A study of nucleotide acceptor specificity of photophosphorylation by spinach chloroplasts

ARCHIVES

OF

A Study

BIOCHEMISTRY

ASD

of Nucleotide

BIOPHYSICS

94,

Id-19

Acceptor

Specificity

by Spinach A. R. KRALL*

of Photophosphorylation

Chloroplasts’ AND

From RIa4S, Inc., Received

(1961)

R. PURVIS3

&I.

Baltimore,

January

Photophosphorylation of both pyrimidine and occurs m-ith unwashed whole spinach chloroplasts. pyrimidine derivatives is preferentially removed tonin fragmentation of the chloroplast. Nucleotide cient to account for the rates of phosphorylation not t,he purine derivatives has been demonstrated. INTRODUCTION

In t’he earliest studies on chloroplast photophosphorylation, AMP4 was used as the nucleotide acceptor and was converted to ATP (1). Later it’ was found that, a protein-containing extract of leaves greatly stimulated phosphate uptake (2). Arnon et al. then found that ADP was a much more effective phosphate accept’or than AMP (3). Using bact’erial chromatophores, Frenkel (4) found that washed chromatophores would phosphorylate ADP and that unwashed chromatophores or washed chromatophores and a soluble enzyme fraction would phosphorylate both AMP and ADP. He presumed that, soluble fraction to contain adenylate kinase. Mazelis has shown that 1 Supported in part by grant RG-5583 from the U. S. Public Health Service. 2 Present address: Departments of Psychiatry and Biochemistry, Medical Research Building, Universit,y of Miami School of Medicine, 1600 N. W. 10th Avenue, Miami 36, Florida. 3 Present address: Armour Research Foundation, Chicago, Illinois. 4 Abbreviations used are AMP, IMP, UMP, CMP, and GMP for the monophosphates of adenosine, inosine, uracil, cytosine, and guanosine; ADP etc. for their diphosphates and ATP etc. for their triphosphates; PMS for N-methyl phenazonium sulfate, FMN for flavine mononucleotide, PI for inorganic phosphate, and Tris for tris(hydroxymethyl)aminomethane.

Maryland

20, 1961 purine diphosphoribonucleotides The ability to phosphorylate the by extensive washing or by digidiphosphokinase activity suffiof the pyrimidine derivatives but

chloroplasts contain an adenylate kinase, part of which is removed by washing (5). Frenkel (4) also found that washed chromatophores would phosphorylate IDP without’ the addition of ADP. Simonis et al. have found that’ chloroplasts can phosphorylate IDP and GDP but at lower rates than ADP (6). This paper reports data showing that phosphorylation of purine dinucleotides is probably direct while that of pyrimidine dinucleotides occurs, but involves a soluble enzyme which can be removed by washing the chloroplast. MATERIALS

AND

METHODS

Chloroplasts were prepared from spinach bought at a local market. The leaf blades were ground in a 0.35 M sucrose, 0.05 M Tris, pH 7.8 buffer in a mortar and pestle. The brei was centrifuged according to Arnon et al. (1). Washing was accomplished by resuspending in the grinding medium and resedimenting at 1000 X g for 10 min. This was done as often as indicated in the tables, the chloroplasts as first spun down being termed “unwashed” chloroplasts. Digitonin fragments were prepared according to Koukol et al. (7), and in the experiment’s reported here were used after being frozen several days. React,ions were run in lo-ml. Erlenmeyer flasks suspended in a constanttemperature bath maintained at 15” and illuminated from below with incandescent lamps, which gave a light intensity of about, 1500 ft.-candles at the flasks. Nucleotides were purchased from either Pabst Laboratories or Sigma Chemical Co. Two

NUCLEOTTI)E

ACCEPTOR

difIerent catalyst,s of photophosphorylation were used in the experiments reported here. The P?vzS system of Jagendorf et al. (8) gave the highest rates of PI uptake. Ferricyanide was used as an electron acceptor for noncyclic photophosphorylation in some of the experiments (9). Inorganic phosphat,e uptake was measured by the method of Tausky et al. when the first catalyst was used (10). When ferricyanide was used as the electron acceptor, the reactions were stopped nit,h 109; perchloric acid and dilut,ed to 20 ml. with 107; perchloric acid in a separatory funnel, 5 ml. of 10% ammonium molybdate was added, followed by 25 ml. of 50:50 butanol-benzene (v/v) previously equilibrated with the 10% perchloric acid, the mixture was shaken, the lower, aqueous phase was drawn off and filtered through paper, and an aliyuot was counted. This procedure, essentially a modification of that of Nielsen and Lehninger (II), handles larger amounts of phosphate than theirs. The filtration eliminates contamination of the aqueous phase with PI from the organic phase. In a few experiments the P32-labeled nucleotides were separated on paper strips using an isobutyric acid-ammonia-water, 66: 1:33 (v/v), solvent (12). The positions of the spots were determined with an ultraviolet light, and specific activity of the spots was determined by eluting them, counting an aliquot, and measuring their optical density spect,rophot,ometrically. Ferricyanide reduction was followed continuously at 400 mp in a Cary model 14 spectrophotometer using the machine in its infrared mode of operat,ion. Used in this way the full intensity of the exciting lamp strikes the cuvette with equal intensit,ies impinging 011 both the reference and sample cuvette. The light coming through the cuvette then passes t#hrough the slit into the monochromator syst,em and is analyzed. A cuvette containing all t.he reaction comp0nent.s but the ferricyanide is used as a reference. iZTP32 used was prepared by photophosphorylation and separated and concentrated by ion-exchange chromatography on Dowex 1 (13).

15

SPRCIFICIT~~ TABLE

I

PHOTOPHOSPHORYLATION OF ~V~-~I.EOTII~E ?uk~SOPHOSPH.4TES

The reaction mixture contained 10 rmoles >1gCls , Bl ktrnolcs Tris buffer pH 7.8, 0.03 pmole PMS, 20 pmoles phosphate buffer pH i.8, 20 pmoles acceptor, and 0.2 ml. chloroplasts cont,aining about 0.1 mg. chlorophyll. Total volume 3.0 ml. Illumination was 30 min. at 1500 ft.-candles. Rate +moles Pi,mg.

Times \rashed

0 0 0 0 0 0 2 2 2

Chlorophyll/hr.

Inw

0

UMP

0

(:nw AMP SDP 9MP + 0.5 pmole ATP ,4MP AMP + 10 pmoles ATP ADP

0 52

350 2% 0 0 120

does the addiCon of ATP allow it t,o act, as an acceptor. At, the time this work was in progress, it was found that a soluble extract of chloroplast’s would greatly enhance the rate of photophosphorylation of AMP (14). Our findings led us to the conclusion that the major activity of this extract was probably adenylate kinase, an enzyme already reported to be present in chloroplasts (5), as well as in crude extracts of photosynthetic bacteria (4). We tested for the presence of adenylate kinase by separating the products of the photophosphorylation of ADP with Pia by ion-exchange chromatography. Unwashed which phosphorylate AMP chloroplasts, fairly well, were used, and t,he reactions were run until about) half the ADP was csonr-crted to ATP. In an experiment, to which no supernatant fact,or was added, the isolated RESULTS AND DISCUSSION ATI’ contained 55 times as much 1’“’ as did Table I shows t)he rat,es of phosphorylation ADP. On t’he addition of supernatant facstor,” of mononucleotides compared with ADP which did not st’imulat,e the rate of l’i upas an acceptor. AMP is t.he only mononu(!leotake since AZDI’ w-as being used as an actide effective as an acceptor with unwashed ceptor, t,hc ATI’ contained about the same chloroplasts. AD1 was about seven times as amount of I’?’ as in the control espcriment8, effective in this set’ of experiment,s as AMP. while the dDl’ c~ont,nined five times as mucah The addiCon of a small amount, of ATI’ also 1’32as in the control. J’ery little l’s2 1~:~s found greatly increases the rate of phosphate uptake. If the chloroplast,s are washed twice, j .bi extract of chloroplasts according to Avron AMP is not effrrtive as an acceptor, nor and Jagendorf (II) furnished to us by 11. Avron.

KRALL

16

AND

in either compound in the absence of light and consequent net phosphate uptake. Thus the extract hastened the equilibration of the terminal phosphates of ADP and ATP about fivefold over the rate at which this reaction occurs in the unwashed chloroplast. Washing the chloropl&s twice completely removed their ability to phosphorylate AMP even with a large amount, of ATP as a TABLE

II

EFFECT OF ACCEPTOR AND WASHING ON PHOTOPHOSPHORYLATION OF NUCLEOTIDE DIPHOSPHATES Reaction mixtures contained 2 pmoles acceptor 3 pmoles Pi , 0.015 pmole PMS, 20 pmoles Tris buffer, pH 7.8, 5 pmoles MgCl2 , and chloroplasts containing 0.02-0.03 mg. chlorophyll. Volume was 1.5 ml. Illumination was 20 min. at 1500 ft.candles.

T

Rate ,,moles Pi/mg. chlorophyll/hr.

Times washed ADP

IDP

GDP

UDP

CDP

391 347 299 352 310 170 120

150

210 223

78 40

184 -

310 187 77

44 5 0

144 0 0 -

-I0

2 3 4 6 9 9 (wat)er treated)

201 181 133 108

-

-

TABLE

III

EFFECT OF WASHING ON RATES ON PHOSPHORYLAATION AND FERRICYANIDE REDUCTION Reaction mixtures contained 3 rmoles ferricyanide, 30 pmoles Pia2, 10 Mmoles MgC12, 5.0 pmoles acceptor, 100 pmoles Tris, pH 7.8, and chloroplasts containing approximat)ely 0.1 mg. chlorophyll in 3.0 ml. Reaction time was 3 min. in the light in the Cary spectrophotometer (see text). Rates amoles/mg. chlorophyll/hr. Washed once

Acceptor APi

ADP IDP GDP UDP CDP Base

63 51 46 6 3 0

Ferricyanide reduction

282 220 230 175 161 165

Washed nine times APi

44 45 44 0 0 0

Ferricyanide reduction

167 136 149 125 118 125

PURVIS

primer. Mazelis (5) has reported that chloroplasts contain a soluble (removable by washing) and a bound adenylate kinase. If our washed chloroplasts cont’ain the bound enzyme, AMP and ATP apparently cannot gain access ho it, while ADP has easy access to the site of photophosphorylation. It was found that once-washed chloroplasts phosphorylated all the other ribonucleoside diphosphates available, but t’hat ADP was the most] efficient acceptor, i.e., gave the highest rates of inorganic phosphate disappearance. It was further found that washing the chloroplasts in sucrose buffer solutions removed this activity differentially with respect to the purine and pyrimidine diphosphates. The washing experiments were done separately for each nucleotide tested, comparing, at each washing, t,he rate of Pi uptake wit#h the acceptor under test’, with that found using ADP as an acceptor. Table II presents the results of a typical set of experiments. The activit’y with nine-times washed chloroplast’s with ADP as acceptor was about 30-50 % of t’he rat,e with unwashed chloroplasts. The same was true of the activity with GDP. Activity with CDP or UDP disappeared almost completely aft’er six washings. Thus, some factor making possible phosphorylation of t,he pyrimidine nucleoside diphosphates appears to be removed by the exhaustive washing. It is not certain that closed-chain or cyclic photophosphorylation occurs via the same mechanism as “coupled” or open-chain phosphorylation. Therefore, the washing experiments were repeated using ferricyanide as the electron acceptor and measuring both ferricyanide reduction rates and Pi uptake. Table III shows bhe results of one of those experiments. The pyrimidines CDP and UDP were consistently poorer acceptors with unwashed or once-washed chloroplasts when ferricyanide was used as the electron acceptor than when PMS was used as a catalyst. The pyrimidines were totally ineffective both in stimulation of ferricyanide reduction and in acting as a phosphate acceptor after nine washings. Using purine diphosphates GDP, IDP, and ADP as acceptors, the rate of Pi uptake declined very little on washing and a good stimulation of

SUCLEOTIDE

ACCEPTOR

ferricyanide reduction was observed on addition of the nucleotide. The results with AMP had shown that, adenylate kinase was apparently removed completely after two washes. The results with the diphosphates other than ADP indicated t)hat if something was removed by washing, it was removed much more slowly than was adenylate kinase. Therefore, it was thought that’ a broken chloroplast preparation such as the digitonin preparation of Koukol et al. (7) might be freed of such substance more readily than the whole chloroplast. Table IV shows t,he results of an experiment with fragments from digitonintreated chloroplasts. These fragments were prepared from once-washed chloroplasts, and t,he fragments were not washed aft,er being sedimented out of digitonin solution. It is seen that again the purine diphosphates are phosphorylated at essentially the same rates while the pyrimidine diphosphates are inactive as acceptors. It seemed possible that the chloroplasts contain bound ADP which was convert’ed to ATP and the terminal phosphate of ATP transferred to the other diphosphates by the enzyme, nucleoside diphosphokinase (NUDIKI), first demonstrated by Berg and ,Joklik (15). This possibility was tested by use of P”2-labeled ATP, which was synthesized and isolated from well-washed chloroplast,s. Equimolar amounts of ATP3A and of the diphosphnte acceptors GDP, IDP, UDP, and CDP were added to twice-leashed and six-t,imes-washed chloroplasts. The react,ion mixtures were the same as those used in phot80phosphorylation except t)hat light was omitted. Table V shows the rate/mg. chlorophyll/hr. at which t,he terminal phosphate of ATP was transferred to the dinucleotide under test. Neither of the purine diphosphates was as effectivean acceptor a3 were the pyrimidine diphosphates. The rea&on ran to what was apparently equilibrium with CDP and UDP with twice-washed chloroplasts. These results indicate that there is probably sufficient’ ?;UDIKI present to account for the rat,es at which CDP and IJDP are phosphorylated in the lightly washed chloroplasts, but t,hat this pathway cannot, accsount for t,he high rat)es at which

17

SPECIFICITY TABLE

IV

PHOTOPHOSPHORYLATIOX WITH DIGITONINTREATED PREPARATIONS The reaction mixture was the same as in Table II. Digitonin particles contained 0.16 mg. chlorophyll per vessel; reaction time was 30 min. at 1500 ft.-candles at 15°C. Phosphate

Acceptor

uptake

pmoles

ADP GDP IDP UDP CDP

0.85 0.61 0.55 0.02 0

TABLE XUCLEOTIDE

V

DIPHOSPHOKINASE ACTIVITY SPIXACH CHL~ROPLASTS

OF

Rates in pmoles Pi transferred/mg. chlorophyll/hr. Reaction mixtures contained ‘2 pmoles acceptor, 2 pmoles ATP32, 3 pmoles P, , 3.3 rmoles MgCl* ) 6.7 pmoles Tris pH 7.8, and 0.01 pmole PMS, and chloroplast,s containing 0.042 mg. chlorophyll in 1 ml. volume. Reaction time was 30 min. in dark. Reaction was terminated by heating, and nucleotides mere separat,ed on paper strips using Pabst Solvent 1 (1’2). Rates Acceptor

Times

--~ 2 .~-

--__

GDP IDP CDP UDP

12 18 48 48

washed 6

G 30 “4 30

GDP and IDP are phosphorylated in any chloroplast preparation. An attempt was made to use ATP as a trapping agent and prove that GDP was phosphorylated direct,ly. Radioactive inorganic phosphate, GDP, and nonradioactive ATP were added to a reaction mixture and the mixture was illuminated. The product,s of t’he reaction were separated by paper chromatography (12)) the spots locat’ed with an ultraviolet lamp, eluted, count,ed, and assayed spectjrophot,ometrically. CrTP showed a higher specific act’ivity than ATI’ in all such experiments, and the ratio of specific activities (GTP/ATP) was higher when six-times-washed chloroplasts lvere

18

KRALL

AND

used (5.5: 1) than when twice-washed chloroplasts were used (1.6: 1). These results indicate that GDP is phosphorylated without the intervention of ATP from the trapping pool. It may be that ATP is intermediate between Pi and GTP, but, if it is, such ATP is spatially separated from the ATP-trapping pool. The results of all the experiments shown here demonstrate that t,he purine dinucleotide phosphorylation is different from that of pyrimidine dinucleotides. The activity with pyrimidines is removed differentially by washing and by digitonin fragmentation. The pyrimidine derivatives, CDP and UDP, served as better acceptors for transphosphorylation from ATP than did GDP and IDP. The rate of this react,ion, nucleotide diphosphokinase act’ivity, was high enough to account for all activity observed with the pyrimidine derivatives but very much lower than that required for the activities observed with GDP and IDP, especially in the wellwashed chloroplasts. The test for NUDIKI activity was run with 0.002 M/ATP. This is probably a very much higher ATP concentration t,han would be built up from bound ADP in the chloroplast and would therefore give higher rates than could occur during attempts at phosphorylation of CDP or UDP with well-washed chloroplasts. It is probable that the drop in rates with CDP and UDP as acceptors is a result of leaching out of both bound ADP and the IWDIKI enzyme. The latter appears to be much more firmly bound than adnenylate kinase activity and is probably a normal component of the chloroplast. The chloroplast structure is damaged considerably by repeated washing as shown by electron micrographs of washed chloroplasts in which the outer membrane was highly fragmented even t,hough the lamellar structure is int’act. Further evidence of fragmentation is the continued removal of starch granules from the chloroplast’s on successive washes. It may be t,hat NUDIKI is a part of the stromal protein of the chloroplast’ which is slowly eroded away from the grana after the chloroplast membrane is broken. ADP is a somewhat better acceptor than GDP or IDP in most cases, but not enough

PURVIS

better that it may be termed a specific acceptor. Therefore, the only specificity shown is for purine nucleoside diphosphates, rat’her than pyrimidine nucleoside diphosphates. This is a much wider range of act,ivity than that shown by several other phosphorylating enzymes. The phosphorylation associated with the oxidation of cr-ketoglutaric acid has been shown to be specific for GDP or IDP and would nob utilize ADP (16). That mediated by pyruvic kinase is four t’imes as effective with ADP as with any other acceptor (17). The ADP-ATP exchange reaction, thought to be the terminal react’ion in oxidative phosphorylation, is absolutely specific for ATP (18). Thus, phosphorylating enzymes can apparently vary widely in specificit,y from an absolute requirement such as t’hat] shown by oxidative phosphorylation t’o the relatively low specificity requirement seen here in our results with t’he phosphorylation system of chloroplasts. ACKXOWLEDGMENTS We wish to acknowledge the ance of Miss Olga v. h. Owens Cowin in some of these studies, Mordhay Avron and Dr. A. T. valuable discussions.

technical assistand LMrs. Martha and to thank Dr. Jagendorf for in-

REFERENCES 1. ARNON, D. I., ALLEN, M. B., AND WHATLEY, F. R., Nature 174, 394 (1954). A. T., AND AVRON, M., J. Biol. 2. JAGENDORF, Chem. 222, 453 (1956). D. I., WHATI~EY, F. R., AND ALLEX, 3. ARNON, M. B., Nature 180, 182 (1957). 4. FREXKEL, A. W., J. Biol. Chem. 222,823 (1956). M., Plant Physiol. 31, 37 (1956). 5. MAZELIS, W., ANI) FDCHTBAI-ER, W., Planta 6. SIMOIVIS, 64, 95 (1959). J., CHOIV, C. T., ANI) VENNESLAND, 7. KOUKOL, B., J. Biol. Chem. 234, 2196 (1959). 8. T., XND Av~ox, 11.: J. Biol. 8. JAGEXLIORP, Chem. 231, 277 (1958). D. I., WHATLET~, F. I<., AND ALLEN, 9. ARNOS, M. B., Science 127, 1026 (1958). G:.. 10. TAITSKY, H. H., SHORR: E., AND k’r-RZMASN, J. Biol. Chew&. 202, 675 (1953). 11. NIELSEN, S. O., AND LEHNISGER, A. L., J. Biol. Chem. 216, 555 (1955).

SUCI,EOTII)E

ACCEPTOR

12. PABST LABORBTORIES, Circ. OR-lo, 1956. 13. COHS, W. E., AND CARTER, C. E., J. .tnr. Chenl. sot. 72, $273 (1950). 14. AVRON, >I., ASD JAGENDORF, A. T., A’ature 179, 129 (1957). 15. BERG, P., .~sI) Jowm, W. K., J. 13io/. (‘hem. 210, (ii5 (1954).

SPECIFICITY

10

16. SANADI, D. R., GIBSOS, D. M., ATESGAR, P.: AND JACOB, M., J. Hiol. Chem. 218, 505 (1956). 17. STROMINGER, J. I,., Rio&m. et Riophys. dcfa 16, 6lG (1955). 18. COOIT~R, C., AND LEH~~S(;ER. A. L., J. Bid. f’hem. 224, 561 (1957).