Biochimica et Bir~dwuca Acta. Itl7t) ( 1991 ) 263-267 ~ 1991 El~vicr Science Publishers B.V. All right,, reserved 0167-4X3X/91/$(13.5fl A D O N I S 0167483891 {lfl2X17

263

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A fluorometric study with 1-anilinonaphthalene-8-sulfonic acid (ANS) of the interactions of ATP and ADP with rubisco activase Zhen-Yuan Wang

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and Archie R. Portis, Jr. t--"

I DtTgzrl?rIt'rll ofA"ronrmty. IJhit'ertity of Illinois ai Champargn-Urbarta. Urbana, IL/U.S_4,,t and : Photos~.nthe~z~ ICe,cart h (',hr. /4grwuhura~ Red,earth Service, U,5. Deyartmcnt qf Agrtt~Mture. Urhana. IL ¢/~.S~.4J ~Received 12 April 19ql)

Key words: I-Anilinonaphthalene-te.,,u|fomc arid: Fluoromelry: Nucleotide binding, Enzyme rcguk)tion: RubJ~,c~ act~va,.c

The interactions of ATP and ADP with rubisco activase purified from spinach were investigated by measuring enhanced fluorescence due to ANS-binding to the prolein. Evidence of conformational changes was observed from the differences in the interaction of ANS with rubisco activase in the presence of excess ATP and ADP. Fluorescent changes associated with the titration of a rubisco activase-A,NS mixture with ATP and ADP indicated that the binding of ADP to rubisco activase was much tighter .ban that of ATP. The concentration of Mg 2" and pH had significant effects on the affinities of ruhisco activase for ATP and ADP, with higher pH and Mg ~ ÷ concentration facilitating the binding of ATP to rubisco activase in the presence of ADP. The physiological implications of the binding characteristics of ATP and ADP with rubisco activase on the light-dark regulation of rubisco are discussed.

Introduction Rubisco activase is a chloroplast protein which mediates the activation of ribulose 1,5-biphosphate carboxylase/oxygenase (rubisco) [1]. The activation of rubisco involves the formation of a cataly~icMly component enzyme-earbamate-magnesium complex, but this process does not occur either rapidly in the presence of RuBP or extensively at ambient C O : unless rubisco activase is also present [2]. In vitro, activation with rubisco activase is dependent on ATP and inhibited by A D P [3]. Accordingly, purified rubi~o activa~ Ix~ssesses an intrinsic ATPase activity which is highly specific for ATP and Mg 2" and is inhibited by ADP as well [4]. However, the direct interaction of ATP and ADP with rubisco activase has not been characteri,_ed at present. l-Anilinonaphthalene-8-suifonic acid fANS) is a useful fluorescent reporter group for detecting confor-

Abbreviations: Rubisco. rihuh~-l,5-bipho~phate car N ~ l a s c / o ~ genase; RuBP. ribulose 1.5-biphosphate; ANS. i-anihnonaphthaIene-8-sulfonic acid Correspondence: A . R Portis~ Jr.. USDA/'ARS. l t ~ Plant and Amreal Biotechnotogy Laboratory, 12Hl W. GregoD A~e., Url~ana. IL 61801-.'~38, U~SA.

mational alterations in proteins and monitoring ligand binding [5]. The correlation between the optical propert=es of ANS in ~ivent systems of varying polarity and when adsorbed to the heine crevice of apomyoglobin indicates that the change in its fluorescent properties is the result of the noncovalent interaction of ANS with the nonpolar surface of proteins [6]. Changes in ANS fluore~ence can be used to study interactions between enzymes and metabolites under phys=otogical conditions [7]. In this report, the interaction of ATP and ADP with rubisco activase purified from ,,pinach was investigated by monitoring the fluorement changes of ANS-rubisco acthase complex. Materials and Methods Rubi~o activase was purified from spinach leaves as de~ribed by Robinson et al. [8]. and desalted by gel filtration using a column of Sephadex j G-.q) equilibrated with 920 mM 1.3-bis[tris(hydroxymethyl)methylamino]propane (BTP) (pH 7.0), 20 mM KC1. ANS ,~as purchased from Molecular Probes.

J Menti~m cff ~- ~radcmark.. p r o p n e l a ~ product or vendor does nol con,ditu~c ~ guarantee or ~arrant~ of the p n ~ u c l b3 the U,S.

Department of Agric~dturc and doe,, nol imply its appr¢~al to, the exc|u~qon t~t"other pr¢~uct~, or vend~rs thai mat al,~ be suilable.

264 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Hydroly~,is of ATP b~. rubisco activase was measured as described previously [4]. All fluoresccnce measurements were conducted at 4 ° C. using a Perkir,-Elmer LS-5 fluorescence spectrophotometcr vdth the excitation wavelength at 380 nm and the emission wavelength at 48(1 nm. All titrations (except where specifically indicated) were performed in a buffer of 5(I mM Tricine (pH 8.0). 0.I mM EDTA and 2 mM DTT. In the titration of rubi~o activase with ANS, correction was made for free ANS by the method of Thompmn and Yielding [9]. Dissociation constants (K a) and goodness of fit for ATP and ADP under the various experimental conditions were estimated by using a nonlinear regression program [10] with untransformed data. However the data are premnted in standard Eadie-Hofstee plots of ;iF (fluorescence) versus AF/[ATP] or ..XF/IADP] to emphasize the differences in binding.

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Fig. 2. Effect of preincub:~tion at dilferent temlxrature~ on ATPas¢ activity ot rubi~o activase and change of ANS fluorescence by additio, n of ATP. 0.2 m g / m i of rubisco activase was incubated ior 10 rain at each temperature. Then an Miquot was removed (25 i.d/ml final concentration) for the measurement of the change of ANS (40 u M ) fluore~ence after adding 0.2 mM ATP. ATPase activity was a l ~ assayed as d e ~ r i b e d 14]

Results

Similar to othcr proteins, ANS bound to rubisco activase but not with ligands, forming a fluorescent complex with a maximum emission at 480 nm as compared to 530 nm for free ANS. Titration curves obtained by adding small increments of ANS to the protein alone, the protein with 5 mM Mg ~ , with 0.2 mM ATP, and with 0.2 mM ADP, are shown in Fig. 1. Significantly different fluorescent responses were o~ tained in each case. Compared to rubisco activase alone, the presence of MgCI2 resulted in a greater fluorescent responm, whereas the presence of either ATP or ADP reduced the fluorescent response. These results suggested that the conformational change in the rubisco activase protein induced by Mg 2~ was quite different from that in the nucleotide-binding form. Rubisco activase-ATP and -ADP also had different fluore~ence yield when they bound with ANS. r

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Fig. I. Titration of rubisco activase, rubi~o activa,e-Mg, rubi~o activase-ATP and rubisco activase-ADP by ANS. Conce'ttrations ~¢re: r'ubi~o activism, 25 V.~/ml; Mg "~" 5 triM: ATP, (1,2 raM: and ADP, 0 2 raM. Fluore~-ence ).~ in arbitr;~ry unil~.

Using gel-filtration chromatography, disaggregation of the rubisco activase protein was observed after incubation with free Mg 2~'. but not if it was in the nucleotide-binding form (data not shown). However, in the absence of information on whether all ANS-binding sites of the protein are independent and identical, it was not possible to conclude whether the conformational difference in the liganded proteins characterized by fluorescent responses was associated with changes in the number of ANS binding sites, the binding efficiency of ANS or the quantum yield of bound ANS. Rubisco activase is a heat labile enzyme [4]. A compari~n of the effect of a temperature pretreatment on the fluorescence emission of rubisco activase and its ATPase activity revealed (Fig. 2) that the change of fluorescence induced by ATP binding was reduced in a similar manner as the ATPase activity. The parallel changes indicated that the differences in the titration curves in Fig. I were a characteristic of active protein. The distinctive fluorescent responses observed above provide a means to quantitate the interaction of the protein with the ligands. Fig. 3a shows the results of the tttra~,(m_ of .~-b:sco actb, asc in the presence of 40 /aM ANS with ATP and ADP. The fluorescence was reduced much more rapidly by ADP than by ATP, indicating that the dissociation constant of ADP was less than 1 /aM and that of ATP was about 40 v.M. Similarly, the response of fluorescence upon addition of MgCI: indicated that the dissociation constant of Mg ~+ was about 2 mM (Fig. 3b). Since the hydrolysis of ATP by nabisco activase is reduced by more than 95% at 4 °C (Lilley, R.M. and Portis. A.R., unpublished data), the interactions of Mg :'~ with both ATP and ADP could be examined. The results of the titration showed that the affinity of

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cent yields of rubisco acti~a~+ANS ~a~ ob,~crxcd at ,,;

TABLE i

7 I) versus p|-! N,O, lion,ever, nuclcotidc titralion,, indicated that the affinity ~t rubb, c~ acti~a,,c h+z A T P Condilion

K~; { # M

ATP ATP+4rnMMgCi: ADP A ~ P + i mm MgCI z A D P + 4 mM MgCI:

pH 7.0

pH 8.0

57 ± t,4 11.8 +0.4 0.59 ± 0.02 1.00±1L04 1.65 ± 0,07

41 ±. 2+4 8.~ +0.2 0.76 ± (t.1t3 138 ± fl.l~ 2.76 ~+0.24

increased by about 35-4(1e; and the af!inity l

A fluorometric study with 1-anilinonaphthalene-8-sulfonic acid (ANS) of the interactions of ATP and ADP with rubisco activase.

The interactions of ATP and ADP with rubisco activase purified from spinach were investigated by measuring enhanced fluorescence due to ANS-binding to...
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