- Email: [email protected]
[8] Light-influenced ATPase activity: Bacterial
96
METHODOLOGY
[8]
Illumination at 100,000 lux for 20 minutes is provided by the same arr a n g e m e n t as described for light-triggered ATPase. T h e reaction is terminated by turning the light off followed by the addition of 0.1 ml TCA. T h e chloroplasts are removed by centrifugation at 500 g for 10 minutes at 0-4°c. Aliquots from the supernatant solutions are taken for [32p] Pi determination. [32p] Pl is separated from [z2p] ATP by the isobutanol-benzene extraction method, 8 and Pi is determined from the radioactivity in the organic phase. Specific activity is defined as micromoles of Pi released per milligram of chlorophyll per hour. Properties
Light-dependent ATPase activity has substrate specificity for ATP, GTP, and ITP with the highest rate obtained with ATP (apparent K,, = 4 X 10 -5 M). ADP is a competitive inhibitor with an apparent Ki = 3 x 10-5 M ? T h e specific activity is in the order of 10-30. With white light and PMS as cofactor, saturation is approached at 200,000 lux. 9 T h e activity is inhibited by Mg 2+ ions and by uncouplers of photophosphorylation such as 5 x 10 -6 M atebrin, 1.6 x 10 -5 M CCP, and 7 x 10 -6 M gramicidin S. 7 SM. Avron, Anal. Biochem. 2, 535 (1961). 9M. Avron,J. Biol. Chem. 237, 2011 (1962).
[8] Light-Influenced ATPase Activity: Bacterial By T. HORIO, Y. HORIUTI, N. YAMAMOTO,and K. NISHIKAWA
Chromatophores prepared from RhodospiriUum rubrum are able to synthesize ATP from ADP and Pi coupled with photosynthetic electron flow in a cyclic fashion? Besides photosynthetic ATP formation, chromatophores can catalyze the oxidation of N A D H by molecular oxygen coupled with A T P formation, z In addition, chromatophores have activities for ATP-Pi exchange and ATPase either in the light or in dark1T. Horio and M. D. Kamen, Biochemistry 1, 144 (1962). 2.].Yamashita,S. Yoshimura,Y. Matuo, and T. Horio, Biochim.Biophys.Acta 143, 154 (1967).
[8]
LIGHT-INFLUENCED ATPase ACTIVITY: BACTERIAL
97
hess, which are at least in part brought about because of the reversibility of the ATP-forming reaction? ,4 ATPase activity by chromatophores is firmly associated with particle structure and influenced by illumination, redox dyes, and extraction of the quinones from chromatophores. Conceivably, the ATPase activity is catalyzed at least in part by an enzyme system containing redox components associated with chromatophores.
Preparations and Assay Method The culture of R. rubrum cells and preparation of chromatophores from them are performed in the same manner as described elsewhere in this series. 5 ATPase activity is assayed according to the methods described elsewhere, 5 except for the following addition. In some cases, the reactions are carried out under continuous illumination incident on the reaction test tubes of approximately 1000 ft-c from a bank of tungsten lamps. Reactions in the dark are carried out in test tubes covered with aluminum foil.
ATPase Activity Influenced in the Light 6 The rate of ATPase reaction by chromatophores is slower in the light than in the dark, with most preparations of chromatophores, and without addition of redox dyes. On the contrary, ATPase activity is significantly stimulated by 4 × l0 -4 M phenazine methosulfate (PMS) in the light. When a Lineweaver-Burk plot is made of the reciprocals of the ATP concentrations and the rates of the ATPase reaction, the light appears to be a competitive inhibitor against ATP in the absence of PMS and to be a noncompetitive stimulant against ATP in the presence of PMS. The inhibition of ATPase activity in the light is significantly neutralized by adding antimycin A, whereas the inhibitor hardly influences the activity in the dark. The neutralization is maximal at 0.1 tzg/ml of antimycin A, where the extent of the light inhibition is diminished by approximately 70%. It has been shown that antimycin A at 0.1 t~g/ml completely inhibits photosynthetic ATP formation, unless PMS is present. 7 The inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (0.67 /~g/ml), 3T. Horio, K. Nishikawa, M. Katsumata, and J. Yamashita, Biochim. Biophys. Acta 94, 371 (1965). 4T. Horio, K. Nishikawa, Y. Horiuti, and T. Kakuno, in "Comparative Biochemistry and Biophysics of Photosynthesis" (K. Shibata et al., eds.), p. 408. Tokyo University Press, Tokyo, 1968. 5See Vol. 23 [63], 6y. Horiuti, K. Nishikawa, and T. Horio, J. Biochem. 64, 577 (1968). ~T. Horio and J. Yamashita, Biochim. Biophys. Acta 88, 237 (1964).
98
METHODOLOGY
[8]
shows the same effects as antimycin A, both in the dark and in the light. Either in the presence or the absence of PMS, ATPase activity decreases when chromatophores are illuminated with monochromatic light at 880 nm, which is the absorption peak of bacteriochlorophyll associated with the chromatophores from wild-type cells. It has been shown that PMS is reduced in white light but not in 880-nm light, s an observation indicating that the stimulation of ATPase activity by white light in the presence of PMS is caused by the photochemical reduction of the dye. This is confirmed by the observation that ATPase activity in the presence of PMS in the dark increases and then is decreased by the addition of increasing concentration of ascorbate, capable of reducing PMS, as shown in Fig. 1.
Eh-Dependent ATPase Activity When 2,6-dichlorophenolindophenol (DCPI) in the oxidized form (Ema = +0.217 V) is added to the reaction mixture for the ATPase activity assay, it is reduced by chromatophores at a slow rate, and the Eh 40C
'
II
~
i
~x,o-~M
~
'
"1'
'\
0-
I00
0
~
i
i
log (ascorbateM
i
)
Fie. 1. Effect o f p h e n a z i n e m e t h o s u l f a t e (PMS) a n d ascorbate on A T P a s e activity in the dark, E x p e r i m e n t a l conditions are as described in the text, except that 4.0 x 10 -4 M or 6.7 X 10 -5 M PMS a n d various concentrations o f ascorbate are a d d e d as indicated. Circles, + PMS (4.0 × 10 -~ M); triangles, + PMS (6.7 x 10 -5 M).
SD. M. Geller, Ph.D. Dissertation, H a r v a r d University, C a m b r i d g e , Massachusetts, 1958.
[8]
LIGHT-INFLUENCED ATPase ACTIVITY: BACTERIAL , II
,
99
,
0.3
o o
0.2 ~g
rr"6 0.I "6 E ~L
0.0
II 10g ( DC P I, M)
FIG. 2. Effect o f 2,6-dichlorophenolindophenol (DCPI) on ATPase activities in the light and in the dark. Experimental conditions are as described in the text, except that DCPI is added as indicated. Open symbols, in the light; filled symbols, in the dark.
value of the reaction mixture becomes more positive with increasing concentrations of the dye. As shown in Fig. 2, ATPase activities in the light and in the dark are accelerated with increasing concentrations of DCPI up to 6.7 x 10 -5 M, and retarded at higher concentrations. T h e effect of DCPI concentrations are fairly similar for the ATPase activities in the light and in the dark, except for the reproducible difference as follows: At concentrations of DCPI lower than 6.7 × 10-5 M, ATPase activity in the light is lower than in the dark, and vice versa at higher concentrations. Antimycin A (0.33 /~g/ml) does not influence ATPase activity in the dark in the presence of DCPI at any concentration tested. Further, when ascorbate is added in the presence of 6.7 x 10 -4 M DCPI, where ATPase activity is significantly lowered, the activity increases and then decreases with increasing concentrations of ascorbate; the m a x i m u m rate is obtained at 5.0 × 10-4 M. T h e ATPase activity is not altered by repeated washing of chromatophores with various concentrations of DCPI and/or ascorbate, an indication that the effect of redox dyes is reversible. T h e Eh values of the reaction mixtures containing 6.7 × 10 -4 M DCPI and various concentrations of ascorbate are measured with a pH and millivolt meter (Model PHM4) with a platin u m electrode (type P 101) available from Radiometer Co., Copen-
1O0
METHODOLOGY
[8]
hagen. T h e assay system is calibrated relative to 10-2 M ferri- plus ferrocyanide in the assay buffer mixture. For this r e d o x couple, the value Er~,7 = +0.423 was used. T h e m e a s u r e d Eh value o f a reaction mixture containing 6.7 × 10 -4 M DCPI plus 5.0 × 10 -4 M ascorbate is approximately +0.15 V, where the rate o f the ATPase reaction in the dark is maximal. Half-maximal ATPase reaction in darkness is obtained at approximately either +0.1 V or +0.2 V. Although the Eh value required for the m a x i m u m rate o f ATPase reaction is i n d e p e n d e n t o f the concentration o f the r e d o x buffer, the m a x i m u m rate attained in the presence o f 6.7 x 10-4 M DCPI and an a p p r o p r i a t e concentration (5.0 x 10_4 M) o f ascorbate is approximately one-fifth the m a x i m u m rate attained in the presence o f 6.7 × 10 -5 M DCPI and an a p p r o p r i a t e concentration o f ascorbate. Probably two adjacent different oxidationreduction c o m p o n e n t s in the electron transport system are functional c o m p o n e n t s o f one o f the coupling sites which lead to A T P formation, and ATPase activity at the site appears when one o f the two c o m p o n e n t s is in the oxidized form and the o t h e r is in the r e d u c e d form.
Ubiquinone-lO-Dependent ATPase Activity 9
Method for Extraction of Quinones from Chromatophores and Their Reconstitution by Addition of Quinones a° C h r o m a t o p h o r e s f r o m the blue-green m u t a n t o f R. rubrum (G-9) are washed with an excess o f water and then lyophilized. Lyophilized chrom a t o p h o r e s are suspended in a volume o f isooctane such that they would show As73 nm/ml = 5 if suspended in the same volume o f water, and then shaken moderately at 4 ° for 80 minutes, followed by centrifugation. T h e resulting precipitate is dried at 4 ° u n d e r vacuum (extracted chromatophores). For reconstitution, the extracted c h r o m a t o p h o r e s are suspended in isooctane containing an a p p r o p r i a t e a m o u n t o f p u r e q u i n o n e ; and then dried at 4 ° u n d e r vacuum. T h e y are then suspended in a volume o f 0.1 M glycylglycine-NaOH buffer containing 10% sucrose (pH 8.0) such that the resulting suspension would show As73 nm o f approximately 50. Essentially the same results are obtainable with chrom a t o p h o r e s f r o m wild-type cells; the extraction o f quinones is significantly m o r e difficult with c h r o m a t o p h o r e s f r o m wild-type cells than with c h r o m a t o p h o r e s f r o m the m u t a n t cells. Quinones present in c h r o m a t o p h o r e s are d e t e r m i n e d as follows. An aqueous c h r o m a t o p h o r e suspension (As73nm/ml = 50) is extracted three times with 10 volumes o f a mixture o f acetone and methanol (8:2, v/v). ON. Yamamoto, H. Hatakeyama, K. Nishikawa, and T. Horio, J. Biochem. 67, 587 (1970). 1°S. Okayama, N. Yarnamoto, K. Nishikawa, and T. Horio,J. Biol. Chem. 243, 2995, (1968).
[8]
LIGHT-INFLUENCED ATPase ACTIVITY: BACTERIAL
101
The three extracts are combined in a separatory funnel, and an equal volume of petroleum ether plus an equal volume of water saturated with NaCI are added thereto. The petroleum ether should be redistilled before use; the fraction boiling between 40 ° and 60 ° is collected. After gentle swirling, the petroleum ether phase is collected and then washed with an excess of water to remove the acetone and methanol. The resulting petroleum ether phase is shaken three times in succession with an equal volume of 95% methanol; the bacteriochlorophyll and phospholipids present are almost completely extracted into the methanol phase. The petroleum ether phase is evaporated in vacuum with a rotary evaporator. The dried material is dissolved in a small volume of isooctane, spotted on a silica gel thin-layer plate (TLC-plates Silica Gel, E. Merck AG., Darmstadt) and developed with a mixture of chloroform and benzene (1 : 1, v/v) at 20 ° for 2 hours. During chromatography, four zones are formed: a yellow zone with an Rsvalue of 0.5 (ubiquinone-10), a purple zone with an Rf value of 0.4 (rhodoquinone), a pink zone with an Rf value of 0.2 (bacteriopheophytin), and a pale yellow-green zone with an Rs value of 0 (bacteriochlorophyll). The quinones are eluted separately with spectroscopically pure ethanol. The absorption spectra of the eluates are measured before and after addition of NaBH4. Concentrations of quinones are calculated from the absorption changes for which the molar difference extinction coefficients ("oxidized" minus "reduced") of ubiquinone-10 and rhodoquinone are 12.25 × 103 at 275 nm n and 7.2 × 103 at 283 nm 12 respectively. Recovery by this procedure is approximately 80% when pure quinones are used as the starting materials. Further identification of the quinones is achieved by means of cochromatography with authentic quinones on a silicone-impregnated paper. 13
Properties of Extracted and Reconstituted Chromatophores The chromatophore activities for photosynthetic ATP formation and for ATPase are fairly stable against lyophilization; they are 30-50% of those of untreated chromatophores. Chromatophores contain approximately 3.5-4.0 nmoles of ubiquinone- 10 and 0.7 nmole of rhodoquinone p e r A873 nm unit. Other quinone derivatives are not detectable in comparable amounts. Almost all the quinones associated with chromatophores are extracted by shaking in isooctane at 4 ° for a period longer than 45 minutes. The activity for photosynthetic ATP formation, whether measured in the presence of ascorbate or in the presence of PMS, is com11See Vol. X [68]. l*W. Parson and H. RudneyJ. Biol. Chem. 240, 1855 (1965). 13See Vol. VI [36].
102
METHODOLOGY
[8]
pletely depressed when all the quinones are extracted, and is restored to the original level when ubiquinone-10 is added back at the same level as that originally present. When lyophilized chromatophores are freed of the quinones, the activities in the dark for ATPase reaction in the presence and absence of either DNP or DCPI are significantly, but not completely, reduced in rate, in contrast to the activity for ATP formation, as shown in Fig. 3. The activities thus reduced are restored to the original level when ubiquinone-10 is provided. The ATPase activity is therefore composed of two types, one dependent and the other independent of the quinone. With lyophilized and reconstituted chromatophores, the activity is approximately 80% inhibited by oligomycin (3.3 /ag/ml). The oligomycin-insensitive activity is not affected by extraction of the quinones. Most of the oligomycin-sensitive activity is dependent on the quinone. When ubiquinone-10 is added to quinone-free chromatophores, ATPase activities in the presence and in the absence of DNP
150
A
/
o~
I
-
+None
~ Added ubiquinone-[0 (nrnoles/Aa73nm)
FIG. 3. Restoration o f d a r k A T P a s e activity by addition o f various a m o u n t s o f ubiquin o n e - 1 0 to extracted c h r o m a t o p h o r e s . E x p e r i m e n t a l conditions are as described in the text, except for the following: C h r o m a t o p h o r e s which have been extracted with isooctane for 60 m i n u t e s are reconstituted by addition of various a m o u n t s o f u b i q u i n o n e - 1 0 as indicated. In s o m e cases, the reactions are carried o u t in the presence o f 6.7 x 10 -5 M DCPI or 2.0 x 10 -3 M D N P or 3.3/xg/ml oligomycin. O p e n circles, + n o n e (no addition); squares, + DNP; triangles, + DCPI; closed circles, + oligomycin. T h e rates o f A T P a s e reaction by n o n t r e a t e d c h r o m a t o p h o r e s are 0.064, 0.185, a n d 0.163/zmole o f A T P hydrolyzed/ds~a ,m/ h o u r for "+ None," "÷ DNP," a n d "+DCPI," respectively.
[9]
TWO-STAGEPHOSPHORYLATION
103
or DCPI increase with increasing amounts of added ubiquinone-10 and reach a maximum at approximately 4 nmoles/As73 ,m, the same amount as originally present. With quinone-free chromatophores, the quinone-independent activity is markedly stimulated by DNP, but hardly by DCPI, indicating that DNP stimulates both quinone-dependent and independent activities, whereas DCPI stimulates the former, but hardly the latter. Of various quinones (4 nmoles/As73 nm) added to the extracted chromatophores, ubiquinone-9, -7, -6, hexahydroubiquinone-4, rhodoquinone, plastoquinone, o~-tocopherolquinone, and phylloquinone are as effective as ubiquinone-1 0 for the restoration of ATPase activity. Menadione, 2,3-dimethoxy-5,6-dimethylbenzoquinone, 4-amino-l,2-naphthoquinone, 2-amino-l,4-napthoquinone, 2-acetoamino-l,4-naphthoquinone, and S-(2-methyl-l,4-naphthoquinonyl-3)-fl-mercaptopropionic acid are not effective.
[9] Two-Stage Phosphorylation Techniques: Light-toDark and Acid-to-Base Procedures B y ANDRE T .
JAGENDOaE
Light-to-Dark Technique 1,2 The rationale of the experiment is to separate in time the immediate effects of light, especially electron transport, from steps in the phosphorylation reaction itself. The procedure described below can be used to demonstrate the point of action of given reaction mixture components, and of activators or inhibitors of phosphorylation. Procedure. Chloroplasts may be isolated from spinach leaves ground in 0.4 M sucrose, 10 mM NaCI, and 50 mM Tris, pH 8.0; or in any of the other usual buffered osmotic media. The chloroplasts (with or without an optional wash in the same medium) are resuspended to a concentration of 0.05 mg of chlorophyll per milliliter or less in 10 mM NaCI, kept for 20 minutes, collected by centrifugation at 10,000 g, washed once, and finally resuspended to a final concentration of 0.50 mg of chlorophyll per milliliter in 10 mM NaCI. All operations are at 0°C. W. K. Shen and G. M. Shen, Sci. Sinica 1 l, 1097 (1962). 2G. Hind and A. T. Jagendorf, Proc. Nat. Acad. Sci. U.S. 49, 715 (1963).
METHODOLOGY
[8]
Illumination at 100,000 lux for 20 minutes is provided by the same arr a n g e m e n t as described for light-triggered ATPase. T h e reaction is terminated by turning the light off followed by the addition of 0.1 ml TCA. T h e chloroplasts are removed by centrifugation at 500 g for 10 minutes at 0-4°c. Aliquots from the supernatant solutions are taken for [32p] Pi determination. [32p] Pl is separated from [z2p] ATP by the isobutanol-benzene extraction method, 8 and Pi is determined from the radioactivity in the organic phase. Specific activity is defined as micromoles of Pi released per milligram of chlorophyll per hour. Properties
Light-dependent ATPase activity has substrate specificity for ATP, GTP, and ITP with the highest rate obtained with ATP (apparent K,, = 4 X 10 -5 M). ADP is a competitive inhibitor with an apparent Ki = 3 x 10-5 M ? T h e specific activity is in the order of 10-30. With white light and PMS as cofactor, saturation is approached at 200,000 lux. 9 T h e activity is inhibited by Mg 2+ ions and by uncouplers of photophosphorylation such as 5 x 10 -6 M atebrin, 1.6 x 10 -5 M CCP, and 7 x 10 -6 M gramicidin S. 7 SM. Avron, Anal. Biochem. 2, 535 (1961). 9M. Avron,J. Biol. Chem. 237, 2011 (1962).
[8] Light-Influenced ATPase Activity: Bacterial By T. HORIO, Y. HORIUTI, N. YAMAMOTO,and K. NISHIKAWA
Chromatophores prepared from RhodospiriUum rubrum are able to synthesize ATP from ADP and Pi coupled with photosynthetic electron flow in a cyclic fashion? Besides photosynthetic ATP formation, chromatophores can catalyze the oxidation of N A D H by molecular oxygen coupled with A T P formation, z In addition, chromatophores have activities for ATP-Pi exchange and ATPase either in the light or in dark1T. Horio and M. D. Kamen, Biochemistry 1, 144 (1962). 2.].Yamashita,S. Yoshimura,Y. Matuo, and T. Horio, Biochim.Biophys.Acta 143, 154 (1967).
[8]
LIGHT-INFLUENCED ATPase ACTIVITY: BACTERIAL
97
hess, which are at least in part brought about because of the reversibility of the ATP-forming reaction? ,4 ATPase activity by chromatophores is firmly associated with particle structure and influenced by illumination, redox dyes, and extraction of the quinones from chromatophores. Conceivably, the ATPase activity is catalyzed at least in part by an enzyme system containing redox components associated with chromatophores.
Preparations and Assay Method The culture of R. rubrum cells and preparation of chromatophores from them are performed in the same manner as described elsewhere in this series. 5 ATPase activity is assayed according to the methods described elsewhere, 5 except for the following addition. In some cases, the reactions are carried out under continuous illumination incident on the reaction test tubes of approximately 1000 ft-c from a bank of tungsten lamps. Reactions in the dark are carried out in test tubes covered with aluminum foil.
ATPase Activity Influenced in the Light 6 The rate of ATPase reaction by chromatophores is slower in the light than in the dark, with most preparations of chromatophores, and without addition of redox dyes. On the contrary, ATPase activity is significantly stimulated by 4 × l0 -4 M phenazine methosulfate (PMS) in the light. When a Lineweaver-Burk plot is made of the reciprocals of the ATP concentrations and the rates of the ATPase reaction, the light appears to be a competitive inhibitor against ATP in the absence of PMS and to be a noncompetitive stimulant against ATP in the presence of PMS. The inhibition of ATPase activity in the light is significantly neutralized by adding antimycin A, whereas the inhibitor hardly influences the activity in the dark. The neutralization is maximal at 0.1 tzg/ml of antimycin A, where the extent of the light inhibition is diminished by approximately 70%. It has been shown that antimycin A at 0.1 t~g/ml completely inhibits photosynthetic ATP formation, unless PMS is present. 7 The inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (0.67 /~g/ml), 3T. Horio, K. Nishikawa, M. Katsumata, and J. Yamashita, Biochim. Biophys. Acta 94, 371 (1965). 4T. Horio, K. Nishikawa, Y. Horiuti, and T. Kakuno, in "Comparative Biochemistry and Biophysics of Photosynthesis" (K. Shibata et al., eds.), p. 408. Tokyo University Press, Tokyo, 1968. 5See Vol. 23 [63], 6y. Horiuti, K. Nishikawa, and T. Horio, J. Biochem. 64, 577 (1968). ~T. Horio and J. Yamashita, Biochim. Biophys. Acta 88, 237 (1964).
98
METHODOLOGY
[8]
shows the same effects as antimycin A, both in the dark and in the light. Either in the presence or the absence of PMS, ATPase activity decreases when chromatophores are illuminated with monochromatic light at 880 nm, which is the absorption peak of bacteriochlorophyll associated with the chromatophores from wild-type cells. It has been shown that PMS is reduced in white light but not in 880-nm light, s an observation indicating that the stimulation of ATPase activity by white light in the presence of PMS is caused by the photochemical reduction of the dye. This is confirmed by the observation that ATPase activity in the presence of PMS in the dark increases and then is decreased by the addition of increasing concentration of ascorbate, capable of reducing PMS, as shown in Fig. 1.
Eh-Dependent ATPase Activity When 2,6-dichlorophenolindophenol (DCPI) in the oxidized form (Ema = +0.217 V) is added to the reaction mixture for the ATPase activity assay, it is reduced by chromatophores at a slow rate, and the Eh 40C
'
II
~
i
~x,o-~M
~
'
"1'
'\
0-
I00
0
~
i
i
log (ascorbateM
i
)
Fie. 1. Effect o f p h e n a z i n e m e t h o s u l f a t e (PMS) a n d ascorbate on A T P a s e activity in the dark, E x p e r i m e n t a l conditions are as described in the text, except that 4.0 x 10 -4 M or 6.7 X 10 -5 M PMS a n d various concentrations o f ascorbate are a d d e d as indicated. Circles, + PMS (4.0 × 10 -~ M); triangles, + PMS (6.7 x 10 -5 M).
SD. M. Geller, Ph.D. Dissertation, H a r v a r d University, C a m b r i d g e , Massachusetts, 1958.
[8]
LIGHT-INFLUENCED ATPase ACTIVITY: BACTERIAL , II
,
99
,
0.3
o o
0.2 ~g
rr"6 0.I "6 E ~L
0.0
II 10g ( DC P I, M)
FIG. 2. Effect o f 2,6-dichlorophenolindophenol (DCPI) on ATPase activities in the light and in the dark. Experimental conditions are as described in the text, except that DCPI is added as indicated. Open symbols, in the light; filled symbols, in the dark.
value of the reaction mixture becomes more positive with increasing concentrations of the dye. As shown in Fig. 2, ATPase activities in the light and in the dark are accelerated with increasing concentrations of DCPI up to 6.7 x 10 -5 M, and retarded at higher concentrations. T h e effect of DCPI concentrations are fairly similar for the ATPase activities in the light and in the dark, except for the reproducible difference as follows: At concentrations of DCPI lower than 6.7 × 10-5 M, ATPase activity in the light is lower than in the dark, and vice versa at higher concentrations. Antimycin A (0.33 /~g/ml) does not influence ATPase activity in the dark in the presence of DCPI at any concentration tested. Further, when ascorbate is added in the presence of 6.7 x 10 -4 M DCPI, where ATPase activity is significantly lowered, the activity increases and then decreases with increasing concentrations of ascorbate; the m a x i m u m rate is obtained at 5.0 × 10-4 M. T h e ATPase activity is not altered by repeated washing of chromatophores with various concentrations of DCPI and/or ascorbate, an indication that the effect of redox dyes is reversible. T h e Eh values of the reaction mixtures containing 6.7 × 10 -4 M DCPI and various concentrations of ascorbate are measured with a pH and millivolt meter (Model PHM4) with a platin u m electrode (type P 101) available from Radiometer Co., Copen-
1O0
METHODOLOGY
[8]
hagen. T h e assay system is calibrated relative to 10-2 M ferri- plus ferrocyanide in the assay buffer mixture. For this r e d o x couple, the value Er~,7 = +0.423 was used. T h e m e a s u r e d Eh value o f a reaction mixture containing 6.7 × 10 -4 M DCPI plus 5.0 × 10 -4 M ascorbate is approximately +0.15 V, where the rate o f the ATPase reaction in the dark is maximal. Half-maximal ATPase reaction in darkness is obtained at approximately either +0.1 V or +0.2 V. Although the Eh value required for the m a x i m u m rate o f ATPase reaction is i n d e p e n d e n t o f the concentration o f the r e d o x buffer, the m a x i m u m rate attained in the presence o f 6.7 x 10-4 M DCPI and an a p p r o p r i a t e concentration (5.0 x 10_4 M) o f ascorbate is approximately one-fifth the m a x i m u m rate attained in the presence o f 6.7 × 10 -5 M DCPI and an a p p r o p r i a t e concentration o f ascorbate. Probably two adjacent different oxidationreduction c o m p o n e n t s in the electron transport system are functional c o m p o n e n t s o f one o f the coupling sites which lead to A T P formation, and ATPase activity at the site appears when one o f the two c o m p o n e n t s is in the oxidized form and the o t h e r is in the r e d u c e d form.
Ubiquinone-lO-Dependent ATPase Activity 9
Method for Extraction of Quinones from Chromatophores and Their Reconstitution by Addition of Quinones a° C h r o m a t o p h o r e s f r o m the blue-green m u t a n t o f R. rubrum (G-9) are washed with an excess o f water and then lyophilized. Lyophilized chrom a t o p h o r e s are suspended in a volume o f isooctane such that they would show As73 nm/ml = 5 if suspended in the same volume o f water, and then shaken moderately at 4 ° for 80 minutes, followed by centrifugation. T h e resulting precipitate is dried at 4 ° u n d e r vacuum (extracted chromatophores). For reconstitution, the extracted c h r o m a t o p h o r e s are suspended in isooctane containing an a p p r o p r i a t e a m o u n t o f p u r e q u i n o n e ; and then dried at 4 ° u n d e r vacuum. T h e y are then suspended in a volume o f 0.1 M glycylglycine-NaOH buffer containing 10% sucrose (pH 8.0) such that the resulting suspension would show As73 nm o f approximately 50. Essentially the same results are obtainable with chrom a t o p h o r e s f r o m wild-type cells; the extraction o f quinones is significantly m o r e difficult with c h r o m a t o p h o r e s f r o m wild-type cells than with c h r o m a t o p h o r e s f r o m the m u t a n t cells. Quinones present in c h r o m a t o p h o r e s are d e t e r m i n e d as follows. An aqueous c h r o m a t o p h o r e suspension (As73nm/ml = 50) is extracted three times with 10 volumes o f a mixture o f acetone and methanol (8:2, v/v). ON. Yamamoto, H. Hatakeyama, K. Nishikawa, and T. Horio, J. Biochem. 67, 587 (1970). 1°S. Okayama, N. Yarnamoto, K. Nishikawa, and T. Horio,J. Biol. Chem. 243, 2995, (1968).
[8]
LIGHT-INFLUENCED ATPase ACTIVITY: BACTERIAL
101
The three extracts are combined in a separatory funnel, and an equal volume of petroleum ether plus an equal volume of water saturated with NaCI are added thereto. The petroleum ether should be redistilled before use; the fraction boiling between 40 ° and 60 ° is collected. After gentle swirling, the petroleum ether phase is collected and then washed with an excess of water to remove the acetone and methanol. The resulting petroleum ether phase is shaken three times in succession with an equal volume of 95% methanol; the bacteriochlorophyll and phospholipids present are almost completely extracted into the methanol phase. The petroleum ether phase is evaporated in vacuum with a rotary evaporator. The dried material is dissolved in a small volume of isooctane, spotted on a silica gel thin-layer plate (TLC-plates Silica Gel, E. Merck AG., Darmstadt) and developed with a mixture of chloroform and benzene (1 : 1, v/v) at 20 ° for 2 hours. During chromatography, four zones are formed: a yellow zone with an Rsvalue of 0.5 (ubiquinone-10), a purple zone with an Rf value of 0.4 (rhodoquinone), a pink zone with an Rf value of 0.2 (bacteriopheophytin), and a pale yellow-green zone with an Rs value of 0 (bacteriochlorophyll). The quinones are eluted separately with spectroscopically pure ethanol. The absorption spectra of the eluates are measured before and after addition of NaBH4. Concentrations of quinones are calculated from the absorption changes for which the molar difference extinction coefficients ("oxidized" minus "reduced") of ubiquinone-10 and rhodoquinone are 12.25 × 103 at 275 nm n and 7.2 × 103 at 283 nm 12 respectively. Recovery by this procedure is approximately 80% when pure quinones are used as the starting materials. Further identification of the quinones is achieved by means of cochromatography with authentic quinones on a silicone-impregnated paper. 13
Properties of Extracted and Reconstituted Chromatophores The chromatophore activities for photosynthetic ATP formation and for ATPase are fairly stable against lyophilization; they are 30-50% of those of untreated chromatophores. Chromatophores contain approximately 3.5-4.0 nmoles of ubiquinone- 10 and 0.7 nmole of rhodoquinone p e r A873 nm unit. Other quinone derivatives are not detectable in comparable amounts. Almost all the quinones associated with chromatophores are extracted by shaking in isooctane at 4 ° for a period longer than 45 minutes. The activity for photosynthetic ATP formation, whether measured in the presence of ascorbate or in the presence of PMS, is com11See Vol. X [68]. l*W. Parson and H. RudneyJ. Biol. Chem. 240, 1855 (1965). 13See Vol. VI [36].
102
METHODOLOGY
[8]
pletely depressed when all the quinones are extracted, and is restored to the original level when ubiquinone-10 is added back at the same level as that originally present. When lyophilized chromatophores are freed of the quinones, the activities in the dark for ATPase reaction in the presence and absence of either DNP or DCPI are significantly, but not completely, reduced in rate, in contrast to the activity for ATP formation, as shown in Fig. 3. The activities thus reduced are restored to the original level when ubiquinone-10 is provided. The ATPase activity is therefore composed of two types, one dependent and the other independent of the quinone. With lyophilized and reconstituted chromatophores, the activity is approximately 80% inhibited by oligomycin (3.3 /ag/ml). The oligomycin-insensitive activity is not affected by extraction of the quinones. Most of the oligomycin-sensitive activity is dependent on the quinone. When ubiquinone-10 is added to quinone-free chromatophores, ATPase activities in the presence and in the absence of DNP
150
A
/
o~
I
-
+None
~ Added ubiquinone-[0 (nrnoles/Aa73nm)
FIG. 3. Restoration o f d a r k A T P a s e activity by addition o f various a m o u n t s o f ubiquin o n e - 1 0 to extracted c h r o m a t o p h o r e s . E x p e r i m e n t a l conditions are as described in the text, except for the following: C h r o m a t o p h o r e s which have been extracted with isooctane for 60 m i n u t e s are reconstituted by addition of various a m o u n t s o f u b i q u i n o n e - 1 0 as indicated. In s o m e cases, the reactions are carried o u t in the presence o f 6.7 x 10 -5 M DCPI or 2.0 x 10 -3 M D N P or 3.3/xg/ml oligomycin. O p e n circles, + n o n e (no addition); squares, + DNP; triangles, + DCPI; closed circles, + oligomycin. T h e rates o f A T P a s e reaction by n o n t r e a t e d c h r o m a t o p h o r e s are 0.064, 0.185, a n d 0.163/zmole o f A T P hydrolyzed/ds~a ,m/ h o u r for "+ None," "÷ DNP," a n d "+DCPI," respectively.
[9]
TWO-STAGEPHOSPHORYLATION
103
or DCPI increase with increasing amounts of added ubiquinone-10 and reach a maximum at approximately 4 nmoles/As73 ,m, the same amount as originally present. With quinone-free chromatophores, the quinone-independent activity is markedly stimulated by DNP, but hardly by DCPI, indicating that DNP stimulates both quinone-dependent and independent activities, whereas DCPI stimulates the former, but hardly the latter. Of various quinones (4 nmoles/As73 nm) added to the extracted chromatophores, ubiquinone-9, -7, -6, hexahydroubiquinone-4, rhodoquinone, plastoquinone, o~-tocopherolquinone, and phylloquinone are as effective as ubiquinone-1 0 for the restoration of ATPase activity. Menadione, 2,3-dimethoxy-5,6-dimethylbenzoquinone, 4-amino-l,2-naphthoquinone, 2-amino-l,4-napthoquinone, 2-acetoamino-l,4-naphthoquinone, and S-(2-methyl-l,4-naphthoquinonyl-3)-fl-mercaptopropionic acid are not effective.
[9] Two-Stage Phosphorylation Techniques: Light-toDark and Acid-to-Base Procedures B y ANDRE T .
JAGENDOaE
Light-to-Dark Technique 1,2 The rationale of the experiment is to separate in time the immediate effects of light, especially electron transport, from steps in the phosphorylation reaction itself. The procedure described below can be used to demonstrate the point of action of given reaction mixture components, and of activators or inhibitors of phosphorylation. Procedure. Chloroplasts may be isolated from spinach leaves ground in 0.4 M sucrose, 10 mM NaCI, and 50 mM Tris, pH 8.0; or in any of the other usual buffered osmotic media. The chloroplasts (with or without an optional wash in the same medium) are resuspended to a concentration of 0.05 mg of chlorophyll per milliliter or less in 10 mM NaCI, kept for 20 minutes, collected by centrifugation at 10,000 g, washed once, and finally resuspended to a final concentration of 0.50 mg of chlorophyll per milliliter in 10 mM NaCI. All operations are at 0°C. W. K. Shen and G. M. Shen, Sci. Sinica 1 l, 1097 (1962). 2G. Hind and A. T. Jagendorf, Proc. Nat. Acad. Sci. U.S. 49, 715 (1963).