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A soluble component of the hill reaction in Anacystis nidulans
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 104, 3 9 - 4 9
(1964)
A Soluble Component of the Hill Reaction in Anacystis nidulans 1"2's W A L T E R W. F R E D R I C K S 4 AND A N D R E T. J A G E N D O R F From the McCollum-Pratt Institute and Biology Department, The Johns Hopkins University, Baltimore Maryland Received May 20. 1963 Cell-free preparations (fragments of photosynthetic lamellae) from Anacystis nidulans catalyze the Hill reaction using ferricyanide, indophenol dyes or cytochrome c as electron acceptor. A high molecular weight polymer (Carbowax or dextran) not only must be present during preparation of the particles to preserve Hill reaction activity, but gives a marked stimulation if present in the reaction mixture. Calcium salts also are stimulatory. Sodium or potassium salts were inhibitory, as were all buffer anions that were tested. Extraction of the cell-free particles by water leads to a loss of their phycocyanin and of the Hill reaction activity. With ferricyanide as electron acceptor, adding back the extract led to a reconstitution of photolysis by washed particles. The active component of the extract was partially purified and was found not to be identical with phycocyanin. Information is reported concerning the presence of an endogenous reducing material in the cell-free extracts. INTRODUCTION The accessory pigment, phycoeyanin, of the blue-green algae is dffierent from that of higher plants, chlorophyll b, in that the former is water soluble. Because of this, the bhJe-green algae lend themselves to the study of the function of the accessory pigment by some of the more traditional biochemical procedures. Cell-free preparations of red (1) and bluegreen algae (2, 3) have been shown to catalyze the Hill reaction provided certain 1 Contribution number 406 from the McCollure-Pratt Institute. 2 These studies constitute part of the dissertation submitted by W. Fredricks in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This investigation was supported in part by a pre-doctoral fellowship (HF-9259-C1) from the National Heart Institute, U. S. Public Health Service and by a research ~rant (RG-3923) from the National Institutes of Health, U. S. Public Health Service. 4 Present address: National Heart Institute, National Institutes of Health, Bethesda 14, Md.
precautions are taken in the preparation of the material. Of particular importance is the presence in the isolation medium of a high molecular weight polymer (e.g., Carbowax or dextran). Should this polymer be deleted, the accessory pigments are extracted from the particles and the Hill activity is immediately lost. I t is, therefore, tempting to infer that the loss of Hill activity is a direct consequence of the extraction of the accessory pigment. Thomas and DeRover (2) offered evidence which supported this idea but added, "though the experiments make it unlikely that the activity loss should be attributed to the release of enzyme systems, the possibility of a linkage of these systems to the phycocyanin molecules themselves cannot be excluded with certainty." Petrack (4) and Petrack and Lipmann (5) observed that cell-free preparations of Anabaena (a blue-green alga) catalyze "cyclic photophosphorylation." However, these preparations were made in the absence of any polymer and were essentially free of phycocyanin. 39
40
FREDRICKS AND JAGENDORF
These facts suggested to us a possible e x p l a n a t i o n of the E m e r s o n e n h a n c e m e n t effect, viz., light absorbed b y chlorophyll a is sufficient for "cyclic p h o t o p h o s p h o r y l a t i o n , " b u t a d d i t i o n a l a b s o r p t i o n b y a a accessory p i g m e n t is necessary for oxygen e v o l u t i o n . T o s u p p o r t this hypothesis it would be necessary to establish t h a t the loss of Hill a c t i v i t y is due to the loss of p h y c o c y a n i n a n d n o t some other c o m p o n e n t extracted a t the same time. This p a p e r reports the characteristics of the Hill reaction in cell-free p r e p a r a t i o n s from Anacystis nidulans, a blue-green alga. A q u e o u s e x t r a c t i o n of the active particles leads to a loss of this a c t i v i t y . I t was f o u n d possible to r e c o n s t i t u t e at least a p a r t of the a c t i v i t y b y a d d i n g back the extract, a n d accordingly a n i n v e s t i g a t i o n was m a d e into the relationship b e t w e e n the factor(s) active i n r e c o n s t i t u t i o n a n d the p h y c o c y a n i n comp o n e n t of the extract. MATERIALS AND METHODS The organism used in this study was the unicellular blue-green alga, Anacystis nidulans, of the family Chroococcaceae. The cultures were kindly supplied by Dr. B. Kok, RIAS, Inc., Baltimore, Md. The algae were cultured in l-liter Roux culture bottles containing 500 ml. ; or in 12liter carboys containing 9 liters of Medium C of Kratz and Myers (6) with the following modifications: the micronutrient solution was essentially that proposed by Brody and Emerson (7) and 5 ml. of a 10% Na~CO3 solution per liter of medium was added to bring the pH to about 8 to 9. In addition several marble chips were added to each bottle to help maintain the alkaline pH. No attempt was made to redissolve the precipitate formed on autoclaving the medium. In the case of the small volume cultures, the temperature was maintained at 39 =t= I~ Continuous illumination was provided by three Sylvania Daylight fluorescent lights (F20T12D) at a distance of about 12 cm. from the cultures. Each culture was aerated at a rate of 0.50-0.75 liters per minute. The air was cleaned by passage through concentrated H2SO4 and humidified by passage through water. Under these conditions the cultures yielded about 2.5 g. of cells (wet weight) per liter after 5-7 days growth. In the case of the large volume cultures the temperature was maintained at 38~ in a Percival Plant Growth Chamber. Continuous illumination was provided at about 500 foot-candles from Daylight fluorescent lights. As the cultures
became more dense additional light was supplied. Aeration was essentially that described for the small volume cultures, except at a proportionally higher rate. Stock cultures were maintained on slants of the above medium solidified by 1.5% agar. PREPARATION OF CELL EXTRACTS Cells from 2 liters of culture (about 5 g. wet weight) were harvested by centrifugation. They were suspended in 20 ml. of grinding medium, and 16 g. of glass powder (about 200 mesh) was added with stirring. The grinding medium consisted of a solution 30% in Carbowax 4000, 0.02 M in Tris ~ and 0.001 M in EDTA adjusted to pH 7.2. The suspension was blended in an Omnimixer for 5 minutes. An additional 20 ml. of grinding medium was added, followed by 2 minutes agitation. The homogenate was centrifuged for 10 minutes at 7710g to remove glass powder and whole cells. The supernatant fluid was centrifuged for 21/~ hours at 105,0009. Approximately 90% of the chlorophyll was sedimented during this operation. The supernatant fluid was discarded, and the residue which consisted of particles containing both chlorophyll and phycocyanin was stored frozen until use. This residue is designated as lamellar fragments. All operations were carried out from 0~ to 5 ~ For assay of Hill activity a portion of the residue was thawed and suspended in a small volume of a solution 20% in Carbowax 4000, 0.02 M in Tris, and 0.001 M in EDTA adjusted to pH 5.8. This Carbowax concentration is near the lower limit necessary for retention of Hill activity, e.g., suspension in 15% Carbowax results in decreased activity. The suspension was frozen and thawed to yield a very fine dispersion of particles. Both residue and resuspended material were very stable and could be kept frozen for weeks without loss of activity. They are also stable to repeated freezing and thawing. (For an extensive study of the stabilizing properties of Carbowax 4000 cf. Clendenning et al. (8), 1956.) Lamellar fragments catalyzed the reduction of electron accepters at a rate of between 80 and 150 microequivalents per milligram chlorophyll per hour. Some preparations exhibited rates as low as 30 or as high as 200. 5Abbreviations used: Tris, tris (hydroxymethyl) aminomethane; TPN, triphosphopyridine nucleotide; DCPIP, 2,6-dichlorophenolindophenol; TCPIP, 2,3',6 trichlorophenolindophenol; EDTA, ethylenediaminetetraacetate; DEAEcellulose, diethylaminoethylcellu!ose; PPNR, photosynthetic pyridine nucleotide reductase; ATP, adenosine triphosphate.
HILL REACTION IN ANACYSTIS Several attempts were made to improve the activity of the material by different preparative methods. None of the variations, including the addition of ascorbate or 2-mercapto-ethanol or grinding with alumina instead of glass powder, led to particles with increased activity. To prepare washed lamellar fragments, lamellar fragments equivalent to 2 liters of culture were extracted with 20-30 ml. of cold, deionized water. The suspension was centrifuged for 2 hours at 105,000g; the resulting supernatant fluid is designated as the crude extract. The residue was washed by centrifugation with approximately 60 ml. of cold, deionized water. The final residue contained chlorophyll but was free of phycocyanin. ANALYTICAL PROCEDURES AND ACTIVITY MEASUREMENTS Chlorophyll was measured by the method of Arnon (9), protein was determined according to Lowry et al. (10), and phycocyanin by the optical density at 620 rag. I n spectrophotometric assays of the Hill reaction, euvettes were exposed to light filtered through 10 cm. of water at room temperature, and the optical density of the particular dye was measured before and after illumination. The change in optical density was corrected for any dark reduction of the oxidants. The following oxidants were used, and their reduction assayed at the wavelengths noted: KaFe(CN)8 (420 mg), D C P I P (600 rag), T C P I P (620 mtt), and cytochrome c (550 m#). The millimolar extinction coefficients used to calculate concentrations are: K~Fe(CN)r (1), D C P I P , pH 6.5 (19.1), T C P I P (t8), and cytochrome e, reduced minus oxidized (20). Despite evidence in the literature for photoreduction of pyridine nucleotides in Anacystis in vivo (11) no reduction of T P N could be demonstrated with lamellar fragments or washed lamellar fragments, even with added spinach P P N R (12). Most reactions reported here involved the direct determination of oxygen evolution with the aid of a Clark-type oxygen electrode (13) in a closed reaction vessel without a gas phase (construction and circuit details available upon request). A linear response to oxygen concentration was assumed (see references 14 and 15), and the instrument was calibrated for two oxygen concentrations: that in water in equilibrium with air, and zero oxygen as achieved by the addition of hydrosulfite. A cMibration curve (concentration of oxygen in water at equilibrium with air as a function of temperature) was calculated from available data (16).
41
In the standard assay ferricyanide was the oxidant. The reaction mixture contained, in a final volume of 3 ml., 70 ~moles of NaCl, 5 gmoles of K3Fe(CN)6, 30 ~moles of maleate or phosphate buffer at pH 5.8, 150 or 180 rag. of Carbowax 4000, 10 or 20 ~moles of CaCI~, and lamellar fragments or washed lamellar fragments containing about 200 ~g. of chlorophyll. Light from a 100 watt Bausch and Lomb microscope lamp was filtered through 6 em. of water. Each reaction mixture was allowed 7 minutes to come to temperature equilibration at 35 ~ and recording was started 2 minutes prior to illumination. Oxygen evolution was linear with time over the period of measurement (5 minutes). The usual control contained all components except ferricyanide, and dark controls gave the same values. Rates are reported as microequivalents of oxidant reduced per milligram chlorophyll per hour. Oxygen evolution as measured with the electrode was found to be stoichiometric to ferricyanide reduction as measured with the spectrophotometer, with the expected ratio of 4 ~moles of ferricyanide reduced for each ftmole of oxygen evolved. In chromatography on DEAE-cellulose approximately 100 mg. of protein, dissolved in 0.02 M phosphate buffer, pH 7.0, was adsorbed to a 2 X 12 era. column previously washed with the same buffer. Elution was by a 200 ml. linear KC1 gradient, from 0.0 to 0.5 M, in 0.02 M phosphate, pI-I 7.0. Carbowax 4000 (a polyethylene glycol of approx. 3600 molecular weight) was obtained from the Union Carbide and Chemical Corp. Clinical grade dextran (a branched glucose polymer having a molecular weight of 60,000-90,000) was from the Nutritional Biochemicals Corporation. I t should be noted that attempts to measure photophosphorylation in the present study were completely foiled by the presence of Carbowax in the reaction mixtures. I t was found t h a t Carbowax interferes with (a) various modifications of the inorganic phosphate determination of Taussky and Shorr (17), (b) separation of inorganic from organic phosphates by the method of Lindberg and Ernster (18), and (c) adsorption of A T P onto Norite (19). In the latter case as little as 0.6% Carbowax caused complete inhibition, with partial interference at 0.1%. Dextran did not cause the same interference with the analytical methods for phosphate. RESULTS CHARACTERISTICS OF THE HILL REACTION WITH LAMELLAR FRAGMENTS In the standard reaction using ferricyanide as o x i d a n t , t h e r a t e of o x y g e n e v o l u t i o n w a s
42
FREDRICKS AND JAGENDORF
other experiments using either chloride, sulfate, or nitrate salts of the divalent metals. It can be seen (Table I) that, in the abFsence of CaC12, low concentrations of NaC1 o 0.25 -3 were stimulatory but higher concentrations inhibitory. Only inhibition was seen in the ,a) 0 . 2 0 presence of CaC12. Almost identical results 7 were obtained with KC1 in place of NaC1. It is not clear whether the stimulation was a LU 0.15 partial replacement of calcium by sodium, 0 > or a chloride effect similar to that reported L~ by Warburg (20) and Arnon (21). ON O. JO In experiments with buffer anions, the buffer to be studied was varied over a cong~ centration range in the presence of a minimal ~L 0 . 0 5 but constant level of a second buffer necessary to control the pH. Figure 2c illustrates that all the anions tested inhibit the Hill I I I O* 0 I00 200 300 reaction to some degree. Similar effects were ,u.g. C H L O R O P H Y L L found when the reactions were run in the F1o. I. Chlorophyll concentration series. absence of CaC12. Of particular interest are Standard assay, except that the Carbowax con- the curves for phosphate and for citrate, centration was 15%. where the quantitative effect negates the entire amount of simulation due to calcium. a linear function of chlorophyll concentra- However, the per cent inhibition by a given tion from 0 to 200 #g. of chlorophyll (Fig. 1). concentration of citrate was the same at all At higher concentrations light became limit- calcium concentrations; in other words adding, and there was little change in rate with ing extra calcium did not reverse the citrate increasing chlorophyll concentration. The inhibition. Consistent with the idea that pH curve shows an optimum centered this is an inhibition by the anion is the fact around p H 5.8, falling to very low values at that Tris hydroehloride showed only a 13 % p H 8. This was true also for the reconstituted inhibition at a concentration as high as system (see below). The p H optima for the 0.055 M. T C P I P Hill reaction was 6.5 and for the In the absence of calcium, increasing cytochrome c Hill reaction, 7.5. concentrations of E D T A gave increasing As shown in Fig. 2a, addition of Carbo- amounts of inhibition up to 0.001 M (Fig. wax to the reaction mixture stimulated the 2d). In the presence of calcium, E D T A inferricyanide Hill reaction up to an optimal hibition increased progressively at concenlevel, after which inhibition set in due to trations even higher than 0.001 M. At the excess polymer. These effects were also seen highest concentration of E D T A only about using T C P I P as the electron acceptor. A half the calcium would be complexed, so similar phenomenon occurs when dextran that the inhibition cannot be due simply to was the polymer used, although neither the a removal of calcium from the lamellar stimulation nor the inhibition was as pro- fragments. From the data in Fig. 2b it can nounced in this case. It can be noted that be seen that the rate was little affected by a the concentration of Carbowax used for change in calcium concentration in this preparation of active particles (30%) was range (between 4 and 7 raM). These results inhibitory when present in the reaction mixsuggest that the calcium-EDTA complex ture. Figure 2b shows that calcium chloride, was more inhibitory than E D T A alone, and to a lesser extent magnesium chloride, since E D T A inhibition was the most prostimulated the reaction rate when present at nounced in the presence of high levels of 0.01 M. Identical stimulations were seen in calcium. 0.30
0
'0
80
t/l
60
' ~/
i/
W I-.<~ n-
\\
,20~.
(c)
40
\
W I-,,:r n,-
%.
20
48
24
I
O
~ 20
I
I 40
I 60
CONCENTRATION OF BUFFER(raM) 0
f
I
0
I
I,
I0
I
I
20
CONCENTRATION
I00
50
OF CARBOWAX
150
(d)
(%) (b) 80
120
90
40~
60
20
50
0
I
0
I IO
]
CONCENTRATION
I 20
I
OF S A L T
I 50-
0
0
(mM)
I I
CONCENTRATION
J 2
OF
EDTA
5
(mM)
FIG. 2. The effects of various reaction components on t h e rate of t h e Hill reaction with lamellar fragments. (a) Effect of Carbowax at two concentrations of chlorophyll. S t a n d a r d assay: O O 49 ~g. chlorophyll; 0 - - - 0 185 ~g. chlorophyll. (b) Effect of MgCI~ and CaC12. S t a n d a r d assay: 0 - - 0 CaC12; O O MgC12. T h e point at 0 m M salt is t h e average of t h r e e experiments. (c) Effect of various buffers. S t a n d a r d assay in t h e presence of 6.67 m M CaCl~. T h e p H control in the case of t h e maleate and citrate curves was provided b y a minimal level of phosphate, pH 5.8. I n t h e case of the p h o s p h a t e curve pH control was provided by a minimal level of maleate, p H 5.8. O phosphate, pH 5.8; 9 maleate, pH 5.8; citrate, p H 5.8. (d) Effect of calcium a n d E D T A . S t a n d a r d assay; O 6.67 m M CaC12; 9 no CaC12. R a t e m e a s u r e m e n t s were m a d e b y following oxygen evolution a n d are expressed as microequivalents of oxidant reduced per milligram chlorophyll per hour. 43
44
FREDRICKS AND JAGENDORF RESOLUTION
AND RECONSTITUTION
THE
HILL
OF
REACTION
After extraction of lamellar fragments with water (see Materials and Methods) the Hill reaction activity was reduced to 10 % or less of the original rate. This was true whether ferricyanide, cytochrome c or the indophenol dyes were the oxidants used. The washing procedure which resulted in TABLE I E F F E C T OF NaC1 ON THE H I L L REACTION OF LAMELLAR FRAGMENTS IN THE PRESENCE OR ABSENCE OF CaC12 #moles NaCl added to 3 ml. reaction mixture
Rate a
0 35 70
105 140 350 700 98O
No/CaCI2
6.67 mM CaCI2
13.0 29.1 35.5 39.4 40.3 41.3 35.5 30.4
81.0 84.2 82.9 75.7 80.6 71.7 53.4 47.0
Rates are given as microequivalents of oxidant reduced per milligram chlorophyll per hour.
the loss of Hill activity produced an intense blue extract with red fluorescence characteristic of phycocyanin. Figure 3 shows the spectrum of this extract. Phycocyanin accounts entirely for the absorption at 620 and 350 m~ and is the major contributor to the 280 mu absorption (22). The shoulder at about 410 m~ and the absorption at 265 m~ can possibly be attributed to pteridines (3). Reactivation of the Hill reaction with ferricyanide by increasing concentrations of the above extract is shown in Fig. 4. I n this experiment the extracted lamellar fragments had a rate 9 % t h a t of the original, and with an optimal concentration of added extract 36 % of the original activity was obtained. With occasional preparations of lamellar fragments whose activity was abnormally low to begin with, complete reactivation could be seen after extraction. No added reactivation was obtained b y preincubating particles and extract for various times or at various Carbowax concentrations. Reactivation could not be ascribed to Carbowax in the extract because an optimal amount of Carbowax was added to each reaction mixture regardless of the presence or absence of extract.
1.0 _
Crude e x t r o c t
0.8
~ 0.6 ._1 "~ 0 . 4 f.D
0
0.2
0 22(
300
400
500
600
700
WAVELENGTH ( m/J.) Fi~. 3. Spectrum of crude extract. Lamellar fragments prepared in a buffered Carbowax solution (see Materials and Methods) was extracted with cold, distilled water. The mixture was centrifuged at 105,000g, and the supernatant fluid constituted the crude extract.
HILL REACTION IN ANACYSTIS
45
40
24 30
21
18
t~ 20
r~
15
i'
10
o 0
I
!
]
L
I
]
0.3
0.6
0.9
1.2
i.5
1.8
EXTRACT
ADDED
21
(ml)
FIG. 4. Effect of extract concentration on the Hill reaction of washed lamellar fragments. Standard assay; rates are expressed as microequivalents of oxidant reduced per milligram chlorophyll per hour. Using D C P I P or T C P I P as electron acceptor, the extent of reactivation was usually negligible. Cytochrome c could not be tested, because it caused the extracted lamellar fragments to precipitate under the conditions of reconstitution. Figure 4 shows a definite o p t i m u m curve for reaction rate vs. extract concentration (i.e., inhibition at higher concentrations). The inhibitory effect was even more pronounced with the original lamellar fragments, where the concentration of extract which maximally reactivated washed fragments was actually inhibitory. Optimal reactivation of washed lamellar fragments was provided by an amount of extract very nearly the same as that present in the same a m o u n t of particles prior to extraction. Carbowax had about the same effect on the residual activity of washed lamellar fragments (Fig. 5) as it did on the original, unextracted fragments (Fig. 2a). The situation was a little more complex with the rereconstituted system, in t h a t the reactivation due to added extract (about twofold) was constant from 0 to 12 % Carbowax, but falls to zero at 25 % Carbowax. The salt effects were changed somewhat in the extracted and reconstituted system (Table I I ) . Calcium was still stimulatory
-3 0
4
8
CONCENTRATION
IZ
~ro
20
24
OF CARBOWAX(%)
FIG. 5. Effect of Carbowax concentration on the Hill reaction of washed lamellar fragments arid the reconstituted system. Standard assay with the addition of 0.5 ml. of crude extract for the reconstituted system: 9 reconstituted system; O washed lamellar fragments; [] enhancement (rate of reconstituted system minus rate of washed lamellar fragments). Rates are expressed as microequivalents of oxidant reduced per milligram chlorophyll per hour. but KC1 had essentially no effect (in the presence or absence of calcium), and NaC1 is stimulatory under all circumstances. The stimulation by NaC1 seen here was additive with t h a t of CaC12. Enhancement activity, simply the difference between lines a and b of Table II, was thus best seen in the presence of a mixture of sodium and calcium chloride. CHARACTERISTICS
OF
THE
ACTIVE
COMPONENT
The active component in the water extracts was precipitated at p H 4.8 or b y amraonium sulfate, and could be adsorbed to and eluted from calcium phosphate gel. I t moved as a large molecule in a Sephadex G-25 column, and it is nondialyzable. In addition, Table I I I shows t h a t it is heat
46
FREDRICKS AND JAGENDORF TABLE II
4.0
REACTION
OF WASHED FRAGMENTS
Added extract
+Ca
}
LAMELLAR a
No monovalent salt - Ca
160
9\
EFFECT OF VARIOUS SALTS ON THE HILL
.2
NaCI - Ca
t28
2
KCI
+Ca
o
- Ca+Ca -E 2 . 4
(a) (b) +
0 --6
18 54
12 30
42 84
0 5
18 44
w
(b) - (a)
-6
36
18
42
5
26
~_ ,.6 E
-
-
a This experiment was done with the standard assay except for the addition of 100 ttmoles of NaC1 or KC1 and 20 ttmoles of CaC12 where indicated. The data are expressed as corrected changes in current (muamps)/5 minutes illumination.
OF HEATING OF THE
EXTRACT HILL
ON RECONSTITUTION
REACTION
a
Rate Lamellar fragments Washed lamellar fragments Washed lamellar fragments extract Washed lamellar fragments heated extractb
plus
155 7 56
plus
15
a S t a n d a r d a s s a y e x c e p t for t h e a d d i t i o n of 1 m l . of d i a l y z e d c r u d e e x t r a c t w h e r e i n d i c a t e d . b H e a t e d for 10 m i n u t e s a t 60 ~
labile, losing about 75% of its activity in 10 minutes at 60 ~ These characteristics are consistent with the idea t h a t the active component in the aqueous extracts is a protein. On tile initial assumption that the active component might be phycocyanin, relatively pure phycocyanin was prepared as estimated by the ratio of absorbancy at 620/280 mg (22, 23). This purified phycocyanin did not reactivate washed lamellar fragments. Others a t t e m p t s at fractionation indicated that when the activity was recovered it was not necessarily found in the fraction richest in phycocyanin. This was particularly true in the case of DEAE-cellulose column chromatography using stepwise elution. Here it was found t h a t 70 % of the activity was found
C Z
64 ~
o r
0.8
32
z z X
3 0 18
I
1
19
20
I
I
I
1
I
21
2~
23
24
25
TUBE
TABLE I I I ~]FFECT
96
0 26
NUMBER
FIG. 6. DEAE-cellulose chromatography of extract. Standard assay with 0.5 ml. of extract: 0 protein (mg./ml.); 9 phycocyanin (units/ml.); [] reactivation activity (arbitrary units/ml.). in a fraction containing only 16 % of the original phycoeyanin, while 20 % of the activity was associated with the fraction containing 70 % of the phyeocyanin. The most significant fractionation was achieved on a DEAE-cellulose column with a linear KC1 gradient elution (see Materials and Methods). Figure 6 shows t h a t portion of one chromatogram where the active component was eluted. There is only one peak (tube 18) in the protein profile, which is followed almost point by point by the phycocyanin concentration curve. On the other hand, activity was recovered primarily in tubes 21 to 25. The fact that only one protein peak appears is not too surprising since the phycocyanin represents about 30 % of the total cell protein (22), and the lamellar fragments from which the extract is obtained have already been removed from other cytoplasmic proteins. Generally close to 100 % of the activity could be recovered from the D E A E fractions. Pooling fractions which contained the largest amount of activity (tubes 21 to 25) gave a preparation purified between 5- and 8-fold, in different experiments.
HILL REACTION IN ANACYSTIS TABLE IV RESOLUTION AND RECONSTITUTION ~REDuC1NG MATEtlIAL a~
47
DISCUSSION OF
Lamellar fragments from Anacystis differ in a number of respects from chloroplasts of higher plants, with respect to their Hill Optical density Additions decrease, reaction activity. Besides the previously 580 rr~ noted requirement for a concentrated polymer solution during homogenization and None 0 isolation (1-3), there are a number of ionic Washed lamellar fragments 0.032 Extract 0.047 effects which appear to be unique for the Washed lamellar fragments p l u s 0.272 present system: in particular the marked extract stimulation by calcium ions (Fig. 2b), inWashed lamellar fragments p l u s 0.234 hibition by the cMeium-EDTA complex heated extract b (Fig. 2d), or by very ordinary buffer anions a Each reaction mixture contained in a final (Fig. 2c). In addition the presence of the volume of 3 ml.; 600 t~moles of phosphate buffer, polymer during the Hill reaction is strongly pH 6.5, TCPIP, and where indicated washed stimulatory (Fig. 2a), a phenomenon not lamellar fragments equivalent to 180 t*g. of chloro- noticed with higher plant chloroplasts. It phyll and/or 1 ml. extract (about 4 rag. protein). should be noted that the concentration of bHeated for 10 minutes at 60~ Carbowax needed for best results during the preparation of lamellar fragments is inhibiDARK REDUCTION OF HILL OXIDANTS tory if present in the reaction mixture. Many of these differences from higher When using spectrophotometric assays, it plant chloroplasts are undoubtedly due to was observed that lamellar fragments cause a rapid dark reduction of added oxidants, the differences in physical structure. The presumably because of the presence of endog- photosynthetic apparatus of the blue-green enous reducing compounds. Van Baalen algae is seen to consist of a series of lamellae (3) also reported this observation for cell-free running around the outer edge of the cell preparations of Anacystis. After extraction and enclosing the other cell contents (24) of the lamellar fragments, neither the washed rather than being concentrated in discrete particles nor the extract reduced added plastids. Biocbemieally the present iamellar fragTCPIP to any great extent (Table IV). ments differ from the usual chloroplasts in Surprisingly, the two in combination caused being unable to reduce TPN, and in showing a considerable reduction of dye. This reduca more vigorous reduction of ferricyanide tion is virtually instantaneous and no further than of the indophenol dyes. The concept reaction is seen with time. Heating the exthat reduction of indophenol dyes occurs tract sufficiently to cause loss of reconstitu- by a shorter and simpler series of reactions, tion activity has no effect on the dark reduc- than does the reduction of ferricyanide detion phenomenon (Table IV). veloped from studies of spinach chloroplasts In other experiments one component was (25, 26), may not be true for this system. held constant and the amount of the other The reason for the failure to reduce TPN was varied. In both cases the amount of dark is not apparent from the experiments so reduction was a linear function of the com- far undertaken, since the addition of both ponent being varied, whether it was the PPNR (12) and transhydrogena~e (27) amount of washed lamellar fragments or the failed to restore activity. This is especially amount of extract. It should also be noted surprising because the in vivo photoreduction of pyridine nucleotides by Anacystis has been that the material in the extract responsible reported by Duysens et al. (11)and in vitro for dark dye reduction is nondialyzable. The by Van Baalen (3) and by Black et al. (28). relation between this reduction of dyes in the As would be anticipated from the work of dark and the light-dependent Hill reaction is McClendon and Blinks (1) and Thomas entirely unknown. and DeRover (2), aqueous extraction of the
48
FREDRICKS AND JAGENDORF
lamellar fragments resulted in the loss of Hill activity for all oxidants tested. A partial reconstitution was observed in the case of ferricyanide as the Hill oxidant, but not with any of the other electron acceptors. T h e degree of reactivation was variable, running up to 45 % of the original activity of the lamellar fragments but usually being 20-30 %. The unsupplemented washed lamellar fragments showed approximately 5-10 % of the original activity. The fact that there is inhibition at higher concentrations of extract (Fig. 4) possibly is one factor in the failure to demonstrate full reactivation. Another factor may be some degree of irreversible physical damage to the lamellar fragments; this might also account for the variability in the extent of reactivation. This is suggested by the differences between lamellar fragments and washed lamellar fragments with respect to stability and the facility for being resuspended. The washed lamellar fragments are only stable when kept at 0 ~ (not frozen) as pellets, and they cannot be obtained as a fine dispersion (even with freeze-thaw treatments) in the "resuspending medium" used for the lamellar fragments. T h e y can, however, be suspended readily in water. Several authors have observed a simultaneous loss of Hill activity and of phycobilin pigments when cell-free material from red (1) and blue-green (2, 3) algae are extracted by buffers in the absence of polymers. On the other hand, loss of phycobilins did not result in a loss of the ability to catalyze cyclic electron flow and phosphorylation b y particles of blue-green algae (4, 5). These facts suggest that the phycobilins are specifically concerned in the oxidation of water and evolution of oxygen in the Hill reaction. Our own data tend to support this hypothesis only up to a point. Loss of activity is correlated with loss of the phycobilin pigment in our experiments. Restoration of activity depends on a non-dialyzable, heatlabile, acid precipitable factor, but our evidence suggests that it is some enzyme or enzyme system other than phycocyanin (Fig. 6). None of our reaction systems, even with the purified extract, were entirely free of
phycocyanin. The reconstituted Hill reaction may have been dependent on residual phycocyanin present in both the washed lamellar fraction and the partially purified extracts. Even if a Hill reaction were possible in the complete absence of phycocyanin, it might be dependent on the presence and functioning of the "form" of chlorophyll a with peak absorption at 670 rag, found in many plants (29-32). This altered chlorophyll functions in the same way as accessory pigments both for complete photosynthesis and for the Hill reaction, as shown by Govindjee and Rabinowitch (32, 33). The active component described here could be functioning either in oxygen evolving steps, or in electron transport to the Hill oxidant. Since reconstitution appeared to be relatively specific for ferricyanide as the electron acceptor, the latter seems to be the more likely site of action. To our knowledge, this is the first report of an extractable protein required in the Hill reaction with ferricyanide as the electron acceptor. ACKNOWLEDGMENT The photosynthetic pyridine nuceotide reductase and transhydrogenase were the kind gift of Dr. D. Keister. REFERENCES 1. MCCLENDON, J. H., AND BLINKS, R., Nature 170, 577 (1952). 2. THOMAS,J. B., AND D~ROVER, W., Biochim. et Biophys. Acta 16, 391 (1955). 3. "VAN BAALEN, C., Ph.D. dissertation, The University of Texas, pp. 1-52 (1957). 4. PETRACK,B., Federation Proc. 18, 302 (1959). 5. PETRACK, B., AND LIPMANN, F., in "Life and Light" (W. McElroy and B. Glass, eds.). Johns Hopkins Press, Baltimore, Maryland, 1960. 6. KRATZ, A., AND MYERS, J., Am. J. Bot. 42, 282 (1955). 7. BRODY, M., AND EMERSON, R., Am. J. Bol. 46, 433 (1959). 8. CLENDENNINO, K. A., BROWN, T. E., AND WALLDOV, E. E., Physiol. Plantarum 9,
519 (1956). 9. ARNON, D. I., Plant Physiol. 24, 1 (1949). 10. LownY, O. H., ROSESROUO~,N. J., ANDFARR, A. L., J. Biol. Chem. 193, 265 (1951). 11. DUYSENS,L. N. M., AND SWEEP, G., Biochim. et Biophys. Acta 25, 12 (1957).
HILL REACTION IN ANACYSTIS IVN PIETRO, A., AND LANG, H. M., J. Biol. hem. 231, 211 (1958). ARK, L. C., Trans. Am. Soc. Artificial IInternal Organs 2, 41 (1956). rAVB, N. C., J. Appl. Physiol. 16,192 (1961). *AVIES, P. W., in "Physical Techniques in Biological Research" (W. L. Nastuk, ed.), Vol. IV, pp. 137-179. Academic Press, New ork, 1962. andbook of Chemistry and Physics," 38th Edition. (D. D. Hodgman, R. C. Weast and S. M. Selby, eds.), p. 1608. Chemical Rubber Publishing Co., Cleveland, Ohio, 1956. 'AUSSKY, H. H., AND SI-IORR, E., Jr. Biol. Chem. 202, 675 (1953). !INDBERG, O., AND ERNSTER, L., in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. I I I , pp. 1-22. Wiley (Interscience), New York, 1956. IRANE, R. •., AND LIPMANN, F., J. Biol. Chem. 201, 235 (1953). IARBURG, O., "Heavy Metal Prosthetic Groups and Enzyme Action," p. 213. Oxford Univ. Press, Amen House, London, 1949. LRNON, E. I., Nature 184, 10 (1959).
~
49
22. HATTORI, A., AND FUJITA, Y., J. Biochem. 46, 633 (1959). 23. BANNISTER, T. T., Arch. Bioehem. Biophys. 49, 222 (1954). 24. BERGERON, J. A., AND SMILLIE, R. M., Plant Physiol. Suppl. 36, xlviii (1961). 25. KROGMANN, D. W., AND JAGENDORF, A. T., Plant Physiol. 34, 277-282 (1959). 26. LOSADA, M., WHATLEY, F. R., AND ARNON, D. I., Nature 190, 607-610 (1961). 27. KEISTER, D. L., SAN PIETRO, A., AND STOLZENBACH, F., J. Biol. Chem. 235, 2989 (1960). 28. BLACK, C. C., FEWSON, C. A., AND GIBES, M., Nature 198, 88 (1963). 29. FRENCH, C. S., in "Life and Light" (W. McE1roy and B. Glass, eds.), pp. 447-474. Johns Hopkins Press, Baltimore, Maryland, 1960. 30. MYERS, J., AND FRENCH, C. S., J. Gen. Physiol. 43, 723 (1960). 31. GOVINDJEE, R., AND RABINOWITCH,E., Science 132, 355 (1960). 32. GOVINDJEE, R., AND RABINOWITCH, E., Biophys. J. 1,377 (1961). 33. GOVINDJEE, R., THOMAS, J. B., AND RABINOWITCH, E., Science 132, 421 (1960).
(1964)
A Soluble Component of the Hill Reaction in Anacystis nidulans 1"2's W A L T E R W. F R E D R I C K S 4 AND A N D R E T. J A G E N D O R F From the McCollum-Pratt Institute and Biology Department, The Johns Hopkins University, Baltimore Maryland Received May 20. 1963 Cell-free preparations (fragments of photosynthetic lamellae) from Anacystis nidulans catalyze the Hill reaction using ferricyanide, indophenol dyes or cytochrome c as electron acceptor. A high molecular weight polymer (Carbowax or dextran) not only must be present during preparation of the particles to preserve Hill reaction activity, but gives a marked stimulation if present in the reaction mixture. Calcium salts also are stimulatory. Sodium or potassium salts were inhibitory, as were all buffer anions that were tested. Extraction of the cell-free particles by water leads to a loss of their phycocyanin and of the Hill reaction activity. With ferricyanide as electron acceptor, adding back the extract led to a reconstitution of photolysis by washed particles. The active component of the extract was partially purified and was found not to be identical with phycocyanin. Information is reported concerning the presence of an endogenous reducing material in the cell-free extracts. INTRODUCTION The accessory pigment, phycoeyanin, of the blue-green algae is dffierent from that of higher plants, chlorophyll b, in that the former is water soluble. Because of this, the bhJe-green algae lend themselves to the study of the function of the accessory pigment by some of the more traditional biochemical procedures. Cell-free preparations of red (1) and bluegreen algae (2, 3) have been shown to catalyze the Hill reaction provided certain 1 Contribution number 406 from the McCollure-Pratt Institute. 2 These studies constitute part of the dissertation submitted by W. Fredricks in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This investigation was supported in part by a pre-doctoral fellowship (HF-9259-C1) from the National Heart Institute, U. S. Public Health Service and by a research ~rant (RG-3923) from the National Institutes of Health, U. S. Public Health Service. 4 Present address: National Heart Institute, National Institutes of Health, Bethesda 14, Md.
precautions are taken in the preparation of the material. Of particular importance is the presence in the isolation medium of a high molecular weight polymer (e.g., Carbowax or dextran). Should this polymer be deleted, the accessory pigments are extracted from the particles and the Hill activity is immediately lost. I t is, therefore, tempting to infer that the loss of Hill activity is a direct consequence of the extraction of the accessory pigment. Thomas and DeRover (2) offered evidence which supported this idea but added, "though the experiments make it unlikely that the activity loss should be attributed to the release of enzyme systems, the possibility of a linkage of these systems to the phycocyanin molecules themselves cannot be excluded with certainty." Petrack (4) and Petrack and Lipmann (5) observed that cell-free preparations of Anabaena (a blue-green alga) catalyze "cyclic photophosphorylation." However, these preparations were made in the absence of any polymer and were essentially free of phycocyanin. 39
40
FREDRICKS AND JAGENDORF
These facts suggested to us a possible e x p l a n a t i o n of the E m e r s o n e n h a n c e m e n t effect, viz., light absorbed b y chlorophyll a is sufficient for "cyclic p h o t o p h o s p h o r y l a t i o n , " b u t a d d i t i o n a l a b s o r p t i o n b y a a accessory p i g m e n t is necessary for oxygen e v o l u t i o n . T o s u p p o r t this hypothesis it would be necessary to establish t h a t the loss of Hill a c t i v i t y is due to the loss of p h y c o c y a n i n a n d n o t some other c o m p o n e n t extracted a t the same time. This p a p e r reports the characteristics of the Hill reaction in cell-free p r e p a r a t i o n s from Anacystis nidulans, a blue-green alga. A q u e o u s e x t r a c t i o n of the active particles leads to a loss of this a c t i v i t y . I t was f o u n d possible to r e c o n s t i t u t e at least a p a r t of the a c t i v i t y b y a d d i n g back the extract, a n d accordingly a n i n v e s t i g a t i o n was m a d e into the relationship b e t w e e n the factor(s) active i n r e c o n s t i t u t i o n a n d the p h y c o c y a n i n comp o n e n t of the extract. MATERIALS AND METHODS The organism used in this study was the unicellular blue-green alga, Anacystis nidulans, of the family Chroococcaceae. The cultures were kindly supplied by Dr. B. Kok, RIAS, Inc., Baltimore, Md. The algae were cultured in l-liter Roux culture bottles containing 500 ml. ; or in 12liter carboys containing 9 liters of Medium C of Kratz and Myers (6) with the following modifications: the micronutrient solution was essentially that proposed by Brody and Emerson (7) and 5 ml. of a 10% Na~CO3 solution per liter of medium was added to bring the pH to about 8 to 9. In addition several marble chips were added to each bottle to help maintain the alkaline pH. No attempt was made to redissolve the precipitate formed on autoclaving the medium. In the case of the small volume cultures, the temperature was maintained at 39 =t= I~ Continuous illumination was provided by three Sylvania Daylight fluorescent lights (F20T12D) at a distance of about 12 cm. from the cultures. Each culture was aerated at a rate of 0.50-0.75 liters per minute. The air was cleaned by passage through concentrated H2SO4 and humidified by passage through water. Under these conditions the cultures yielded about 2.5 g. of cells (wet weight) per liter after 5-7 days growth. In the case of the large volume cultures the temperature was maintained at 38~ in a Percival Plant Growth Chamber. Continuous illumination was provided at about 500 foot-candles from Daylight fluorescent lights. As the cultures
became more dense additional light was supplied. Aeration was essentially that described for the small volume cultures, except at a proportionally higher rate. Stock cultures were maintained on slants of the above medium solidified by 1.5% agar. PREPARATION OF CELL EXTRACTS Cells from 2 liters of culture (about 5 g. wet weight) were harvested by centrifugation. They were suspended in 20 ml. of grinding medium, and 16 g. of glass powder (about 200 mesh) was added with stirring. The grinding medium consisted of a solution 30% in Carbowax 4000, 0.02 M in Tris ~ and 0.001 M in EDTA adjusted to pH 7.2. The suspension was blended in an Omnimixer for 5 minutes. An additional 20 ml. of grinding medium was added, followed by 2 minutes agitation. The homogenate was centrifuged for 10 minutes at 7710g to remove glass powder and whole cells. The supernatant fluid was centrifuged for 21/~ hours at 105,0009. Approximately 90% of the chlorophyll was sedimented during this operation. The supernatant fluid was discarded, and the residue which consisted of particles containing both chlorophyll and phycocyanin was stored frozen until use. This residue is designated as lamellar fragments. All operations were carried out from 0~ to 5 ~ For assay of Hill activity a portion of the residue was thawed and suspended in a small volume of a solution 20% in Carbowax 4000, 0.02 M in Tris, and 0.001 M in EDTA adjusted to pH 5.8. This Carbowax concentration is near the lower limit necessary for retention of Hill activity, e.g., suspension in 15% Carbowax results in decreased activity. The suspension was frozen and thawed to yield a very fine dispersion of particles. Both residue and resuspended material were very stable and could be kept frozen for weeks without loss of activity. They are also stable to repeated freezing and thawing. (For an extensive study of the stabilizing properties of Carbowax 4000 cf. Clendenning et al. (8), 1956.) Lamellar fragments catalyzed the reduction of electron accepters at a rate of between 80 and 150 microequivalents per milligram chlorophyll per hour. Some preparations exhibited rates as low as 30 or as high as 200. 5Abbreviations used: Tris, tris (hydroxymethyl) aminomethane; TPN, triphosphopyridine nucleotide; DCPIP, 2,6-dichlorophenolindophenol; TCPIP, 2,3',6 trichlorophenolindophenol; EDTA, ethylenediaminetetraacetate; DEAEcellulose, diethylaminoethylcellu!ose; PPNR, photosynthetic pyridine nucleotide reductase; ATP, adenosine triphosphate.
HILL REACTION IN ANACYSTIS Several attempts were made to improve the activity of the material by different preparative methods. None of the variations, including the addition of ascorbate or 2-mercapto-ethanol or grinding with alumina instead of glass powder, led to particles with increased activity. To prepare washed lamellar fragments, lamellar fragments equivalent to 2 liters of culture were extracted with 20-30 ml. of cold, deionized water. The suspension was centrifuged for 2 hours at 105,000g; the resulting supernatant fluid is designated as the crude extract. The residue was washed by centrifugation with approximately 60 ml. of cold, deionized water. The final residue contained chlorophyll but was free of phycocyanin. ANALYTICAL PROCEDURES AND ACTIVITY MEASUREMENTS Chlorophyll was measured by the method of Arnon (9), protein was determined according to Lowry et al. (10), and phycocyanin by the optical density at 620 rag. I n spectrophotometric assays of the Hill reaction, euvettes were exposed to light filtered through 10 cm. of water at room temperature, and the optical density of the particular dye was measured before and after illumination. The change in optical density was corrected for any dark reduction of the oxidants. The following oxidants were used, and their reduction assayed at the wavelengths noted: KaFe(CN)8 (420 mg), D C P I P (600 rag), T C P I P (620 mtt), and cytochrome c (550 m#). The millimolar extinction coefficients used to calculate concentrations are: K~Fe(CN)r (1), D C P I P , pH 6.5 (19.1), T C P I P (t8), and cytochrome e, reduced minus oxidized (20). Despite evidence in the literature for photoreduction of pyridine nucleotides in Anacystis in vivo (11) no reduction of T P N could be demonstrated with lamellar fragments or washed lamellar fragments, even with added spinach P P N R (12). Most reactions reported here involved the direct determination of oxygen evolution with the aid of a Clark-type oxygen electrode (13) in a closed reaction vessel without a gas phase (construction and circuit details available upon request). A linear response to oxygen concentration was assumed (see references 14 and 15), and the instrument was calibrated for two oxygen concentrations: that in water in equilibrium with air, and zero oxygen as achieved by the addition of hydrosulfite. A cMibration curve (concentration of oxygen in water at equilibrium with air as a function of temperature) was calculated from available data (16).
41
In the standard assay ferricyanide was the oxidant. The reaction mixture contained, in a final volume of 3 ml., 70 ~moles of NaCl, 5 gmoles of K3Fe(CN)6, 30 ~moles of maleate or phosphate buffer at pH 5.8, 150 or 180 rag. of Carbowax 4000, 10 or 20 ~moles of CaCI~, and lamellar fragments or washed lamellar fragments containing about 200 ~g. of chlorophyll. Light from a 100 watt Bausch and Lomb microscope lamp was filtered through 6 em. of water. Each reaction mixture was allowed 7 minutes to come to temperature equilibration at 35 ~ and recording was started 2 minutes prior to illumination. Oxygen evolution was linear with time over the period of measurement (5 minutes). The usual control contained all components except ferricyanide, and dark controls gave the same values. Rates are reported as microequivalents of oxidant reduced per milligram chlorophyll per hour. Oxygen evolution as measured with the electrode was found to be stoichiometric to ferricyanide reduction as measured with the spectrophotometer, with the expected ratio of 4 ~moles of ferricyanide reduced for each ftmole of oxygen evolved. In chromatography on DEAE-cellulose approximately 100 mg. of protein, dissolved in 0.02 M phosphate buffer, pH 7.0, was adsorbed to a 2 X 12 era. column previously washed with the same buffer. Elution was by a 200 ml. linear KC1 gradient, from 0.0 to 0.5 M, in 0.02 M phosphate, pI-I 7.0. Carbowax 4000 (a polyethylene glycol of approx. 3600 molecular weight) was obtained from the Union Carbide and Chemical Corp. Clinical grade dextran (a branched glucose polymer having a molecular weight of 60,000-90,000) was from the Nutritional Biochemicals Corporation. I t should be noted that attempts to measure photophosphorylation in the present study were completely foiled by the presence of Carbowax in the reaction mixtures. I t was found t h a t Carbowax interferes with (a) various modifications of the inorganic phosphate determination of Taussky and Shorr (17), (b) separation of inorganic from organic phosphates by the method of Lindberg and Ernster (18), and (c) adsorption of A T P onto Norite (19). In the latter case as little as 0.6% Carbowax caused complete inhibition, with partial interference at 0.1%. Dextran did not cause the same interference with the analytical methods for phosphate. RESULTS CHARACTERISTICS OF THE HILL REACTION WITH LAMELLAR FRAGMENTS In the standard reaction using ferricyanide as o x i d a n t , t h e r a t e of o x y g e n e v o l u t i o n w a s
42
FREDRICKS AND JAGENDORF
other experiments using either chloride, sulfate, or nitrate salts of the divalent metals. It can be seen (Table I) that, in the abFsence of CaC12, low concentrations of NaC1 o 0.25 -3 were stimulatory but higher concentrations inhibitory. Only inhibition was seen in the ,a) 0 . 2 0 presence of CaC12. Almost identical results 7 were obtained with KC1 in place of NaC1. It is not clear whether the stimulation was a LU 0.15 partial replacement of calcium by sodium, 0 > or a chloride effect similar to that reported L~ by Warburg (20) and Arnon (21). ON O. JO In experiments with buffer anions, the buffer to be studied was varied over a cong~ centration range in the presence of a minimal ~L 0 . 0 5 but constant level of a second buffer necessary to control the pH. Figure 2c illustrates that all the anions tested inhibit the Hill I I I O* 0 I00 200 300 reaction to some degree. Similar effects were ,u.g. C H L O R O P H Y L L found when the reactions were run in the F1o. I. Chlorophyll concentration series. absence of CaC12. Of particular interest are Standard assay, except that the Carbowax con- the curves for phosphate and for citrate, centration was 15%. where the quantitative effect negates the entire amount of simulation due to calcium. a linear function of chlorophyll concentra- However, the per cent inhibition by a given tion from 0 to 200 #g. of chlorophyll (Fig. 1). concentration of citrate was the same at all At higher concentrations light became limit- calcium concentrations; in other words adding, and there was little change in rate with ing extra calcium did not reverse the citrate increasing chlorophyll concentration. The inhibition. Consistent with the idea that pH curve shows an optimum centered this is an inhibition by the anion is the fact around p H 5.8, falling to very low values at that Tris hydroehloride showed only a 13 % p H 8. This was true also for the reconstituted inhibition at a concentration as high as system (see below). The p H optima for the 0.055 M. T C P I P Hill reaction was 6.5 and for the In the absence of calcium, increasing cytochrome c Hill reaction, 7.5. concentrations of E D T A gave increasing As shown in Fig. 2a, addition of Carbo- amounts of inhibition up to 0.001 M (Fig. wax to the reaction mixture stimulated the 2d). In the presence of calcium, E D T A inferricyanide Hill reaction up to an optimal hibition increased progressively at concenlevel, after which inhibition set in due to trations even higher than 0.001 M. At the excess polymer. These effects were also seen highest concentration of E D T A only about using T C P I P as the electron acceptor. A half the calcium would be complexed, so similar phenomenon occurs when dextran that the inhibition cannot be due simply to was the polymer used, although neither the a removal of calcium from the lamellar stimulation nor the inhibition was as pro- fragments. From the data in Fig. 2b it can nounced in this case. It can be noted that be seen that the rate was little affected by a the concentration of Carbowax used for change in calcium concentration in this preparation of active particles (30%) was range (between 4 and 7 raM). These results inhibitory when present in the reaction mixsuggest that the calcium-EDTA complex ture. Figure 2b shows that calcium chloride, was more inhibitory than E D T A alone, and to a lesser extent magnesium chloride, since E D T A inhibition was the most prostimulated the reaction rate when present at nounced in the presence of high levels of 0.01 M. Identical stimulations were seen in calcium. 0.30
0
'0
80
t/l
60
' ~/
i/
W I-.<~ n-
\\
,20~.
(c)
40
\
W I-,,:r n,-
%.
20
48
24
I
O
~ 20
I
I 40
I 60
CONCENTRATION OF BUFFER(raM) 0
f
I
0
I
I,
I0
I
I
20
CONCENTRATION
I00
50
OF CARBOWAX
150
(d)
(%) (b) 80
120
90
40~
60
20
50
0
I
0
I IO
]
CONCENTRATION
I 20
I
OF S A L T
I 50-
0
0
(mM)
I I
CONCENTRATION
J 2
OF
EDTA
5
(mM)
FIG. 2. The effects of various reaction components on t h e rate of t h e Hill reaction with lamellar fragments. (a) Effect of Carbowax at two concentrations of chlorophyll. S t a n d a r d assay: O O 49 ~g. chlorophyll; 0 - - - 0 185 ~g. chlorophyll. (b) Effect of MgCI~ and CaC12. S t a n d a r d assay: 0 - - 0 CaC12; O O MgC12. T h e point at 0 m M salt is t h e average of t h r e e experiments. (c) Effect of various buffers. S t a n d a r d assay in t h e presence of 6.67 m M CaCl~. T h e p H control in the case of t h e maleate and citrate curves was provided b y a minimal level of phosphate, pH 5.8. I n t h e case of the p h o s p h a t e curve pH control was provided by a minimal level of maleate, p H 5.8. O phosphate, pH 5.8; 9 maleate, pH 5.8; citrate, p H 5.8. (d) Effect of calcium a n d E D T A . S t a n d a r d assay; O 6.67 m M CaC12; 9 no CaC12. R a t e m e a s u r e m e n t s were m a d e b y following oxygen evolution a n d are expressed as microequivalents of oxidant reduced per milligram chlorophyll per hour. 43
44
FREDRICKS AND JAGENDORF RESOLUTION
AND RECONSTITUTION
THE
HILL
OF
REACTION
After extraction of lamellar fragments with water (see Materials and Methods) the Hill reaction activity was reduced to 10 % or less of the original rate. This was true whether ferricyanide, cytochrome c or the indophenol dyes were the oxidants used. The washing procedure which resulted in TABLE I E F F E C T OF NaC1 ON THE H I L L REACTION OF LAMELLAR FRAGMENTS IN THE PRESENCE OR ABSENCE OF CaC12 #moles NaCl added to 3 ml. reaction mixture
Rate a
0 35 70
105 140 350 700 98O
No/CaCI2
6.67 mM CaCI2
13.0 29.1 35.5 39.4 40.3 41.3 35.5 30.4
81.0 84.2 82.9 75.7 80.6 71.7 53.4 47.0
Rates are given as microequivalents of oxidant reduced per milligram chlorophyll per hour.
the loss of Hill activity produced an intense blue extract with red fluorescence characteristic of phycocyanin. Figure 3 shows the spectrum of this extract. Phycocyanin accounts entirely for the absorption at 620 and 350 m~ and is the major contributor to the 280 mu absorption (22). The shoulder at about 410 m~ and the absorption at 265 m~ can possibly be attributed to pteridines (3). Reactivation of the Hill reaction with ferricyanide by increasing concentrations of the above extract is shown in Fig. 4. I n this experiment the extracted lamellar fragments had a rate 9 % t h a t of the original, and with an optimal concentration of added extract 36 % of the original activity was obtained. With occasional preparations of lamellar fragments whose activity was abnormally low to begin with, complete reactivation could be seen after extraction. No added reactivation was obtained b y preincubating particles and extract for various times or at various Carbowax concentrations. Reactivation could not be ascribed to Carbowax in the extract because an optimal amount of Carbowax was added to each reaction mixture regardless of the presence or absence of extract.
1.0 _
Crude e x t r o c t
0.8
~ 0.6 ._1 "~ 0 . 4 f.D
0
0.2
0 22(
300
400
500
600
700
WAVELENGTH ( m/J.) Fi~. 3. Spectrum of crude extract. Lamellar fragments prepared in a buffered Carbowax solution (see Materials and Methods) was extracted with cold, distilled water. The mixture was centrifuged at 105,000g, and the supernatant fluid constituted the crude extract.
HILL REACTION IN ANACYSTIS
45
40
24 30
21
18
t~ 20
r~
15
i'
10
o 0
I
!
]
L
I
]
0.3
0.6
0.9
1.2
i.5
1.8
EXTRACT
ADDED
21
(ml)
FIG. 4. Effect of extract concentration on the Hill reaction of washed lamellar fragments. Standard assay; rates are expressed as microequivalents of oxidant reduced per milligram chlorophyll per hour. Using D C P I P or T C P I P as electron acceptor, the extent of reactivation was usually negligible. Cytochrome c could not be tested, because it caused the extracted lamellar fragments to precipitate under the conditions of reconstitution. Figure 4 shows a definite o p t i m u m curve for reaction rate vs. extract concentration (i.e., inhibition at higher concentrations). The inhibitory effect was even more pronounced with the original lamellar fragments, where the concentration of extract which maximally reactivated washed fragments was actually inhibitory. Optimal reactivation of washed lamellar fragments was provided by an amount of extract very nearly the same as that present in the same a m o u n t of particles prior to extraction. Carbowax had about the same effect on the residual activity of washed lamellar fragments (Fig. 5) as it did on the original, unextracted fragments (Fig. 2a). The situation was a little more complex with the rereconstituted system, in t h a t the reactivation due to added extract (about twofold) was constant from 0 to 12 % Carbowax, but falls to zero at 25 % Carbowax. The salt effects were changed somewhat in the extracted and reconstituted system (Table I I ) . Calcium was still stimulatory
-3 0
4
8
CONCENTRATION
IZ
~ro
20
24
OF CARBOWAX(%)
FIG. 5. Effect of Carbowax concentration on the Hill reaction of washed lamellar fragments arid the reconstituted system. Standard assay with the addition of 0.5 ml. of crude extract for the reconstituted system: 9 reconstituted system; O washed lamellar fragments; [] enhancement (rate of reconstituted system minus rate of washed lamellar fragments). Rates are expressed as microequivalents of oxidant reduced per milligram chlorophyll per hour. but KC1 had essentially no effect (in the presence or absence of calcium), and NaC1 is stimulatory under all circumstances. The stimulation by NaC1 seen here was additive with t h a t of CaC12. Enhancement activity, simply the difference between lines a and b of Table II, was thus best seen in the presence of a mixture of sodium and calcium chloride. CHARACTERISTICS
OF
THE
ACTIVE
COMPONENT
The active component in the water extracts was precipitated at p H 4.8 or b y amraonium sulfate, and could be adsorbed to and eluted from calcium phosphate gel. I t moved as a large molecule in a Sephadex G-25 column, and it is nondialyzable. In addition, Table I I I shows t h a t it is heat
46
FREDRICKS AND JAGENDORF TABLE II
4.0
REACTION
OF WASHED FRAGMENTS
Added extract
+Ca
}
LAMELLAR a
No monovalent salt - Ca
160
9\
EFFECT OF VARIOUS SALTS ON THE HILL
.2
NaCI - Ca
t28
2
KCI
+Ca
o
- Ca+Ca -E 2 . 4
(a) (b) +
0 --6
18 54
12 30
42 84
0 5
18 44
w
(b) - (a)
-6
36
18
42
5
26
~_ ,.6 E
-
-
a This experiment was done with the standard assay except for the addition of 100 ttmoles of NaC1 or KC1 and 20 ttmoles of CaC12 where indicated. The data are expressed as corrected changes in current (muamps)/5 minutes illumination.
OF HEATING OF THE
EXTRACT HILL
ON RECONSTITUTION
REACTION
a
Rate Lamellar fragments Washed lamellar fragments Washed lamellar fragments extract Washed lamellar fragments heated extractb
plus
155 7 56
plus
15
a S t a n d a r d a s s a y e x c e p t for t h e a d d i t i o n of 1 m l . of d i a l y z e d c r u d e e x t r a c t w h e r e i n d i c a t e d . b H e a t e d for 10 m i n u t e s a t 60 ~
labile, losing about 75% of its activity in 10 minutes at 60 ~ These characteristics are consistent with the idea t h a t the active component in the aqueous extracts is a protein. On tile initial assumption that the active component might be phycocyanin, relatively pure phycocyanin was prepared as estimated by the ratio of absorbancy at 620/280 mg (22, 23). This purified phycocyanin did not reactivate washed lamellar fragments. Others a t t e m p t s at fractionation indicated that when the activity was recovered it was not necessarily found in the fraction richest in phycocyanin. This was particularly true in the case of DEAE-cellulose column chromatography using stepwise elution. Here it was found t h a t 70 % of the activity was found
C Z
64 ~
o r
0.8
32
z z X
3 0 18
I
1
19
20
I
I
I
1
I
21
2~
23
24
25
TUBE
TABLE I I I ~]FFECT
96
0 26
NUMBER
FIG. 6. DEAE-cellulose chromatography of extract. Standard assay with 0.5 ml. of extract: 0 protein (mg./ml.); 9 phycocyanin (units/ml.); [] reactivation activity (arbitrary units/ml.). in a fraction containing only 16 % of the original phycoeyanin, while 20 % of the activity was associated with the fraction containing 70 % of the phyeocyanin. The most significant fractionation was achieved on a DEAE-cellulose column with a linear KC1 gradient elution (see Materials and Methods). Figure 6 shows t h a t portion of one chromatogram where the active component was eluted. There is only one peak (tube 18) in the protein profile, which is followed almost point by point by the phycocyanin concentration curve. On the other hand, activity was recovered primarily in tubes 21 to 25. The fact that only one protein peak appears is not too surprising since the phycocyanin represents about 30 % of the total cell protein (22), and the lamellar fragments from which the extract is obtained have already been removed from other cytoplasmic proteins. Generally close to 100 % of the activity could be recovered from the D E A E fractions. Pooling fractions which contained the largest amount of activity (tubes 21 to 25) gave a preparation purified between 5- and 8-fold, in different experiments.
HILL REACTION IN ANACYSTIS TABLE IV RESOLUTION AND RECONSTITUTION ~REDuC1NG MATEtlIAL a~
47
DISCUSSION OF
Lamellar fragments from Anacystis differ in a number of respects from chloroplasts of higher plants, with respect to their Hill Optical density Additions decrease, reaction activity. Besides the previously 580 rr~ noted requirement for a concentrated polymer solution during homogenization and None 0 isolation (1-3), there are a number of ionic Washed lamellar fragments 0.032 Extract 0.047 effects which appear to be unique for the Washed lamellar fragments p l u s 0.272 present system: in particular the marked extract stimulation by calcium ions (Fig. 2b), inWashed lamellar fragments p l u s 0.234 hibition by the cMeium-EDTA complex heated extract b (Fig. 2d), or by very ordinary buffer anions a Each reaction mixture contained in a final (Fig. 2c). In addition the presence of the volume of 3 ml.; 600 t~moles of phosphate buffer, polymer during the Hill reaction is strongly pH 6.5, TCPIP, and where indicated washed stimulatory (Fig. 2a), a phenomenon not lamellar fragments equivalent to 180 t*g. of chloro- noticed with higher plant chloroplasts. It phyll and/or 1 ml. extract (about 4 rag. protein). should be noted that the concentration of bHeated for 10 minutes at 60~ Carbowax needed for best results during the preparation of lamellar fragments is inhibiDARK REDUCTION OF HILL OXIDANTS tory if present in the reaction mixture. Many of these differences from higher When using spectrophotometric assays, it plant chloroplasts are undoubtedly due to was observed that lamellar fragments cause a rapid dark reduction of added oxidants, the differences in physical structure. The presumably because of the presence of endog- photosynthetic apparatus of the blue-green enous reducing compounds. Van Baalen algae is seen to consist of a series of lamellae (3) also reported this observation for cell-free running around the outer edge of the cell preparations of Anacystis. After extraction and enclosing the other cell contents (24) of the lamellar fragments, neither the washed rather than being concentrated in discrete particles nor the extract reduced added plastids. Biocbemieally the present iamellar fragTCPIP to any great extent (Table IV). ments differ from the usual chloroplasts in Surprisingly, the two in combination caused being unable to reduce TPN, and in showing a considerable reduction of dye. This reduca more vigorous reduction of ferricyanide tion is virtually instantaneous and no further than of the indophenol dyes. The concept reaction is seen with time. Heating the exthat reduction of indophenol dyes occurs tract sufficiently to cause loss of reconstitu- by a shorter and simpler series of reactions, tion activity has no effect on the dark reduc- than does the reduction of ferricyanide detion phenomenon (Table IV). veloped from studies of spinach chloroplasts In other experiments one component was (25, 26), may not be true for this system. held constant and the amount of the other The reason for the failure to reduce TPN was varied. In both cases the amount of dark is not apparent from the experiments so reduction was a linear function of the com- far undertaken, since the addition of both ponent being varied, whether it was the PPNR (12) and transhydrogena~e (27) amount of washed lamellar fragments or the failed to restore activity. This is especially amount of extract. It should also be noted surprising because the in vivo photoreduction of pyridine nucleotides by Anacystis has been that the material in the extract responsible reported by Duysens et al. (11)and in vitro for dark dye reduction is nondialyzable. The by Van Baalen (3) and by Black et al. (28). relation between this reduction of dyes in the As would be anticipated from the work of dark and the light-dependent Hill reaction is McClendon and Blinks (1) and Thomas entirely unknown. and DeRover (2), aqueous extraction of the
48
FREDRICKS AND JAGENDORF
lamellar fragments resulted in the loss of Hill activity for all oxidants tested. A partial reconstitution was observed in the case of ferricyanide as the Hill oxidant, but not with any of the other electron acceptors. T h e degree of reactivation was variable, running up to 45 % of the original activity of the lamellar fragments but usually being 20-30 %. The unsupplemented washed lamellar fragments showed approximately 5-10 % of the original activity. The fact that there is inhibition at higher concentrations of extract (Fig. 4) possibly is one factor in the failure to demonstrate full reactivation. Another factor may be some degree of irreversible physical damage to the lamellar fragments; this might also account for the variability in the extent of reactivation. This is suggested by the differences between lamellar fragments and washed lamellar fragments with respect to stability and the facility for being resuspended. The washed lamellar fragments are only stable when kept at 0 ~ (not frozen) as pellets, and they cannot be obtained as a fine dispersion (even with freeze-thaw treatments) in the "resuspending medium" used for the lamellar fragments. T h e y can, however, be suspended readily in water. Several authors have observed a simultaneous loss of Hill activity and of phycobilin pigments when cell-free material from red (1) and blue-green (2, 3) algae are extracted by buffers in the absence of polymers. On the other hand, loss of phycobilins did not result in a loss of the ability to catalyze cyclic electron flow and phosphorylation b y particles of blue-green algae (4, 5). These facts suggest that the phycobilins are specifically concerned in the oxidation of water and evolution of oxygen in the Hill reaction. Our own data tend to support this hypothesis only up to a point. Loss of activity is correlated with loss of the phycobilin pigment in our experiments. Restoration of activity depends on a non-dialyzable, heatlabile, acid precipitable factor, but our evidence suggests that it is some enzyme or enzyme system other than phycocyanin (Fig. 6). None of our reaction systems, even with the purified extract, were entirely free of
phycocyanin. The reconstituted Hill reaction may have been dependent on residual phycocyanin present in both the washed lamellar fraction and the partially purified extracts. Even if a Hill reaction were possible in the complete absence of phycocyanin, it might be dependent on the presence and functioning of the "form" of chlorophyll a with peak absorption at 670 rag, found in many plants (29-32). This altered chlorophyll functions in the same way as accessory pigments both for complete photosynthesis and for the Hill reaction, as shown by Govindjee and Rabinowitch (32, 33). The active component described here could be functioning either in oxygen evolving steps, or in electron transport to the Hill oxidant. Since reconstitution appeared to be relatively specific for ferricyanide as the electron acceptor, the latter seems to be the more likely site of action. To our knowledge, this is the first report of an extractable protein required in the Hill reaction with ferricyanide as the electron acceptor. ACKNOWLEDGMENT The photosynthetic pyridine nuceotide reductase and transhydrogenase were the kind gift of Dr. D. Keister. REFERENCES 1. MCCLENDON, J. H., AND BLINKS, R., Nature 170, 577 (1952). 2. THOMAS,J. B., AND D~ROVER, W., Biochim. et Biophys. Acta 16, 391 (1955). 3. "VAN BAALEN, C., Ph.D. dissertation, The University of Texas, pp. 1-52 (1957). 4. PETRACK,B., Federation Proc. 18, 302 (1959). 5. PETRACK, B., AND LIPMANN, F., in "Life and Light" (W. McElroy and B. Glass, eds.). Johns Hopkins Press, Baltimore, Maryland, 1960. 6. KRATZ, A., AND MYERS, J., Am. J. Bot. 42, 282 (1955). 7. BRODY, M., AND EMERSON, R., Am. J. Bol. 46, 433 (1959). 8. CLENDENNINO, K. A., BROWN, T. E., AND WALLDOV, E. E., Physiol. Plantarum 9,
519 (1956). 9. ARNON, D. I., Plant Physiol. 24, 1 (1949). 10. LownY, O. H., ROSESROUO~,N. J., ANDFARR, A. L., J. Biol. Chem. 193, 265 (1951). 11. DUYSENS,L. N. M., AND SWEEP, G., Biochim. et Biophys. Acta 25, 12 (1957).
HILL REACTION IN ANACYSTIS IVN PIETRO, A., AND LANG, H. M., J. Biol. hem. 231, 211 (1958). ARK, L. C., Trans. Am. Soc. Artificial IInternal Organs 2, 41 (1956). rAVB, N. C., J. Appl. Physiol. 16,192 (1961). *AVIES, P. W., in "Physical Techniques in Biological Research" (W. L. Nastuk, ed.), Vol. IV, pp. 137-179. Academic Press, New ork, 1962. andbook of Chemistry and Physics," 38th Edition. (D. D. Hodgman, R. C. Weast and S. M. Selby, eds.), p. 1608. Chemical Rubber Publishing Co., Cleveland, Ohio, 1956. 'AUSSKY, H. H., AND SI-IORR, E., Jr. Biol. Chem. 202, 675 (1953). !INDBERG, O., AND ERNSTER, L., in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. I I I , pp. 1-22. Wiley (Interscience), New York, 1956. IRANE, R. •., AND LIPMANN, F., J. Biol. Chem. 201, 235 (1953). IARBURG, O., "Heavy Metal Prosthetic Groups and Enzyme Action," p. 213. Oxford Univ. Press, Amen House, London, 1949. LRNON, E. I., Nature 184, 10 (1959).
~
49
22. HATTORI, A., AND FUJITA, Y., J. Biochem. 46, 633 (1959). 23. BANNISTER, T. T., Arch. Bioehem. Biophys. 49, 222 (1954). 24. BERGERON, J. A., AND SMILLIE, R. M., Plant Physiol. Suppl. 36, xlviii (1961). 25. KROGMANN, D. W., AND JAGENDORF, A. T., Plant Physiol. 34, 277-282 (1959). 26. LOSADA, M., WHATLEY, F. R., AND ARNON, D. I., Nature 190, 607-610 (1961). 27. KEISTER, D. L., SAN PIETRO, A., AND STOLZENBACH, F., J. Biol. Chem. 235, 2989 (1960). 28. BLACK, C. C., FEWSON, C. A., AND GIBES, M., Nature 198, 88 (1963). 29. FRENCH, C. S., in "Life and Light" (W. McE1roy and B. Glass, eds.), pp. 447-474. Johns Hopkins Press, Baltimore, Maryland, 1960. 30. MYERS, J., AND FRENCH, C. S., J. Gen. Physiol. 43, 723 (1960). 31. GOVINDJEE, R., AND RABINOWITCH,E., Science 132, 355 (1960). 32. GOVINDJEE, R., AND RABINOWITCH, E., Biophys. J. 1,377 (1961). 33. GOVINDJEE, R., THOMAS, J. B., AND RABINOWITCH, E., Science 132, 421 (1960).