Enzymes of the carbon cycle of photosynthesis

ARCHIVES

OF

BIOCHEMISTRY

AND

Enzymes I. Isolation

of the Carbon

and

Properties

KATHLEEN Department

117, 650-659 (1966)

BIOPHYSICS

of Photosynthesis

of Spinach

BROOKS

of Biochemistry

Cycle

Chloroplast

RICHARD

AND

and Biophysics,

California

University

Aldolase’

S. CRIDDLE of California,

Davis,

95616

Received July 15, 1966 Aldolase from spinach chloroplasts has been isolated and its properties have been investigated. A single aldolase enzyme was found in the chloroplasts which catalyzes the cleavage of both the carbon cycle intermediates fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate. The kinetic values of V,,, and K, for the enzymic cleavage of the two sugar diphosphates are of the same order of magnitude. The ratio of the rate of cleavage of fructose diphosphate to that of sedoheptulose diphosphate is 1.7. The plant enzyme resembles rabbit muscle aldolase in size and enzymic properties. Its activity is sensitive to carboxypeptidase digestion and it has no observed metal ion requirement for activity.

Aldolase activity has been postulated to function in two reactions of the carbon reduction cycle of photosynthesis (1). Both the synthesis of sedoheptulose diphosphate and of fructose diphosphate in this cycle presuppose aldolase activity. However, the nature of the enzymes involved in these reactions is still quite obscure. Even the existence of fructose diphosphate aldolase in all photosynthetic organisms was a matter of controversy for some time. Tewfik and Stumpf (2) showed fructose diphosphate cleavage activity in all the higher plants they examined,

although

they

could not find ac-

tivity in spinach chloroplasts prepared by their method. Fewson et al. (3) verified the existence of fructose diphosphate aldolase activity in extracts of all classes of photosynthetic organisms except the blue-green algae. Rutter (4) has recently reported that the blue-green algae, unlike other photosynthetic organisms studied, possess an aldolase with enzymic properties resembling those of the yeast enzyme. 1 Supported in part by Public Health Service grant CM 10017.

Benson e2 al. (5) reported the isolation of labeled sedoheptulose phosphate from i4C02 photosynthesis products of Chlorella, Scenedeamus, and Rhodospirillum rubrum. Both fructose and sedoheptulose phosphates appeared to be labeled very nearly simultaneously in these photosynthesizing organisms. Distribution of radioactive carbon atoms within the hexose and heptulose molecules is consistent with the occurrence of both a Ca + Cs and a Cz + C4 addition reaction (6). Hough and Jones (7) identified sedheptulose as a product of incubation of smaller sugars with extracts of pea seeds. These observations, together with labeling data and patterns in whole chlorella, have led to the current belief that an aldolase catalyzes formation of sedoheptulose diphosphate from dihydroxyacetone phosphate and erythrose 4-phosphate in the cycle which regenerates ribulose 1,5-diphosphate (6). Little is known about the properties of aldolases of higher plants beyond the fact of their existence. Stumpf purified an aldolase from pea seeds some 92-fold and described its general properties (8). Prasad 650

SPIlVACH

(9) studied the aldolase of mung beans but could not effect, purification without loss of activity. Recently, Clark and Pearce (10) partially purified an aldolase from corn leaves. However, no aldolase has been purified from chloroplasts prior to the work reported here, and it is not known whether one or two aldolases catalyzes the synthesis of fructose diphosphate and sedoheptulose diphosphate in chloroplasts. This paper described the purification and some propert8irla of a spinach chloroplast sldolasc that, ac+s on fructose diphosphate and on sedoheptulosc diphosphate. I~;YPBHlbIENTAL

PROCEl)URE

I>PN and DPNH were purchased front Sigma and Soehn. Chemical Co. and from Boehringer The sugar phosphates used, fructose 1,6-diphosphate, fructose l-phosphate, erythrose 4-phosphate, and dihydroxyacetone phosphate were obt,ained from California Biochemical Corporation. Sucrose was purchased from Mallinckrodt Chemical. Ammonium sulfate, enzyme grade, was obiained from Mann Research Laborat,ories. Hydranine hydrate was purchased from Matheson, Colernan and Bell. Aquacide II was obtained from California Biochemical Corporation. Fresh spinach was purchased at local markets and was used withiln 24 hours of purchase. All other chemicals used were reagent grade. I’rofein &fernGnation. Protein determinations were made by the binret method and by the techtliqtte of Warburg and Christian in the crude ext.racts. The ronceutration of protein in the purified preparations was estimated using absorbance at, 280 rnp and the approximation that a solution of one mg per milliliter yields an absorbance of one. n nlino acid analysis. Petrified spinach aldolase was hydrolyzed in evacuated tubes for periods of 24 and 48 hottrs with 6 N HCl. Amino acid analyses were run on a Phoenix automatic amino arid analyzer ttsillg t hc 3-hour accelerated run on a 60.cm column and the buffer system of Moore et al. (11). Density gradient centrifugation. Sucrose density gradient centrifugalions were performed at, 3” using a SW 39 rotor in a Spinco model L-2 preparative tdtraccntrifuge. The procedure followed was that of Martill and Ames (12) except that gradien tx of 5-205; sllrrose were produced by layering of SIIUOW solrttiotrs, and sample sizes differed in differetlt experiments. The details of each cent,rifugation are indicated in the results section. Sedinlmtution studies. Sedimentation velocity and sedimentation cqtlilibrium experiments were performed i tt :I Spinco model E ultracentrifuge.

ti.?l

ALI)OI,ASE

I)oltble sector cells with a 12.mm opt icnl path and sapphire windows were used in molerular weight determinations. Sedimentation velocity experiment,s were conducted at, 8” in 0.025 x Tris buffer containing 0.1 .M KC1 and 0.001 1~dithioerythritol with a rot.or speed of 59,780 rpm. Observed valrtes of sedimetttation coefficients were corrected to the v:tl~~c!s corresponding t)o sedimentat,ion in a solvettt, with the viscosity and densit,y of water at 20” isZo,Wj. Sedimentation equilibrium experiments were run Itsing the short,-column (3 mm) equilibrirtm procedure of Van Holde and Baldwin (13) alld plotted as log of cortcentration vs distance from t,he cent,er of rotation squared (In c vs. ~2). blolecular weights were calculated from the slope of such plots. The partial specific volume of aldolase was assumed IO be 0.74 cm3 per gram for these calculatiolls. Gel clrctrophoresis. Acrylamide gel electrophoresis was run io a Canalco model 12 “Disc Electroplioresis” apparatus; the procedure oi Davis was used (14). A 5!$% acrylamidc gel rnouomer concentration was Itsed alotlg with the Tris buffer system of Jovin et al. (I.?). I’Xecrrophoretic rtms were rnade at, 5 1~4 per {Itbe for approximately 11,$ hours or tuttil the t,racking dye approached t.he lower end of the tube. The proteius were st,aitted on the colttrnu itsing amide schwaltz dye in 7.5
of Substmtcs

The bari~m~ salt of fructose 1 ,G-diphosphate was converted to the sodium salt, by passage through Uowex-50 resin, II+ cycle, followed by netttralizat,iort of the free acid eluate with 2 s NaOII. The sodium salt, pH 7.5, was apportioned int.o 5-ml aliqrlots :tlrti was stored frozen until it was used. The concentration of the fructose diphosphate solution used for the determinatiotl of kinetic parameters was measttred by means of the rcsorcinol method of Roe et al. (16) as described by Ashwell (17). Fructose solutions wrprc employed as standards, since this method gives the same extinction for fructose and frltctose phosphates. Fructose I-phosphaic. Fntctose l-phosphate, barium salt,, was prepared attd assayed in the same manner as fructose 1 ,6-diphosphat c. Bruthrose .J-phosphate. 125 mg (260 pntolcsi of I)-erythrose 4-phosphate dimrth~l:tc~etal, dicyclohexylammonium salt, ntotlohydratr, was dissolved in 12.4 ml deionized water aud swirled for 1 minute with 2.5 ml dry Dowex 50, II’ ~yclc. The resin was removed by sllctiotl filt,rat.iorr. The flask containing the filtrate was stoppered tight,ly and incubated at 40” for 18 holtrs. The erythroscs $-phosphate was t~eulr:tlizcd to pl1 6 with molar KOFT. Fructose

I ,6-diphosphate.

652

BROOKS

AND

Dihydroxyacetone phosphate (DHAP). 113 mg (260 rmoles) of dihydroxyacetone phosphate, dicyclohexylamine salt, dimethylketal monohydrate, was dissolved in 11.3 ml of water and swirled for 1 minute with 2.5 ml dry Dowex 50, H+ cycle. The resin was removed by suction filtration. The filtrate was placed in a tightly stoppered flask and incubated for 6 hours at 40”. The dihydroxyacetone phosphate was neutralized to pH 6 with molar KOH. Sedoheptulose 1,7-diphosphate. Sedoheptulose 1,7-diphosphate was prepared enzymically from erythrose 4-phosphate and dihydroxyacetone phosphate by a variation of the method of Horecker (18) using isomerase-free rabbit muscle aldolase. Assay for Aldolase

Activity

The triose phosphates produced in the aldolase reaction react with hydrazine to give products absorbing at 240 mp. This reaction serves to displace the equilibrium by trapping the triose products; it also enables a spectrophotometric measurement of the rate of the cleavage reaction (19). The reaction mixtures contained l&12 rmoles of NaFDP, pH 7.5, 7 rmoles of hydrazine, and 150 pmoles Tris chloride, pH 7.5, in a volume of 3 ml. The reaction mixture was kept at 30” in a Zeiss spectrophotometer for 5 minutes to observe the basal reaction of the assay components in the absence of enzyme. Since the rate of t’he blank reaction decreased sharply with time in the first few minutes, the difference between the third and fifth minutes was used to determine an average blank value. At the end of this period a suitable enzyme sample was added and the change in absorbance at 240 rnp observed. The change in absorbance between the fifth and tenth minutes after addition of enzyme, minus the value of the blank reaction, was used to calculate enzyme activity. A unit of activity is a net change in absorbance of 1.00 at 240 rnp under the above assay conditions. The same method was used to assay for sedoheptulose diphosphate (SDP) aldolase activity, except that the substrate was 5 pmoles of NaSDP in place of the NaFDP. Isolation

of Aldolase

Preparation of chloroplasts. Fresh spinach was obtained locally; the midribs were cut out and the leaves were washed in two changes of ice water and shaken to remove excess water; the cleaned spinach was then weighed. All subsequent operations were performed in a 5” cold room. A typical preparation is described below. Each of three 700-gm batches of spinach was

CRIDDLE extracted with 1050 ml of sucrose phosphate solution (0.5 M sucrose, pH 7.5, 0.03 M K2HPOI) in a 5-quart large stainless steel Waring blendor operated at medium speed for a total of 1 minute (three 20.second intervals). The mixture, pH 7.4, was passed through two thicknesses of cheesecloth to remove fibrous material. The extract was passed through a pad of glass wool on a Buchner funnel to remove unbroken cells and cellular debris. Chloroplasts were sedimented from the filtrate by centrifugation at 4009 for 5 minutes at 0”. The combined sediments were washed by suspension in 1200 ml of fresh sucrose phosphate solution and centrifuged at 750s for 10 minutes. The supernatant fluids were discarded. Preparation of chloroplast soluble protein. The chloroplasts so obtained were osmotically lysed by suspending them in 60 ml deionized water at 0” for 30 minutes, then centrifuged for 15 minutes at 23,OOOg, 0”. The residue was suspended in an additional 40 ml of water for 20 minutes and then centrifuged as before. The two water extracts were combined and dialyzed overnight against a pH 7.5, 0.025 M Tris buffer containing 0.001 M MgS04, 0.005 M CaCl2, and 0.0002 M MgC12. This buffer is used throughout the remainder of the isolation procedure and will be referred to simply as Tris buffer. The dialyzed extract (90 ml) was concentrated to 42 ml by placing the dialysis casings in Aquacide II for several hours. Column chromatography on Sephadex G-200. A portion (20 ml) of the concentrated extract was placed on a 4 X 45-cm column of Sephadex G-200 equilibrated with Tris buffer which was 0.1 M in KCl, and the column was eluted overnight. The remaining one-half of the sample was dialyzed overnight against the same buffer, and chromatography was performed the following day. Fractions of approximately 6.5 ml were collected at 20minute intervals and assayed for aldolase activity. Fractions having a specific activity of 7 units per milligram or greater in the hydrazine assay were pooled as SPA and subjected to ammonium sulfate fractionation. Ammonium sulfate fractionation. Saturated ammonium sulfate solution, pH 7.5, was added to approximately 100 ml of pooled SPA to bring the solution to 457, saturation. The slightly turbid solution was stirred gently for 2 hours in the cold and then centrifuged for 15 minutes at 23,000g. The supernatant fraction was then brought to 557, saturation by adding additional saturated ammonium sulfate solution. The solution was stirred for 2 hours and then centrifuged as before. The sediment (ASII) was dissolved in a small volume of Tris buffer and reprecipitated by bringing the solution to 607, saturation with saturated

SPINACII

ALlNLrlSIS

y-globultn

.,&,

.4-

,I -

FRACTION

NUMBER

FIG. 1. %hchmatkJll of aldolase from the solltble protein of spinach chloroplasts on Sephadox (;-ZOO. Chromatograpny was run in pH 7.5, 0.025 M Tris buffer with 0.1 M KCI. Column dimensions were -1 X 45 cm. Flow rate was adjusted to approximately 0.5 ml per minute. Protein concentration was followed by measuring absorbance at 280 mp (0). Measurement of aldolase activity in the variotts fractiotrs was made using the hydrazine assay and observing absorbance change :I t 210 rnp per minrtte (X)

ammonitml sulfate solution. Aldolase activity of this srtspension was stable for at least 2 weeks. l’rcparations were routinely st,ored at this step for a few days until enough AS11 had been accumulated for usein the next step of thepurification. DEdE-cellulose column chromatography. Three ilSII preparations having specific activities of 1719 units per milligram protein were dialyzed in the cold agaittst Tris buffer, pII 7.5, and centrifrtged at 12,oOOg for 15 minutes to remove traces of insolrtble material. The solution was placed on a washed column of I)EAE-cellulose, 1.5 X 12 cm, washed in with 100 ml of Tris buffer, and then washed with 100 1111 of Tris buffer which was 0.1 ,M in KCl. No aldolase activity was elnted in these steps. Elrtt ion was effected by means of a linear salt gr,adirnt from 0.1 SI KC1 (250 ml) to 0.3 >f KC1 (250 ml) it1 the same Tris buffer, pll 7.5. Every fottrth fraction was assayed for aldolase activity. Fractiotls having a specific activity of 44-X wrre pooled. The pooled sample was concentrated and precipit,ated by overnight dialysis against 75’:c satttntted ammonium srtlfate. The precipitate was again dissolved in a small volume of Tris httffer and reprecipitated with saturated ~ammor~itnn sttlfate.

RESULTS Pigurc

1 shows

the distributiorr

of ;LIdol:Lso

activity on Sephadex chromatography rclative to the total protein of the soluble extract of chloroplast proteins. It can be seen that under these conditions, aldolasc activity is readily separable from the bulk of the soluble protein. In the absence of added KU, chromatography resuks in considerable overlap of aldolasc activity in the region corresponding to high 2SO rnp :tbsorpt8ion. Sucrose gradient sefhmcnt~at~ions of 0.1 ml of soluble chloroplast proteins also indicate a similar distribution of aldolase activity in the prcparat ion (I’ig. 2). The pattern of optical density (280 mK) shows the presence of major sedimcnting bands with scdimemation coetficients of 16 8 and 3.2 S (relative to gamma globulin selected as :I 7.0 S standa,rd) in qualitative agreement, wit,h t,he vahres obtained initially by Lyt8tlcton and T’so using the analytical ult~raccntrifugc (20). All aldolase activity is observed jr, :I single 6.7 S hard.

654

BROOKS AND CRIDDLE

Q

I IO

I

I 20 FRACTION

40

30 NUMBER

FIG. 2. Sucrose density gradient of chloroplast soluble protein. Sedimentation was run in a 5-20% sucrose gradient at 3” in a Spinco model L-2. A swinging bucket rotor was run at 39,006 rpm for 6.25 hours. Fractions were collected by counting drops from the punctured bottom of the ce.4and were monitored for protein at 280 mp in a Zeiss spectrophotometer (0). Aliquots were removed for activity determination using the hydrazine assay, and absorbance change at 240 rnp was followed (X). The specific activity of the peak fractions from the column was approximately 10. Gamma globulin was run under identical conditions to serve as a 75 standard.

TABLE PURIFICATION

OF SPINACH

I CHLOROPLAST

ALDOLME

step

1. Initial extract from 21009 spinach 26 1165 100 54 1404 2. Sephadex G-200 eluate (SPA) 57 536 46 1.17 67 3. Ammonium sulfate fractionation 45% sup 96 504 43 0.37 36 45Y0 sed 5 <40 55Y* sup 111 139 0.14 15.5 45%-55% (ASII) 7.3 313 27 2.26 16.5 The following step was performed on the pooled AS11 from 7700g spinach. 4. Applied to DEAE-cellulose 7.9 1008 7.14 56 column DEAE-cellulose eluate 34 312 9 0.21 7.1

The same pattern was observed when the extraction and density gradient centrifugation were performed in the presence of the sulfhydryl-group protecting reagents cysteine and dithioerythritol, respectively. No significant change in sedimentation coeEi-

100 4.6 2.6 1.1 1.2

0.83 8.0 14.0 <2 9.0 19.0 18.0

0.23

44.0

cient or total aldolase activity was observed under these conditions. Table I shows the purification obtained at each major step of the isolation procedure. Approximately a 70- to 80-fold purification of aldolase is obtained from the soluble

SPINACH

ALI)OLASE

FIG. 3. Sedimentation of purified spinach aldolase, specific activit,y 44. Phot.ographs are shown at 8.minute intervals for sedimentation at 59,780 rpm in 0.1 M KCl, 0.025 M Tris buffer, pH 7.5, containing 0.001 M iUgSOd, 0.005 M CaClp, and 0.0002 M illgClz and 0.001 M dithioerythritol. Temperat IIre was WIItrolled at 8”. The schlieren diaphragm angle was 70”.

chloroplast protein with an overall yield of 9% of the initially measured aldolase activit,y. The final spinach aldolase preparations contained no measurable glyceraldehydephosphate dehydrogenase, triose phosphate isomerase, 3-phosphoglyceric acid kinase, ribose phosphate isomerase, or phosphoribulokinasc activity.

FIG. 4. Sedimentation equilibrium of spinach aldolase. Sediment,ation was run at 6166 rpm for 26 hours in 0.025 M t,ris, pII 7.5, 0.1 M KC1 buffer. A single determination was made at a protein concentration of 5 mg/ml, the column height 3.5 min, and the temperature 14”. C is protein concentration and X is the distance from the center of rotation. Molecular weight was calculated from t,he slope of t,he curve by the method of Van Holde and Baldwin (13) and found to be 140,000 gm per molt TABLE AMINO

Amino Acid Alanine Arginine Aspurtic acid Cystine (Glutamic acid Glycine Histidine Isoleucine Leucine ii-H,

ACID

Sedimentation. Sedimentation of spinach aldolase indicated the presence of only a single migrating boundary (Fig. 3). A value of s20,Wof 6.65 S was obtained. 111 the absence of thiol reagent,, a small amount of rapidly sedimenting material, which was presumed to be a dimer, was observed. Such a dimer

has beer1 observed

gm protein

125.8 54.6 103 .o 134.2 84.1 37.7 54.5 117.0 19.3

rnusc~le

II

COMPOSITION OF SPIN.WH

M&s/140,000

in rnhbit

aldolase (21). Sedimentation equilibrium analysis (Fig. 4) shows a plot of log c vs x2 for spinach aldolasc in JIH 7.5 Tris buffrr and 0.1 11

ALDOL.WP

amino

acid

Lysille lLIe!hioninr Phenyl:tlanir~e Proline Serillr> Threonine TryptophaG Tyrosine V:ilinc

M&s/‘140,000

gm protein

70.ti 3 ,8 36. -4 15.4 5!) -1 GO.1 1x 33.1 !L%.1

a The values reported are the averages of two runs, except for serine and threonine, which polated values t,o zero time of hydrolysis. * Determined spectrophotometrically by the method of (Goodwin and ZGrton (22).

are ext,r:t-

656

BROOKS

AND

CRIDDLE

Glycylglycine

Phosphate

Tris

FIG. 5. Polyacrylamide gel disc electrophoresis of spinach aldolase. Migration of sample proceeded from top to bottom in the gel. Conditions are as listed in text.

KCl. For homogeneous material such plots give straight lines. It can be seen from the figure that the data for aldoIase deviate slightly from a straight line and suggest some heterogeneity. A weight average molecular weight of 142,000 gm per mole was obtained. Amino acid composition. Table II shows the amino acid composition of spinach aldolase. Gel electrophoresis. Figure 5 shows the observed electrophoretic pattern for purified spinach aldolase run on disc electrophoresis. ?Sote that most of the protein present moves as a single sharp band, but that a faint, slower moving band is also detectable. This may be a contaminant band or may again reflect the tendency of the aldolase to dimerize as noted in the velocity sedimentation runs. This question has not been investigated further. E$ect of pH on activity. The relationship of pH to enzymic activity was examined in three different buffers (Tris, glycylglycine and phosphate) ; fructose diphosphate was

I

6.5

7.0

I

7.5 6.0 PH

,

6.5

9D

I

9.5

FIG. 6. The pH optimum for spinach aldolase in various assay buffers. A preparation of spinach aldolase was assayed using fructose diphosphate as substrate as described in Experimental Procedures except that the following buffers were used: glycylglycine, 0.12 M; potassium phosphate, 0.05 M; and Tris-chloride, 0.12 M.

used as substrate. A sample of AS11 having a specific activity of 10 was dialyzed against Tris buffer, pH 7.5, and used for all assays in the series. The effect of pH on the FDPcleavage activity is shown in Fig. 6. In glycylglycine, spinach aldolase exhibits a marked optimum at pH 8.25, while in phosphate and Tris buffers, the optimum range is much broader and centers about pH 7.5. On the basis of these results, Tris buffer at pH 7.5 was selected for use in further kinetic studies. E$ect of incubation with EDTA. Spinach chloroplast aldolase shows no dependence on metal cofactors. Prolonged dialysis against 1O-3 M sodium EDTA, pH 7.5, resulted in no significant loss of activity.

SPINACH

ALDOLASE

TABLE EFFECT OF CARBOSYPEPTIDASE

6.7

III

ON SPINACH ALDOLASE ACTIVITY Activitv/j

minutes

Spinach

Rabbit -

FDP

F-l-P

__~_

Illitial 15 min 90 min

0.420

ControP

FDP

~~~~~ .~~-~ --

,101 ,357

0.010

. 000

muscle

__

,043

FDP

F-I-P

,841 ,032 ,055

,173

ControP

FDI’

-~~~ ~~~ ~~-.~~.~~ ,811

.18fi

n Controls were composed of the same components as the samples except that 105;. 1,iCl was added instead of carboxypeptidase dissolved in 107; LiCl. The procedure used was ident,ical t,o that, of Westhead et al. (23). Carboxypeptidase (0.06 mg) was dissolved in 2 ml 107;) LiCl and t#hepH was adjusted to 8 with 0.1 N NaOH. 0.15 ml of this solution was added to each incubation tube which contained 2 mg aldolase ill Tris buffer, pH 7.5, which was lo-” M in EDTS. Incltbation was at room temperatltre. TABLE

IV

REL.ITIVE ACTIVITY OF SPINMH CHLOROPLMT ALI)OL.ME TOW.\HI) FIVJCTOSE DIPHOSPH.~TE .\NI) SJWOHEITT:LOSE I>IPIIOSPIL~TE~

Inif.i:ll extrwt SPA AS11 SOllltion Purified a!dolase

Activity/S

min I_

DFP

SDP

.372

,217

,310 ,428

.187 .221

,216

,118

Specific activity t;w&

.55

9.5 44

Ratio FDP/SDP

1.72 1.65 1.91 1.78

o These assays were run in 0.15 M Tris, pH 7.5. Similar measurements using 0.15 M glycylglycine, pH 7.5, were also run on another preparation. Uue to somewhat different baseline correction factors in the blanks for the t>wo buffers, the ratios are slightly lower but nearly constant. The values observed were SPR = 1.54; ANTI = 1.56; and purified nldolase = 1.58.

Under these conditions, muscle aldolase activity is also unaffected, while the activity of yeast aldolase is completely inhibited. Addit’ion of ZnSOa to a concentration of IO-” A\I gave no enhancement of enzyme artivity. Incubation of spinach alclolase with ccwboxypeptidase. When spinach aldolase was subjected to carboxypeptidase degradation using the method of Westhead et al. (23), it rapidly lost activity, but the pattern of the decline was somewhat different from that of the rabbit muscle aldolase run at) the same time. It, ran be seen in Table III that there

is no residual fructose diphosphatc :u*tivit.y of spinach aldolase and t,hat the fructostl Iphosphate activit,y is less than half t,hc initial activity. Thus, spinach c~hloroplast, uldolase appears to be more sensitive to cnrboxypeptidasr action than docks tho rabbit muscle enzyme, although in both C%5W, t,he frucatose I -phosphate :rc*tivity persists. Relatiue actioitg oJ’ spinach aldodnsr toward two substrates. The ac+ivity of spin:&1 aldolase in cat,alyzing the cleavage of hot h fructose diphosphate and sedoheptulose diphosphat)c was examined at all st,:tgcs in t,htt purification proc,edure. Identical amounts OI enzyme source were added to parallel rea+ tion mixtures which were identical in every respect> except the substrate used. Table IV shows that the rat,io of activities toward thr~ two substrates dots not’ vary during t>hr purification. Sodium borohytlndc reduction of dihydroxyacetone phosphate onto the act.ivc site of aldolase specifically inhibits the enzyme (23). When the C~CYI of t8his blocking on activit,y was tested, it) \Y;LSfound that both act,ivities are inhibited to t ho s;lme ext,ent as is shown in Table 1’.

Specific activity Specific activity Ratio

(Fl>P) (SDP)

Before reduction

i\fk!r reduction

!)(5 5.0

0.75 0.41

1 .I)

1 x:Jl

658

BROOKS AND CRIDDLE TABLE KINETIC

Enzyme.. Substrate.

K, (molar/liter) l/F vs. l/S S/V’vs. s v vs. v/s yrn?~ :!

VI

COIWT+NTS FOR CHLOROPLAST ALDOLASE Chloroplast Aldokw?

Rabbit muscle aldolaseb

I

FDP

SDP

FlP

FDP

SDP

6.8 x 10-S 5.7 x 10-c 6.1 X lO+ 3900

1.7 x 10-s 1.6 X W6 1.7 x 10-b 2300

3.9 x 10-a 3.6 X 1F 4.0 x 10-a 156

6.2 X lo-&

3.1 x 10-b 3.0 x 10-S 3.1 x lo-5 5400

-

6200

FiP

4.3 x 10-a

400

~‘Purified chloroplast aldolase was used for K, determinations using FDP and SDP; AS11 was used ‘for K, determination with FlP, and the V maxcited is based on the fraction of aldolase in this preparation. b Rabbit muscle aldolase, three-times recrystallized, was used for determination of K, with SDP; values for FDP and FlP were taken from Westhead et al. (23). c Moles fructose diphosphate cleaved per minute per mole of enzyme using the hydrazine assay. An optical density change of 0.97 at 240 rnp in the hydrazine assay was assumed to correspond to cleavage of one pmole of FDP (24).

Enzyme kinetics. Michaelis constants for spinach aldolase action on the three substrates studied are listed in Table VI. The best-fitting slopes were determined by the least-squares approximation to determine K,. It can be seen that Km’s for fructose diphosphate and sedoheptulose diphosphate are of the same order of magnitude, while the Michaelis constant for fructose l-phosphate activity is much higher. DISCUSSION

Aldolases from many sources have been isolated and shown to have a broad range of substrate specificity with a high degree of selectivity only for the dihydroxyacetone phosphate moiety and a preferred tram configuration on carbon-3 and carbon-4 (23). Sedoheptulose diphosphate cleavage as well as fructose diphosphate cleavage is therefore readily catalyzed by aldolases, and it is no surprise to find a plant aldolase with this capability. The proposed carbon cycle in the dark reaction of photosynthesis suggests two steps of aldolase activity in which glyceraldehyde 3-phosphate and erythrose 4phosphate are coupled to DHAP for the formation of the six carbon and seven carbon sugar diphosphates, respectively. While one enzyme is capable of participating in both reactions, it would be unique to have a single enzyme function both with two

different substrates and at two different steps in a major metabolic pathway. However, the constant ratio of activities for the cleavage of fructose 1,6-diphosphate and sedoheptulose 1,7diphosphate by aldolase at all stages of the enzyme purification from crude homogenate to near homogenity suggests that a single enzyme is present in the chloroplast for carrying out both of these reactions. Blocking the active site of the enzyme by borohydride reduction in the presence of FDP further strengthens this conclusion, as it was shown that 92 % of the activity may be destroyed with no change in the ratio of activities towards the two substrates. The plant enzyme has been classified as a muscle type aldolase, as opposed to the yeast type aldolase, on the basis of its lack of dependence on divalent metal ions for activity as judged by insensitivity to metal-chelating agents (25). This similarity has been further established in this study by the demonstrations of a molecular weight of 142,000 gm per mole and an appreciable enzyme activity using fructose l-phosphate as substrate, both characteristic of the muscle enzyme. The rapid inactivation with carboxypeptidase is also typical of the muscle type enzymes. The maximum specific activity obtained for the plant aldolase using the hydrazine assay was 44 units per milligram, while activities near 75 units per milligram may be obtained with

SPINACH

good preparations of rabbit’ enzyme using this assay. This difference may indicate a real difference between the activities of the two prot)eins but, as aldolase activity is lost quite rapidly in isolated preparations from all sources, it may simply reflect the tliflkulties in preparation of t’he active form of the> spinach enzyme. The kinetic properties of spinach aldolase using &her FDP or SDP as substrate show the relative values of K, and V,,, t’o be of the same order of magnitude. For one enzyme to act on each of two different substrates during each turn of the carbon cycle might well require similar kinetic properties of the enzyme toward both substrates as sho\vn here. REFERENCES 1. (:.u,vIP\‘,

Science 136, 879 (1962) ANI) STUMPF, P. K., Anl. J. Botany 36, 567 (1949). FEWSON, C. A., AL-HAFIDH, M., .\ND GIBBS, >;I., Plant Physiol. 37, 402 (1962). RUTTER, W. J., Federation Proc. 23, 1248 (1961). BENSON, A. A., BASSHBM, J. A., AND CALVIN RI., J. Am. Chem. Sot. 73, 2970 (1951). B.MZL~M, J. A., AND CALVIN, M., “The Path of Carbon in Photosynthesis.” PrentisHall, Englewood Cliffs, N. J. (1957). HOUGH, L., MD JONES, J. K. N., J. Chem. Sot. 342 (1953).

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ALDOL-\SE 8. 9. 10. 11. 12. 13. vz\\s

lIOLI)E, K. E., .ANl) l~.~LI)\\Ih. Phgs. C’hcm. 62, i.34 (lY58). R. J.. .lnn. .y. lT. .Ictr,l. 14. ~).\vIs,

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T., CIJK.~MB.WK, h.. 1x1) S.\V(:~JTOS, 11. A-l., :Ina/. Riochenc. 9, 351 (lU(i41. 16. ROE, J., J. Hiol. (‘hem. 178, 839 /lWY) (;.. in “ Methods in 1~11z~mology” 17. ASHWELL.

(S’. P. Colowick and N. 0. Kaplatt. etls.) VIJ~. III. .%csdemic Press, KeN- \-r)rk (l!f55). 13. I,., J. Hiol. I’hf ,,I. 218, 745 18. ffOltECKFX, 0951;). 0. C., .\NK RIm'l‘.I.EJi, \\'. .I.. ./. 19. RIcJJ.\RJ)s, Hiol. (“hem. 236, 3185 (1961). d. \I;., .\so T’so. I’. (). L’.. .I rcl,. 20. LYTTLETOX, Biochew. Biophys. 73, 120 (1958). Dissertation. Ilniver21. BROOKS, K.. Ijoctoral sity of California, llavis, C‘alifornia 11966). T. kv., .IND &~ORTON. I(. .\., Hio22. ~~(~DwIN, them. J. 40, 628 (1946). E:. W., BUTLER, L., .XXJ) UOYER. 23. WESTHEBD, P. I)., Biochemistry 2, 927 i1963j. B. M., Doctoral Dissertation, I;ni24. WOODFIX, versity of Illinois, Urbana (1963). IP. Boyer. 25. RI.TTER, W. J., in “The Enzymes” H. Lardy and K. Myrback, eds.j Vol. .i. Academic Press, New York (1961).