a catalytic activity. Fessenden and Racker (4) found that a specific antiserum to coupling factor 1 (5) abolished the oligomycin stimulation of phosphorylation in submitochondrial particles and that optimal rates of phosphorylation with oligomycin were obtained only in the presence of coupling factors.
These results established that residual coupling factor 1 in the particles was required for oxidative phosphorylation even in the presence of oligomycin.
Coupling factor 1 which was inactivated with regard to its adenosine triphosphatase but still combined with submitochondrial particles was found to stimulate phosphorylation like oligomycin. These experiments indicated a dual role for the coupling factor, a catalytic function and a structural one.
The first indication for such a dual role of a coupling factor was obtained in experiments with spinach chloroplasts. It was shown that an antiserum against the chloroplast coupling factor (6, 7) strongly inhibited photophosphorylation (8). However, the light-induced pH rise (9) was completely resistant to antiserum (8). The treatment of chloroplasts with dilute solutions of ethylenediaminetetraacetate resolved the coupling factor from the chloroplasts (6,8) and was found by Neumann and Jagendorf (9) to inhibit the light-induced pH rise. We recently reported that the coupling factor restored the ability of these particles to catalyze the light-induced pH rise (8). Beechey et al. (10) reported that N ,N'-dicyclohexylcarbodiimide inhibited oxidative phosphorylation in a manner similar to that of oligomycin (11). Racker and Horstman (12) have shown that DCCD* at low concentrations stimulated oxidative phosphorylation in particles which were partially depleted in coupling factors. In this paper, it will be reported that DCCD inhibited electron transport and phosphorylation in chloroplasts in a manner distinct from other known inhibitors of photophosphorylation.
It will also be shown that DCCD stimulated photophosphorylation and the light-induced pH rise in EDTAtreated chloroplasts which were partially deficient in the coupling factor.

EXPERIMENTAL METHODS
Preparation of Chloroplasts-Forty-five grams of washed, deveined market spinach were homogenized in a Waring Blendor for 10 to 15 set with a buffer which contained 20 mM Tricine-NaOH (pH S.O), 0.4 M sucrose, and 10 mM NaCl.
The chloroplasts were isolated as described by Avron and Jagendorf (13) and were washed either with the grinding medium or with unbuffered 10 mM NaCl as indicated below.
Preparation of EDTA-treated Chloroplasts and CF1-Chloro- plasts which had been washed with 10 mM NaCl were resuspended in a minimal volume of this solution.
Aliquots of this suspension were diluted to a final chlorophyll concentration of 0.1 mg per ml with cold 0.75 mM EDTA, pH 8. After 5 min at 0", the suspension was centrifuged at 45,000 x g for 20 min. The pellets (EDTA-treated chloroplasts) were resuspended with the aid of gentle homogenization in 10 mM NaCl. The supernatant fluids, which were clear and nearly colorless, served as a source of CS in these experiments and were stored at room temperature prior to use. The protein concentration in the extracts varied from 0.05 to 0.07 mg per ml.
Analytical hfethods-Photophosphorylation was assayed in test tubes (1 x 10 cm) at room temperature, under air, at a light intensity of about 100,000 lux under conditions described in the legends to the tables. "Pi esterification was measured by a modification (14) of the procedure of Lindberg and Ernster (15). Ferrocyanide production was assayed by the method of Punnett,Iyer,and Ellinwood (16) with 0.67 M sodium formate, pH 3.0, as the buffer for color development.
DCCD solutions (10 mM) were prepared in ethanol. Dilutions of this stock solution were made in water immediately before use. Chlorophyll (17) and protein (18) concentrations were determined spectrophotometrically.
Tricine was purchased from General Biochemicals, Chagrin Falls, Ohio.

RESULTS
Effects of DCCD and Dio-9 on Oxygen Evolutian-DCCD strongly inhibited oxygen evolution which accompanied ferricyanide reduction in illuminated chloroplasts ( Table I). The inhibition which occurred in the absence of a phosphate acceptor system was reversed by NH&l (Experiment I). It can be seen, however, that the rate of oxygen evolution was lower than that observed when NH&l was added in the absence of DCCD (Experiment II).
When ADP, Pi, and hIg+f rather than NH&l were used to increase oxygen evolution, the inhibit.ory effect of DCCD was as pronounced as in the absence of a stimulator.
If NH&l was now introduced into the system, only a partial restoration of oxygen evolution was achieved (Experiment III). Atabrine was found to relieve the DCCD inhibition in a manner identical with NH&l.
It thus appears that DCCD partially inhibited oxygen evolution even in the presence of NH&l. This is in contrast to Dio-9 (19), which did not inhibit in the presence of NH&l (Experiment IV). When Dio-9 was added to chloroplasts in the presence of ADP, Pi, and Mg++, a pronounced inhibition of oxygen evolution was noted which was reversed by NH&l.
The addition of DCCD again markedly reduced the rate in the presence of the uncoupler (Experiment V).

Effects of DCCD on Ferricyanide
Reduction and Associated Phosphorylation- Table  II shows the effects of DCCD concentration on ferricyanide reduction and coupled phosphorylation. Both ferricyanide reduction and phosphorylation were inhibited by DCCD.
At low levels of DCCD, ferricyanide reduction and phosphorylation were inhibited to the same extent, and the P/2e ratio remained constant.
At higher concentrations of DCCD, phosphorylation was more severely inhibited than reduction and the P/2e ratio fell. Pyocyanine-dependent cyclic phosphorylation in chloroplasts containing 50 pg of chlorophyll per ml was inhibited about 50% by 25 C(M DCCD. These results resemble those obtained with Dio-9 (19) and phlorizin (20) (Table  III).
Since the action of DCCD is time-dependent, it was not possible to ascertain whether DCCD interfered primarily with the light activation process or the ATI' hydrolysis, per se. It should be pointed out, however, that the Ca++-dependent ATPase activity of soluble CFI was resistant to DCCD.
Little or no inhibit,ion of the Ca++-ATPase activity of trypsin-activated CE (1.5 kg of protein) was observed when the enzyme was incubated for 15 min with 0.25 pmole of DCCD in a volume of 0.5 ml.

Effects
of DCCD on the Light-induced pH Rise-DCCD, like Dio-9 and the antiserum to CF1 (S), was found to have no effect on the light-induced pH rise in chloroplasts which had been washed in 10 mM NaCl (Table IV).
DCCD caused no significant alteration in the apparent first order rate constant for either the pH rise in the light or its decay in the dark. EDTA-treated chloroplasts, which were prepared from the same chloroplast suspension, showed a much smaller total increase in pH on illumination.
DCCD caused a marked increase in the total pH change in the EDTA-treated chloroplasts upon illumination. The rate constant for the pH rise was similar to that of the control chloroplasts, whereas the constant for the pH decay was greatly reduced NH&l at 2 mM inhibited the pH rise in the presence of DCCD and increased the rate of pH decay. It was found that 10 mpmoles of DCCD per 0.06 mg of chlorophyll  (Table V). The maximal stimulation of phosphorylation by DCCD in the presence of pyocyanine was less than that observed with CR.
However, highest rates of phosphorylation were routinely observed only in the presence of both DCCD and CF1. Concentrations of DCCD which gave optimal rates of   Vol. 242,No. 15 photophosphorylation in the absence of CF, actually inhibited phosphorylation in the presence of CFI. The antiserum to CF, inhibited the DCCD-and CF1-stimulated phosphorylation as well as the phosphocylation which occurred in their absence (Table VI). No stimulation of pyocyanine-dependent phosphorylation could be observed at any concentration of Dio-9. In fact, Dio-9 inhibited phosphorylation in the presence of DCCD and CF1 at all concentrations tested. EJects of DCCD on Ferricyanide-dependent Phosphorylation in  Chloroplasts-DCCD also stimulated photophosphorylation catalyzed by EDTA-treated chloroplasts in the presence of ferricyanide (Table VII). The stimulation of phosphorylation by DCCD in the presence of ferricyanide was comparable to that obtained with the coupling factor. Considerable enhancement of phosphorylation was observed, however, when both DCCD and CF, were added.

DISCUSSION
The Inhibition of Phosphorylation and Oxygen Evolution by DCCD-The results presented in this paper show that DCCD is an inhibitor of photophosphorylation as well as oxidative phosphorylation (10, 12). In tightly coupled mitochondria, DCCD, like oligomycin, inhibits electron transport as well as phosphorylation. The inhibition of electron transport by DCCD is relieved by uncouplers (10). The effects of DCCD on photosynthetic electron transport and phosphorylation in chloroplasts are similar to those obtained in mitochondria. DCCD inhibited oxygen evolution in both the presence and absence of a phosphate acceptor system. This inhibition was partially relieved by the addition of an uncoupler such as NH&J. The effect of DCCD appears to differ somewhat from that of  and phlorizin (20). These compounds inhibit electron transport maximally only in the presence of ADP, Pi, and Mg++, and this inhibition is completely reversed by uncouplers. On the basis of this observation and the fact that Dio-9 inhibits the trypsin-activated, Ca++-dependent ATPase of soluble CF, (19), it has been postulated that Dio-9 inhibits photophosphorylation at the terminal transphosphorylation reaction which results in ATP formation.
Effects of DCCD on the Light-induced pH R&e-According to current ideas, the light-induced pH rise represents either a high energy state in chloroplasts (20) or ion translocation driven by a high energy intermediate. Since DCCD had no effect on this process in coupled chloroplasts, it can be concluded that DCCD does not affect the formation of this high energy intermediate or state in chloroplasts. DCCD, then, interferes with the utilization of this high energy intermediate or state to form ATP, and presumably does so at a step prior to the site of action of Dio-9 or phlorizin.
Since DCCD enhances the light-induced pH rise in EDTAtreated chloroplasts while diminishing the rate of its decay, it is probable that DCCD increases the level of a high energy intermediate. If the rate of decay of the pH in the dark can be taken as an indication of the rate of breakdown of a high energy intermediate, the DCCD stimulation of the pH rise could be ascribed to an inhibition of the rate of the breakdown of this intermediate. A similar mechanism has been proposed for the oligomycin stimulation of the succinate-driven transhydrogenase reaction in submitochondrial particles deficient in coupling factors (1, 2), and for the stimulation of phosphorylation by oligomycin (3) and by DCCD (12). At low concentrations, DCCD appears to inhibit the decay of a high energy intermediate; at high concentrations, DCCD inhibits its utilization for ATP synthesis.
CFr may also stimulate the light-induced pH rise in EDTA-treated chloroplasts by preventing the breakdown of the high energy intermediate.
E$ects of DCCD on Photophosphorylation-The fact that DCCD stimulates photophosphorylation in uncoupled chloroplasts in the absence of added CF, is in accord with the idea that DCCD can block the breakdown of a high energy intermediate. The stimulation by DCCD of oxidative phosphorylation in submitochondrial particles which were completely resolved with respect to F1 is dependent upon the addition of F1 (12). Moreover, in partially resolved submitochondrial particles and EDTAtreated chloroplasts, maximal rates of phosphorylation were obtained only in the presence of both DCCD and F1 and CF1, respectively. These results, in combination with the observation that the antiserum to CFI abolished phosphorylation in the presence of DCCD, suggest that CF1 is required for phosphorylation. This view is strengthened by the fact that considerable amounts of CF, are left in the particles after treatment with EDTA. Since CF1 contains a latent, Ca+-dependent ATPase (7) which is unmasked by trypsin, it is possible to evaluate the extent of the resolution of CFI from the chloroplasts. It was found that the EDTA treatment removed from 50 to 70% of the trypsin-activated, Ca++-dependent ATPase activity of the chloroplasts.
These experiments indicate that CF1 fulfills two functions: a catalytic role which is inhibited by the antibody and a structural role which is not affected by the antibody. Evidence for such a dual role of F1 in mitochondria has recently accumulated. Schatz,Penefsky,and Racker (24) found that F1 from yeast mitochondria stimulated phosphorylation in submitochondrial particles from beef heart which were partially deficient in F1 , but did not stimulate phosphorylation in submitochondrial particles that were completely resolved with respect to Fr. Thus, the yeast enzyme, like oligomycin or DCCD, required the presence of a catalytically active beef Fr.
Penefsky (25) showed that iodine treat,ment of beef heart coupling factor 1 inhibited its Mg*-ATPase activity without markedly inhibiting its coupling factor activity. Recently it was found, however (24), that the iodine-treated enzyme, like the yeast enzyme, did not stimulate oxidative phosphorylation in submitochondrial particles which were devoid of residual coupling factor 1. The use of completely resolved particles, as well as immunological methods, permits a functional separation of the structural and the catalytic roles of membrane-bound factors.