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Fructose 1,6-diphosphate aldolase of Candida utilis: Purification and properties
ARCHIVES
OF
RIOCHEMISTRY
Fructose
AND
BIOPHYSICS
13-23 (lQ66)
114,
1,bDiphosphate Purification
J. KOWAL:
Aldolase and
T. CREMONA,
of Candida
utilis:
Properties’ AND B. I,. HORECKER
Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York Received December 30, 1965 Fructose diphosphate aldolase has been purified from Curded ~~~Z~s.The final product is homogeneous by several criteria and stable for several weeks in the presence of a reducing agent. The molecular weight from equilibrium sedimentation measurement is 67,500. The enzyme contains 1 mole of zinc per mole of enzyme and is activated by Kf ions; in these properties it resembles the enzyme from Saccharomyces cereoisiae. It is strongly inhibited by chelating agents such as EDTA, o-phenanthroline, and pyrophosphate. The enzyme catalyzes the cleavage of sedoheptulose 1,7-diphosphate and fructose l-phosphate; the reaction rates with these substrates are 2.5 and 0.06”~0of that with fructose 1,6-diphosphate, respectively. No evidence was obtained for the formation of a Schiff base intermediate with the Candida enzyme. Since transaldolase from C. utilis does form a Schiff base intermediate, this organism represents a ease in which both types of enzyme are present.
Fructose 1,6-~phosp~ate aldolase (FDP aldol~e) catalyzes the reversible cleavage of fructose diphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (1, 2). Two classes of FDP aldolases have been described. Those found in mammalian and plant tissues (Class 1) are characterized by the formation of Schiff base intermediates which are reduced by borohydride to yield inactive secondary amine derivatives (3). On the other hand, the FDP aldolases of yeast and bacteria (Class 2) do not form inactive enzyme-substrate products on reduction with borohydride (7). The Class 2 aldolases appear to be metalloproteins (4, 5) ; they are sensitive to chelating agents and are activated by potassium ions (6). The presence of a different type of FDP aldolase in higher plants and animals, as compared with bacteria and molds, is of
considerable interest in relation to the evolutionary origin of these enzymes, particularly since some microorganisms, e.g. Euglena gracilis, appear to possess both types. Purthermose, although Candida utilis has now been shown to possess an FDP aldolase belonging to Class 2, transaldolase in this organism forms a Schiff base intermediate with its substrate (8) and therefore resembles the FDP aldolases of Class 1. Other aldolases of Class I in microorganisms include the deoxyribose phosphate aldolase of L~to~~ciZl~~ ~~~~ar~~~ (9) and the 2-keto-3-deoxy-6-phosphoglu~onate aldolase of PseudorPtonaa~uorescene (10). The present paper describes the purification and properties of FDP aldolase from C. utilis. It has been identified as a Zn++ protein similar in its properties to the Class 2 aldolase isolated from S. cerevisiae (6, 11) ; unlike transaldolase isolated from C. utilis, it could not be shown to form a Schiff base intermediate with its substrate.
I This work. was supported by grants from the National Institutes of Healt,h (GM 11301) and the National Science Foundation (GB 1465). Communication No. 58 from t.he Joan and Lester Avnet Institute of Molecular Biology. 2 Postdoctoral scholar of the American Cancer Society and the National Institutes of Health.
E~~~I~IMENTAL ~~ate~~aZs. Rabbit
PROCEDURES
muscle FDP Jdolase was purified and crystallized by the method of Taylor 13
14
KOWAL,
CREMONA,
et al. (12). Crystalline transaldolase was prepared according to Pontremoli et al. (13). Triosephosa-glycerophosphat,e dehydrophate isomerase, genase, and a preparation containing both enzymes were obtained from Boehringer and Sons. Glucose G-phosphate dehydrogenase was purchased from the same source. Glucose B-phosphate, DPN, and DPNH were purchased from Sigma Chemical and fructose 1 ,&diphosphate from Corporation, the California Corporation for Biochemical Research. Erythrose 4.phosphate was prepared from glucose G-phosphate by a modification of the procedure of Baxter et al. (14). The final product contained 707% of the total organic phosphate as erythrose 4.phosphate and 27yo as glucose 6-phosphate. Caandida utilis dried at low temperature was purchased from the Lake States Yeast Corporation, Rhinelander, Wisconsin. DEAE-Sephadex (A-50 medium), Sephadex G-25, and Sephadex G-100 were purchased from Pharmacia, Uppsala, Sweden. Analytical methods. Znf+ assays were performed according to the dithizone method of Malmstrom (15), ‘as modified by Cremona and Singer (16). Phosphate assays were performed by the FiskeSubbaRow procedure (17). Protein was determined by the method of Lowry et al. (18), or that of Biicher (19). Crystalline FDP aldolase from rabbit muscle was used as t,he standard in these procedures. Fructose 1,~.diphosphate was determined by a coupled enzymic assay with DPNH, rabbit muscle aldolase, a-glycerophosphate dehydrogenase, and triosephosphate isomerase (20). Sedoheptulose 1,7-diphosph~t,e was measured in the same way, omitting triosephosphate isomerase. Dihydroxyacetone phosphate (DHAP) was determined enzymically wit,h ~-glycerophosphate dehydrogen~e and DPNH. Possible contamination of sedoheptulose 1,7-diphosphat,e with fructose 1,6-diphosphate was checked in these det,erminations by adding triosephosphate isomerase to the assay mixture after the initial reaction had terminated. Sedoheptulose 1,7-diphosphate was also determined with t,he or&no1 reaction (21). Glyceraldehyde 3-phosphate was measured by adding triosephosphate isomerase to the DHAP assay after the reduction of dihydroxyacetone phosphat,e was complete. Erythrose 4-phosphate was determined by a coupled spectrophotometric method with excess fructose 6.phosphate in the presence of transaldolase, or-glycerophosphate dehydrogenase, and triosephosphate isomerase (22). ,Spectrophotometric assays were carried out with a Beckman DU spectrophotometer attached to a Gilford multiple sample absorbance recorder,
ANI)
HORECKER
Polyacrylamide disc gel electrophoresis was performed according to the met,hod of Davis (23), on the model 12 Canalco apparatus (Canal Industrial Corp.). Negative pressure dialysis was carried out with the apparatus obtained from Carl Schleicher and Schuell, Inc., Keene, New Hampshire. Preparation of sedoheptulose 1 ,7-diphosphate. A solution (50 ml) cont.aining 300 rmoles of erythrose 4.phosphate and 125 rmoles of fructose 1,6-diphosphate was adjusted to pH 7.2 and treated wit,h 1 mg of triosephosphate isomerase and 7.5 mg of rabbit muscle aldolase, dialyzed against wat,er. The reaction mixture was incubated for 30 minutes at 25’, at which time analysis by the orcinol reaction showed that fructose 1,6-diphosphate had been converted quantitatively to sedoheptulose 1,7-diphosphate. The reaction mixture was placed directly on a Dowex-1-formate column (0.8 X 38 cm) as described by Smyrniotis and Horecker (24). The column was washed wit’h water, and gradient elution was begun with a mixing chamber containi~~g 140 ml of distilled water fed by a reservoir containing 0.2 M formic acid-O.5 h4 sodium formate mixture. The rate of flow was 1.5 ml per minute. A peak containing sedohept~~lose 1,7diphosphate was eluted between 160 and 300 ml; fructose 1,6-diphosphate which would have followed this peak was not detected. The fractions contair~iI~g sedoheptulose 1,7diphosphate (140 ml) were pooled, adjusted to pH 5.0 with 0.2 ml of sat)urated sodium hydroxide, and treated with 0.2 ml of 1 M barium acetate. The solution was then brought’ to pH 6.3 with saturated barium hydroxide, and the barium salt of sedoheptulose 117-diphosphate was precipitated with an equal volume of absolute ethanol. The suspension was left in the cold for 2-3 hours, and the precipitate was collected by centrifugation and dried under vacuum. The weight of recovered material was approximately 150 mg, containing 163 pmoles of sedoheptulose 1,7-diphosphate, dibarium salt. The yield, based on fructose 1 ,6-diphosphate, was 6550/, of theory, and the purity of the final product was 707,. No other phosphate esters were present. RESULTS
Purification of FDP aldolase from Candida. Dried C. ~~~Z~ (135 gm) was autolyzed in 400 ml of 0.2 M potassium phosphate buffer, pH 7.Fi, containing 1 mM mercaptoethanol, for 40 hours at 4’. The autolysate was centrifuged for 15 minutes in the GSA head of the Servall model RC-2 centrifuge at 9000 rpm, and the precipitate was discarded. This extract (Autolysate, Table I) was stable when stored at -20” and could be
--*I-%-- ‘“~U~-AI.J.IJVLAM!~ TABLE
I
--- CANDIDA UJ!
UTILIS
15
110 gm of ammonium sulfate. After 10 minutes the precipitate was collected and dissolved in a minimal amount of 50 mLV ad Re- pot~siunl phosphate buffer, pH 7.25, conTotal units* Eip. c.*very (~oles/min) (units/ 1%) w) taining 1 m&f B-mercaptoethanol (ammo-.Inium sulfate fraction, 40 ml). This fraction Autolysate 14,000 1.1 100 was stable for several days at -20” and Ammonium sulfate frac8.1 65 9300 retained 80% of its activity after storage tion for 3 weeks at this temperature, provided Sepbadex G-25 effluent 9.7 63 8900 that mercaptoethanol was present. DEAE-Sephadex frac24 3000 73 A column of Sephadex G-25 (3.5 X 30 cm) tion I was prepared at 4” by washing with 50 mlM DEAE-Sephadex frac33 4000 48 potassium phosphate buffer, pH 7.25, contion II taining 1 mM ~-mercaptoethanol. The Sephadex G-100 fraction ! (2100)b 79 j 17 a~onium sulfate fraction (40 ml) was a Assays for C. ut~Z~saldolase were carried out placed on the column and washed through in 1 ml of 50 mM glyeylglycine buffer, pH 7.2, at with the same buffer. The fractions con25” containing 0.1 mM DPNH, 2 m&f fructose 1,6taining aldolase activity were combined diphosphate, 100 mM potassium acetate, and 10 (G-25 Sephadex effluent, 65 ml). pg of a mixture of a-glyeerophosphate dehydroDEAE-Sephadex (A-50 medium grade) genase and triosephosphate isomerase. The was suspended in N KC1 and kept overnight preparation to be assayed was diluted in distilled with mechanical stirring. It was then colwater so that 10 pl would give a change in absorblected on a Buchner funnel and washed ance at 340 In@of 0.01-0.03 per minute. One unit of act,ivity represents the quantity required to successively with distilled water, 0.5 N HCl, cleave 1 @mole of fructose 1,6-diphosphate per water, 0.5 N NaOH, and finally with disminute, when a molar absorptivity coefficient of tilled water until neutral. The gel was then 6200 is used for DPNH (25) and it is assumed that suspended at 4” in 50 mM potassium phos2 moles of DPNH are formed for each mole of phate buffer, pH 7.25, cont~ning 1.0 mM fructose 1,6-diphosphat,e cleaved. mer~aptoethanol and 0.1 mM fructose 1,6b Estimated from the experiments with 1 ml diphosphate. The supernatant solution was aliquots. decanted several times to remove the fine particles. A column (4 X 25 cm) was prekept for several days at 4” with little loss of pared and washed with 25 ml of 10 mM activity. The extract (250 ml) was adjusted EDTA, followed by 2 column volumes of to pH 6.8-7.0 and diluted with 100 ml of 50 m&Y phosphate buffer, containing merwater to bring the protein concentration to captoethanol and fructose 1,6-diphosphate 40-45 mg per milliliter. P-Mercaptoethanol as above. The EDTA wash was essential and fructose 1,6-diphosphate were added in order to obtain stable enzyme preparato final concentrations of 2 and 0.1 miV, tions after elution from the column. The respect~ively, and 525 ml of cold saturated G-25 eluate was placed on the column which ammonium sulfate solution, which had been was then washed with 50 ml of the same adjusted to pH 7.5 with concentrated XHB, mixture of buffer, mercaptoethanol, and was added slowly with &irring. The susfructose 1,6-diphosphate. El&on was begun pension was kept at 0” for 10 minutes and with the same buffer mixture containing the precipitate was removed by centrifugaLion. The supernatant solution (825 ml) was 0.1 M potassium chloride. When the first, protein peak was eluted and the absorbance treated with 55 gm of ammonium sulfate of the effluent at 280 rnp had decreased to added slowly with constant stirring. During about 0.100 (3 column volumes), the eluting these procedures care was taken to maintain the pH above 6.8 to avoid loss of enzyme mixture was replaced by a linear gradient M KC1 in 1300 ml of the buffer activity. The precipitate was again removed of 0.143 mixture (4 column volumes). Enzyme acby centrifugation and the supernatant tivity was eluted at, about 0.15 M KC3 as solution was treated with an additio~l PURIFICATION
OF ~~~d~dn
utiEis ALDOLASE
-1
KOWAL,
Cl~E~~O~A,
1i
i;
VOLUME
OF
ELUATE
hnl)
FIG. 1. Sephadex G-100 chromatography of C. utilis FDP aldolase after purification in DEAESephadex. Open circles, absorbance; closed circles, units/ml. (For details see text.)
the first of two slightly overlapping protein peaks. Fractions in the first half of the enzyme peak, wit,h specific activity greater than 70 units per milligram, were pooled (Fraction I, 140 ml). The pooled eluate was then concentrated by negative pressure dialysis in the cold until the protein concentration reached about 2-3 mg per milliliter (15-20 ml). The remaining fractions from the column containing FDP aldolase were pooled (Fraction II, 220 ml) and concentrated in the same way. Other procedures for concentrating the enzyme at this stage, including precipitation with aInmonium sulfate, resulted in large losses of activity. The dilute column effluent was stable for several days at 0” but was unstable to freezing and thawing. The ~oncel~~~rated enzyme was stable for several weeks at 0” in the presence of 1.0 mM mercaptoethanol. Some further purification was achieved by chromatography on Sephadex G-100. For this purpose l-ml aliquots containing 3 mg of enzyme were placed on Sephadex G-100 CoIumns (0.8 X 30 cm) that had been equilibrated with 10 mM tris-HCl buffer, pH 7.2, containing 1.0 mM mercaptoethanol. The protein was eluted with the same buffer mixture and fractions of about 1 ml volume
AND HORECKER,
were collected. The 4 fractions with highest specific activity were pooled (Fig. 1) (Sephadex G-100 fraction, 3 ml). Puriky and molecular weight. Disc electrophoresis of DEAE-Sephadex fraction I on a~ryl~~ide gel at pH 5.4 prior to Sephadex G-100 chromatography revealed a dark band and a minor component that migrated slightly more rapidly, and a faint band close t,o the origin (Fig. 2). These were partly removed by chromatography on Sephadex G-100. When unstained samples were cut into segments and eluted witah 50 mM glycylglycine buffer, it was found that the major dark band corresponded t,o the location of enzyme activity. When the procedure was repeated with the same G-100 effluent that had been stored at 2” for lo-15 days, it was found t,hat several additional components that migrated more slowly than the original enzyme band had appeared. Solutions of enzyme containing 0.2 and 0.35 mg per milliliter (specific activity, 79 units per l~lli~am), obtained aft,er Sephadex G-100 chromatography, were used for molecular weight determination by the equilibrium ~ont.~fugat,ion method of Yphantis (26). Figure 3 shows the data obtained from a typical solvent-solution pair at equilibORIGIN I)
t-1
____..
,/,, . ..._
(+I MAE-SEPHADEX FRACTION I
~ G-100 EFFLUENT
WOO EFFLUENT (AGED1
FIG. 2. Diagrammatic representation of the disc gel electrophoresia patterns with C. utilis aldolase. Electrophoresis of 75Mg C. ~tilis Jdolase in pH 8.4 buffer was carried out according to the procedure of Davis (24). The gel was stained with amido black. The aged effluent was stored for 10 days at 0” in ice.
FDP-ALDOI,AS~
OF CAiVDiD-4
The average molecular weight calculated from 3 determinations was 67,500 gm per mole. Stability of the purified enzyme preparations. The FDP aldolase of 6. utiZis was found to be sensitive to mild acid conditions. During the purification procedure exposure of the enzyme solutions to pH below 6.5 result,ed in progressive inactivation. The purified enz,yme preparations lost, more than 90% of their activity at, 0” after 10 minut.es at pH 5.0. -Piearly 90% of this activity was recovered IThen the pH was again adjusted to 7.0. Return of activity was not immediate and required incubation at 0” for about 10 minutes. The loss of activity at pH 5.0 became irreversible if the preparations were maintained for longer periods at t’his pH. Effect qf pH and monovalent cations on enzyme activity. The purified enzyme preparations from C. utilis showed maximum activity between pH 7.0 and 7.4 (Fig. 4). The nat,ure of the buffer was important; act>ivity was greatest in glycyIglycine buffer and somewhat less in tris-HCl or triethanolamine buffer. Histidine, malonate, maleate, phosphate, and citrate were found to be inhibitory at t,he concentrations employed; the inhibit,ion by maleate was not reversed by addition of tris buffer. Examination of
rium.
3.0
------l
49
50
51 X2 (cm2)
FIG. 3. Molecular weight determination of C. with t,he high speed equilibrium method (26). Meas~Irement~ were made of the net fringe diapla~ement of an 0.03y0 solution of C. z&is aldolase (specific activity, 79 unite/mg) in 50 m&f tris buffer at pH 7.2. The data are from a single solution-solvent pair run in a 12 mm doublewindowed cell after 17 hours at 24,630 rpm at 10”.
utilis aldolase
UTILIS
17
t,be kinetics of inhibit.ion by phosphate and citrate showed these anions to be competitive with substrate. The values of Kc ealculated from reciprocal plots of the data (27) were I..2 X 10W2M for phosphate and 3.5 X 1O-3 M for citrate. The most poLent anion inhibitor was pyrophosphate, which, at a concentration of 6 mM, reduced the enzyme activity to 40 % of the control value. This inhibition was reversed when pyrophosphat,~ was removed by passa,ge though Sephadex G-25 or by a ZO-fold ~Iutio~~. Inhibition of FDP aldolase from S. cerevisiae by histidine and by high concentrations of tris buffer has been reportsed by Richards and Rutter (6). Tris buffer at 0.1 M had no effect on the Candida enzyme. Like yeast aldolase (6), the Candida enzyme showed a requirement for K*(Fig. 5). At a concentration of lo-20 mM K+ the activity was one half of maximum, while at concentrations above 0.2 M, K+ was inhibitory. K+ could be replaced by N&+, which was active at similar concentrations, but not by Li+ or Na+; however, in the presence of these ions higher concentrations of K+ were required to produce maximal activity. Substrate! speci$city. The FDP aldolase of C. utilis was active with sedoheptulose 1,7-diphosphate as well as with fructose 1,6-diphosphate, although the relative activity with the former substrate was much less than with FDP aldolase from rabbit muscle (28). The value of K, for fructose 1,6-diphosphate was 0.8 X 10F4 M, and the extrapolated value for V,,, w&h the most active preparation was 80 units per milligram (Table II). There was no evidence of inhibition by excess substrate at* concenup to 5 trations of fructose 1,6-diphosphate mM. Sedoheptulose 1,7-diphosphate was cleaved at only 2-3% of the rate observed with fructose 1,6-diphosphate (Table II), compared with 45% for FDP aldolase from rabbit muscle. The muscle and Cundida enzymes showed comparable specific activities with sedohel3tulose 1,7-~phosphate, but the latter was much more active with fructose 1,6-~phosphate than was the enzyme from rabbit muscle. The K, of C. utilis aldolase for sedoheptulose 1,7-diphos-
18
KOWAL,
CRE~O~A,
AND IIORECKER
I
I 7.5
7.0
I
I
8.0
8.5
I
9.
PH
FIG. 4. Effect of pH and anions on enzyme activity. All assays were performed as described in the footnote to Table I, except that the glycylglycine buffer in the standard assay was replaced with other buffers as indicated in the figure. The pH of the reaction mixture was checked after each assay. The enzyme preparation was 0.07 pg of the DEAESephadex Fraction I. The results are expressed as a percentage of the activity observed with glycylglycine buffer at pH 7.2. Glyc-glycine and TEA refer to glycylglycine and triethanolamine, respectively.
phate (1.4 x lo-* M) was comparable to that for fructose 1,6-diphosphate. The enzyme from C. ~~~~~was also shown to be capable of catalyzing the synthesis of sedoheptulose 1,7-diphosphate from erythrose 4-phosphate and dihydroxyacetone phosphate. The rate of the synthetic reaction at maximum was similar for the muscle and Candida enzymes. The effect of K+ on the rate of sedoheptulose 1,7-diphos-
phate cleavage was found to be identical to that observed with fructose 1,6-diphosphate as substrate. Fructose 1-phosphate (1 n&f) was cleaved at a rate which was only 0.05% of the rate with fructose 1 ,6-diphosphate. No activity was detected with fructose 6-phosphate as substrate. The large differences in the rates of cleavage of fructose 1,6-~phosphate and
19
I .05 MOLARITY
I II 0.1 fl OF K+
I 0.2
I 0.3
I 0.4
FIG. 5. Effect of potassium acetate concentratjon on activity of C. utilis aldolase. The assays were performed as described in Table I, except that the coneentra~~onof potassium acetate was varied, as shown in the figure. The enzyme preparation was 0.04 pg of DEAESephadex Fraction I {specific activity, 70 units/mg). The ordinate represents mpmoles fructose 1,6-diphosphate cleaved per minute
sedoheptulose 1,7-diphosphate permitted a study of competitive effects of these substrates. Sedoheptulose 1,7-diphosphate was found to be a competitive inhibitor of fructose 1,6-diphosphate cleavage with a Ki value of 1.5 X lo-* .M (Fig. S}. E.~2~~~.~ ~~~i~it~o~ and reactivation. Addition of EDTA to the assay mixtures at concentrations of 0.5 mM severely inhibited catalytic at:tivit,v. This inhibition was progressive with time and was greater in the presence of mercaptoethanol (Fig. 7). In the presence of 10 mM mercaptoethanol, inhibition with 1 mM EDTA was complete after 15 minutes; 5 mM EDTA produced complete inhibition within 2 minutes. Concentrations of EDTA as low as 0.01 mM were inhibitory after longer incubation. ~~ercap~ethanol alone had little effect. o-PhenanthroIine and pyrophosphate (see above) were also potent inhi~tors of the FDP aldolase of 6. utilis; concentrations of o-phenanthroline as low as 0.5 mM produced
complete inhibition within 1 or 2 minutes. In this case mercaptoethanol (or cysteine) did not alter the rate and degree of inhibition. The enzyme, after treatment with EDTA and mercaptoethanol or with ophenanth~oline, was reactivated by dilution or by the addition of a small excess of a divalent cation such as Zn++ , Mg++, or Mn++ (Table III). The 3 cations tested were equally effective, suggesting that their action was related to their ability to bind EDTA. The results also suggested that inhibition was due to formation of dissociable enzyme-EDTA or enzyme-o-phenanthroline complexes, rather than to removal of metal from the protein. This was confirmed by determination of the Zn++ content of the protein before and after treatment with the chelating agent (see below). Zn* ~0~~~~ oj Candida FLIP a~oZ~e. Analysis of the enzyme prep~atio~ for Znf+ showed this metal to be present in amounts which were proportional to the
KOWAL,
20 TABLE
CREMONA,
AND HORECKER
II
C~MPA~I~O~OFSUBSTRATESFECI~ICITI~~OF ALDOUSE FROM C.utilis AND RABBIT MUSCLE ALDOLASE
Fructose diphosphatea 0.8 X 10-“1 80.0 Sedoheptulose diphos- 1.4 X l(r* 1.9 phates 0.05 Fructose 1-phosphatec
FDP
10.5 4.7 0.19
a For determination of specific activity the reaction mixtures contained 2 m&f fructose 1,6diphosphate, 0.1 m&f DPNH, and 10 pg of the mixture of a-glycerophosphate dehydrogenase and triosephosphate iaomerase in 1 ml of 50 mM glycylglycine buffer, pH 7.2. For the C. utilis FDP aldolase assays potassium acetate was added to 100 mM. For determination of K, for C. utilis aldolase the asssys were as above, except that the concentration of fructose 1 ,Gdiphosphate was varied between 0.02 and 0.4 mM. The concentration of enzyme in each assay was 0.05 &ml for C. u$iZis FDP aldolase, and 0.3 p&/ml for rabbit muscle aldolase. 6 For determination of specific activity the reaction mixtures were the same as above, except that sedoheptulose 1,7-diphosphate, 1 mM, was substituted for fructose 1,Gdiphosphate. For determination of K, the concentration of sedoheptulose 1,7-diphosphate varied between 0.02 and 0.4 m&f. The concentration of enzyme used wa,s 4.0 pg/ml for C. ~~tiZi~ FDP aldolase and 1.2 #g/ml for rabbit muscle aldolase. c The assays were the same as above (a) except that 1 mM fructose l-phosphate was substituted for fructose 1,6-diphosphate. The concentration of enzyme was 75 pg/ml for C. utilis aldolase and 2.0 pg/ml for rabbit muscle aldolase. d Calculated from reciprocal plots according to Lineweaver and Burk (27).
J
FIQ. 6. Effect of sedoheptulose 1,7-diphosphate on the rate of cleavage of fructose 1,6-diphosphate. Enzyme assays were carried out as described in the footnote to Table I, except that the concentration of fructose 1,6-diphosphate varied between 0.02 and 0.4 mM. Sedoheptulose 1,7diphosphate was added to the assays as indicated. The enzyme preparation was 0.04 pg of DEAESephadex Fraction I. Velocity was measured as absorbance change per minute. The data are plotted according to Lineweaver and Burk (28).
catalytic activity (Table IV). On the basis of the Zn++ content of the mo& purified enzyme preparations it was estimated that the protein contained one Zn atom per mole&x weight of 70,000. No change in Zn++ content was detected when the enzyme was treated with a solution containing EDTA and mercaptoethanol and then
atomic absorption spectroscopy.3 A preparation with specific activity equal to 78 units per milligram was found to contain 1.03 pg of Zn++ per milligram protein, corresponding to 1.10 atoms per 70,000 gm (Table V). Exa~nation of the ~rep~atio~ for iron, copper, magnesium, and manganese by atomic absorption spectroscopy showed these metals to be present in much smaller quantities. The reversible loss of activity when enzyme solutions were kept at pH 5.0, described in a previous section, could not be attributed to dissociation of Zn++ from the protein, since activity could be restored to the extent of 80 % by neutraliza-
passed through a Sephadex G-25 column. Analysis for Zn++ was also carried out by
aWe are indebted to Miss Sabina Sprague of the Perkin-Elmer Corporation for these analyses.
FDP-ALDOLASE
OF CANDIDA
U!i”ZLZS TABLE
REACTIVATION AFTER
21 III
OF CANDIDA OR INACTIVATION WITH O-P~ENA~THROLINE
FDP ALDOLASE EDTAo=
ictivity
Conditions
tWOk%/
for reactivation
min)
Dilution Addition
MINUTES
FIG. 7. rnhibition of C. z&&is FDP aldolase by EDTA and mercaptoethanol. Assays were carried out as described in Table I with 0.05 pg of DEAESephadex Fraction I. EDTA (2 X 10-a M) and ~-mercaptoethanol (1.5 X 10-S M) were added as indicated in the figure. The ordinate represents mMmoles of fructose 1,6-diphosphate cleaved per minute. tion after passage through a Sephadex G-25 column. No method has yet been found for the reversible dissociation of Zn++ from the enzyme. E$ect of treut~e~t of ~zy~~-~~~~tru~ mixtures with borohydride. The aldolase of C. utilis does not appear to bind dihydroxyacetone phosphate by a Schiff base mechanism. A number of attempts were made to inactivate the enzyme by reduction with borohydride in the presence of dihydroxyacetone phosphate or fructose 1 ,6-diphosphate. The enzyme was stable to treatment with borohydricie under these conditions, and virtually no loss of activity occurred either in the presence or absence of substrate at pH L&7.0. The t,reatment with borohydride was continued for 10 minutes at the lower pH and for 30-60 minutes at the higher pH values. Treatment at pH 6.0 and 7.5 with radioactive dihydroxyace~ne phosphate and borohydride resulted in the binding of less than 0.2 equivalent of substrate per mole of enzyme. When muscle FDP aldolase was treated under similar conditions, at pH 6.0, there was 90% inactivation with the uptake of about 2 equiva-
of Zn++, 1.0 mM 2.5 mM 3.5 mM of Mg++, 3.5 mM of Mn++, 3.5 mM
Addition Addition Dilution Addition of Zn++, 3.0 mM Addition of MS++, 3.5 m&f Addition of Mn*, 3.5 mM
-
3.0 0 1.1 2.2 2.1 2.1 4.9 4.3 4.3 4.5
91 0 37 75 70 72 100 90 90 94
a The incubation mixture for inactivation (0.2 ml) contained 0.22 mg of FDP aldolase (specific activity, 60 units/mg), 50 mM phosphate buffer, pH 7.2, 2.5 mM EDTA, and 10 mM mercaptoethanol. After 30 minutes at 25” aliquots (10~1 of a 1:2OOdilution) were added to the standard assay mixture. In the experiment with added cations the assay mixture also contained 2.6 mM EDTA and 10 m&f meroaptoethanol. With 2.5 mM EDTA in the assay mixture no activity was detected in the absence of the divalent cations. *The conditions were as in Experiment I, except, that 0.5 mM o-phenanthroline was present instead of EDTA and mercaptoethanol, and the content of enzyme in 0.2 ml was 0.35 mg. After 3-5 minutes, aliquots (1 ~1) were analyzed in the standard assay mixture. In the experiment with added cations the assay mixture also contained 0.5 mM o-phenanthroline.
lents of radioactive ~hydroxyacetoue phate per mole of enzyme (3,29).
phos-
DISCUSSION
Although the FDP aldolase of G. utitis is relatively unstable, particularly to acid conditions, we have obtained preparations of high specific activity that can be stored at low temperature for at least several weeks. Maintenance of activity during storage appears to require a sulfhydryl or other reducing reagent, but the purified enzyme does not require the addition of zinc or cysteine, or both, in the assay mixture, unlike preparations from S. cerevisiae.
22
KOWAL, TABLE
Assay
OF
IV
ALDOLA~EPREP~\RATIONS FORZN++ zn++
sp. act. (units/mg)
30 40 70 70
pmoles
0.37 0.460 0.800 0.740
FROMC.Z&~~
contenta
r&N
CREMONA,
Zn++/mg
moles Zn++/ 7a,ooo gm
0.530 0.705 1.230 1.140
0.390 0.495 0.860 0.800
a The preparations analyzed were obtained by purification through the DEAE-Sephadex step. They were dialyzed for 15 hours against 5 mM tris buffer, pH 7.2, prepared in Zn-free water containing 1 m&f mercapt.oethanol. The last entry in the table represents a preparation which had been treated with 2.5 mM EDTA and 10 mM mercaptoethanol and passed through a Sephadex G-25 column equilibrated with 5 mM tris buffer, pH 7.4. Each value represents the average of 3 determinations which agreed within 10%. Zn++ assays were carried out by a modification of the dithizone method of Malmstrom (15). TABLE
V
ATOMIC ABSORPTION SPECTROGRAPHIC ANALYSIS FORDI~~LENTMET~LSIN YEAST ALDOLASE
content ExperimenP
Cont.(ppm)
Metal
moles/ #dmx 70,000 gm
protein I II III
IV V
Zn++ cuff Fe++ Mn++ Mg++
-
0.635 1; --ii’0.120 0:193 0.106 0.161 None found 0
1.10 0.21 0.20 0 <0.06
5 C. utitis aldolase (specific activity, 79) was prepared for assay by passage through a small Chelex column after concentration by negative pressure dialysis. Traces of zinc, iron, and copper amounting to less than 0.1 ppm were found in the sample of buffer provided for analysis.
The results of our studies clearly place the FDP aldolase of C. ~~i~~ in the category of Class 2 aldolases. It is similar in many respects to the enzyme isolated from X. cercvisiue (4, 6). The molecular weight is approximately 70,000 and the enzyme contains 1 mole of Zn++ per mole of protein. It is strongly inhibited by chelating agents,
AND HORECKER
and evidence has been obtained that this i~hibitio~l is due to formation of an enzymechelator complex, rather than to removal of t,he metal. Inhibition by chelating agents is readily reversed by dilution, dialysis, or passage through Sephadex columns. Attempts to remove the tightly bound zinc with the formation of an active apoenzyme were unsuccessf~~1. Numerous efforts were made to obtain evidence for a Schiff base intermediate with this enzyme; these were uniformly unsuccessful. This result is in agreement with the findings of Rutter (7) that Class 2 aldolases do not form such intermediates. iEtut,ter has proposed a model in which Znf+ serves as a binding site for the substrate. The development of a satisfactory method for removing zinc in a reversible manner with the formation of a metal-free apoenzyme wouId be invaluable for further studies of the active site of this enzyme. unfortunately, no such met,hod is yet available. FDP aldolase from C. z&is, like the enzyme from rabbit muscle, catalyzes the cleavage of sedoheptulose 1,7-diphosphate and fructose l-phosphate, as well as of fructose 1,6-diphosphate. However, the activity with these substrates is low compared to that with fructose 1,6-diphosphate. Thus, the rate of cleavage of sedoheptulose 1,7-diphosphate was found to be less than 3 % of that with fructose 1,6-diphosphate; the low activity with fructose l-phosphate is similar to that reported for the enzyme from S. cerevisiae (30). The lack of significant activity with fructose l-phosphate is not surprising, since there is no evidence for a role of fructose l-phosphate in the intermediary met,aboIism of C. utilis. &md&&r utilis thus contains aldolases of two classes, since it has been demonstrated that transaldolase in this organism does form a Schiff base intermediate (8). The presence of both classes of aldolases in a single cell is of interest in relation to t.he evolutionary origin of these enzymes. REFERENCES 1. MEYEILHOF, O., ANDLOHMANN, K., Bi0chem.Z. 271, 89 (1934). 2. MEYERHOF,O.,B~&SOC.
1345 (1938).
Chim. Biol.20.1033,
FDP-ALI~OLAS~
OF C.4NDIL)B
3. HORECBER, B. L., ROWLEY, P. T., GRAZI, E., CHENG, T., AND TCHOLA, O., Biochem. Z. 338, 36 (1963). 4. WARBURG, O., AND CHRISTIAN, W., Biochem. 2. 314, 149 (1943). BARD, R. C., AND GUNSALUS, I. C., J. Bacteriol. 69, 387 (1950). RICHARDS, 0. C., AND RIJTTER, W. J., J. Biol. Chem. 236, 3177 (1961). Proc. 231, 1248 RUTTER, W. J., Federation (1964). HORECKER, B. L., PO~TREMOLI, S., RICCI, C., AND CHPNG, T., Proc. Xatr. Acad. Sci. U. S. 47, 1949 (1961). 9. HOFFEE, P., R.OSEN, O., AND HORECBER, B. L., J. BioE. Chem. 240, 1512 (1965). 10. MELOCHE, H. P., AND WOOD, W. A., J. Biol. Chem. 239, 3515 (1964). B. S., MEINHART, J. O., 11. VANDERHEIDEN, DODSON, R. G., AND KEEPS, E. G., J. Biol. Chem. 237, 2095 (1962). 12. TAYLOR, J. F., GREEN, A. A., AND CORI, G. T., J. Biol. Chem. 173, 591 (1948). 13. PONTREMOLI, S., PRANDINI, B. D., BONSIGNORE, A., AND HORECKER, B. L., Proc. Natl. Aead. Sci. U. S. 47, 1942 (lQ61). 14. BAXTER, J. N., PERLIN, A. S., AN’D SIMPSON, F. J., Can. J. Bioehem. Physiol. 37, 199
,,nZZZ?\ .
fIJr)J/
15. MALMSTR~M, B., ia “Methods of Biochemical Analysis” (D. Click, ed.), Vol. 3, p. 340. Wiley ([nterscience), New York (1956). 16. CREMONA, T., AND SINGER, T. P., J. Biol. Chem. 239, 1466 (1964).
17. FISKE,
UTILTS C. H.,
23 AND SUBBARO~,
Y., J. Biol.
Chem. 81, 629 (1929). 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 19. B~.!cHER, T., Biochim. Biophys. Acta 1, 292 (1947). 20. RACKER, E., AND SCHROEDER, E. A. R., Arch. Biochem. Biophys. 74, 326 (1958). 21. HORECKER, B. L., in. “Methods in Enzymology” (S. P. Colowiek and N. 0. Kaplan, eds.), Vol. III, p. 105. Academic Press, New York (1955). 22. COOPER, J., SRERE, P. A., TABACHNICK, M., AND RACKER, E., Arch. Biochem. Biophys. 74, 306 (1958). 23. DAVIS, B. J., Ann. N. Y. Acad. Sci. 212, 404 (1964) * 24. SMYRNIOTXS, P. Z., AND HORECKER, B. b., J. Biol. Chem. 218, 745 (1956). 25. HORECKER, B. L., AND KORNBERG, A., J. Biol. Chem. 176, 385 (1948). 26. YPHANTIS, D. A., Biochemistry 3, 297 (1964). 27. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 28. HORECEER, B. L., S~YRNIO~S, P. Z., HEATH, H. H., AND MARKS, P. A., J. Biol. Chem. i@!& 827 (1955). 29. LAI, C. Y., TCHOLA, O., CHENG, T., AND HORECKER, B. L., J. Biot. Chem. 240, 1347 (1965). 30. RICHARDS, 0. C., AND RUTTER, W. J., J. Biol. Chem. 236, 3185 (1961).
OF
RIOCHEMISTRY
Fructose
AND
BIOPHYSICS
13-23 (lQ66)
114,
1,bDiphosphate Purification
J. KOWAL:
Aldolase and
T. CREMONA,
of Candida
utilis:
Properties’ AND B. I,. HORECKER
Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York Received December 30, 1965 Fructose diphosphate aldolase has been purified from Curded ~~~Z~s.The final product is homogeneous by several criteria and stable for several weeks in the presence of a reducing agent. The molecular weight from equilibrium sedimentation measurement is 67,500. The enzyme contains 1 mole of zinc per mole of enzyme and is activated by Kf ions; in these properties it resembles the enzyme from Saccharomyces cereoisiae. It is strongly inhibited by chelating agents such as EDTA, o-phenanthroline, and pyrophosphate. The enzyme catalyzes the cleavage of sedoheptulose 1,7-diphosphate and fructose l-phosphate; the reaction rates with these substrates are 2.5 and 0.06”~0of that with fructose 1,6-diphosphate, respectively. No evidence was obtained for the formation of a Schiff base intermediate with the Candida enzyme. Since transaldolase from C. utilis does form a Schiff base intermediate, this organism represents a ease in which both types of enzyme are present.
Fructose 1,6-~phosp~ate aldolase (FDP aldol~e) catalyzes the reversible cleavage of fructose diphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (1, 2). Two classes of FDP aldolases have been described. Those found in mammalian and plant tissues (Class 1) are characterized by the formation of Schiff base intermediates which are reduced by borohydride to yield inactive secondary amine derivatives (3). On the other hand, the FDP aldolases of yeast and bacteria (Class 2) do not form inactive enzyme-substrate products on reduction with borohydride (7). The Class 2 aldolases appear to be metalloproteins (4, 5) ; they are sensitive to chelating agents and are activated by potassium ions (6). The presence of a different type of FDP aldolase in higher plants and animals, as compared with bacteria and molds, is of
considerable interest in relation to the evolutionary origin of these enzymes, particularly since some microorganisms, e.g. Euglena gracilis, appear to possess both types. Purthermose, although Candida utilis has now been shown to possess an FDP aldolase belonging to Class 2, transaldolase in this organism forms a Schiff base intermediate with its substrate (8) and therefore resembles the FDP aldolases of Class 1. Other aldolases of Class I in microorganisms include the deoxyribose phosphate aldolase of L~to~~ciZl~~ ~~~~ar~~~ (9) and the 2-keto-3-deoxy-6-phosphoglu~onate aldolase of PseudorPtonaa~uorescene (10). The present paper describes the purification and properties of FDP aldolase from C. utilis. It has been identified as a Zn++ protein similar in its properties to the Class 2 aldolase isolated from S. cerevisiae (6, 11) ; unlike transaldolase isolated from C. utilis, it could not be shown to form a Schiff base intermediate with its substrate.
I This work. was supported by grants from the National Institutes of Healt,h (GM 11301) and the National Science Foundation (GB 1465). Communication No. 58 from t.he Joan and Lester Avnet Institute of Molecular Biology. 2 Postdoctoral scholar of the American Cancer Society and the National Institutes of Health.
E~~~I~IMENTAL ~~ate~~aZs. Rabbit
PROCEDURES
muscle FDP Jdolase was purified and crystallized by the method of Taylor 13
14
KOWAL,
CREMONA,
et al. (12). Crystalline transaldolase was prepared according to Pontremoli et al. (13). Triosephosa-glycerophosphat,e dehydrophate isomerase, genase, and a preparation containing both enzymes were obtained from Boehringer and Sons. Glucose G-phosphate dehydrogenase was purchased from the same source. Glucose B-phosphate, DPN, and DPNH were purchased from Sigma Chemical and fructose 1 ,&diphosphate from Corporation, the California Corporation for Biochemical Research. Erythrose 4.phosphate was prepared from glucose G-phosphate by a modification of the procedure of Baxter et al. (14). The final product contained 707% of the total organic phosphate as erythrose 4.phosphate and 27yo as glucose 6-phosphate. Caandida utilis dried at low temperature was purchased from the Lake States Yeast Corporation, Rhinelander, Wisconsin. DEAE-Sephadex (A-50 medium), Sephadex G-25, and Sephadex G-100 were purchased from Pharmacia, Uppsala, Sweden. Analytical methods. Znf+ assays were performed according to the dithizone method of Malmstrom (15), ‘as modified by Cremona and Singer (16). Phosphate assays were performed by the FiskeSubbaRow procedure (17). Protein was determined by the method of Lowry et al. (18), or that of Biicher (19). Crystalline FDP aldolase from rabbit muscle was used as t,he standard in these procedures. Fructose 1,~.diphosphate was determined by a coupled enzymic assay with DPNH, rabbit muscle aldolase, a-glycerophosphate dehydrogenase, and triosephosphate isomerase (20). Sedoheptulose 1,7-diphosph~t,e was measured in the same way, omitting triosephosphate isomerase. Dihydroxyacetone phosphate (DHAP) was determined enzymically wit,h ~-glycerophosphate dehydrogen~e and DPNH. Possible contamination of sedoheptulose 1,7-diphosphat,e with fructose 1,6-diphosphate was checked in these det,erminations by adding triosephosphate isomerase to the assay mixture after the initial reaction had terminated. Sedoheptulose 1,7-diphosphate was also determined with t,he or&no1 reaction (21). Glyceraldehyde 3-phosphate was measured by adding triosephosphate isomerase to the DHAP assay after the reduction of dihydroxyacetone phosphat,e was complete. Erythrose 4-phosphate was determined by a coupled spectrophotometric method with excess fructose 6.phosphate in the presence of transaldolase, or-glycerophosphate dehydrogenase, and triosephosphate isomerase (22). ,Spectrophotometric assays were carried out with a Beckman DU spectrophotometer attached to a Gilford multiple sample absorbance recorder,
ANI)
HORECKER
Polyacrylamide disc gel electrophoresis was performed according to the met,hod of Davis (23), on the model 12 Canalco apparatus (Canal Industrial Corp.). Negative pressure dialysis was carried out with the apparatus obtained from Carl Schleicher and Schuell, Inc., Keene, New Hampshire. Preparation of sedoheptulose 1 ,7-diphosphate. A solution (50 ml) cont.aining 300 rmoles of erythrose 4.phosphate and 125 rmoles of fructose 1,6-diphosphate was adjusted to pH 7.2 and treated wit,h 1 mg of triosephosphate isomerase and 7.5 mg of rabbit muscle aldolase, dialyzed against wat,er. The reaction mixture was incubated for 30 minutes at 25’, at which time analysis by the orcinol reaction showed that fructose 1,6-diphosphate had been converted quantitatively to sedoheptulose 1,7-diphosphate. The reaction mixture was placed directly on a Dowex-1-formate column (0.8 X 38 cm) as described by Smyrniotis and Horecker (24). The column was washed wit’h water, and gradient elution was begun with a mixing chamber containi~~g 140 ml of distilled water fed by a reservoir containing 0.2 M formic acid-O.5 h4 sodium formate mixture. The rate of flow was 1.5 ml per minute. A peak containing sedohept~~lose 1,7diphosphate was eluted between 160 and 300 ml; fructose 1,6-diphosphate which would have followed this peak was not detected. The fractions contair~iI~g sedoheptulose 1,7diphosphate (140 ml) were pooled, adjusted to pH 5.0 with 0.2 ml of sat)urated sodium hydroxide, and treated with 0.2 ml of 1 M barium acetate. The solution was then brought’ to pH 6.3 with saturated barium hydroxide, and the barium salt of sedoheptulose 117-diphosphate was precipitated with an equal volume of absolute ethanol. The suspension was left in the cold for 2-3 hours, and the precipitate was collected by centrifugation and dried under vacuum. The weight of recovered material was approximately 150 mg, containing 163 pmoles of sedoheptulose 1,7-diphosphate, dibarium salt. The yield, based on fructose 1 ,6-diphosphate, was 6550/, of theory, and the purity of the final product was 707,. No other phosphate esters were present. RESULTS
Purification of FDP aldolase from Candida. Dried C. ~~~Z~ (135 gm) was autolyzed in 400 ml of 0.2 M potassium phosphate buffer, pH 7.Fi, containing 1 mM mercaptoethanol, for 40 hours at 4’. The autolysate was centrifuged for 15 minutes in the GSA head of the Servall model RC-2 centrifuge at 9000 rpm, and the precipitate was discarded. This extract (Autolysate, Table I) was stable when stored at -20” and could be
--*I-%-- ‘“~U~-AI.J.IJVLAM!~ TABLE
I
--- CANDIDA UJ!
UTILIS
15
110 gm of ammonium sulfate. After 10 minutes the precipitate was collected and dissolved in a minimal amount of 50 mLV ad Re- pot~siunl phosphate buffer, pH 7.25, conTotal units* Eip. c.*very (~oles/min) (units/ 1%) w) taining 1 m&f B-mercaptoethanol (ammo-.Inium sulfate fraction, 40 ml). This fraction Autolysate 14,000 1.1 100 was stable for several days at -20” and Ammonium sulfate frac8.1 65 9300 retained 80% of its activity after storage tion for 3 weeks at this temperature, provided Sepbadex G-25 effluent 9.7 63 8900 that mercaptoethanol was present. DEAE-Sephadex frac24 3000 73 A column of Sephadex G-25 (3.5 X 30 cm) tion I was prepared at 4” by washing with 50 mlM DEAE-Sephadex frac33 4000 48 potassium phosphate buffer, pH 7.25, contion II taining 1 mM ~-mercaptoethanol. The Sephadex G-100 fraction ! (2100)b 79 j 17 a~onium sulfate fraction (40 ml) was a Assays for C. ut~Z~saldolase were carried out placed on the column and washed through in 1 ml of 50 mM glyeylglycine buffer, pH 7.2, at with the same buffer. The fractions con25” containing 0.1 mM DPNH, 2 m&f fructose 1,6taining aldolase activity were combined diphosphate, 100 mM potassium acetate, and 10 (G-25 Sephadex effluent, 65 ml). pg of a mixture of a-glyeerophosphate dehydroDEAE-Sephadex (A-50 medium grade) genase and triosephosphate isomerase. The was suspended in N KC1 and kept overnight preparation to be assayed was diluted in distilled with mechanical stirring. It was then colwater so that 10 pl would give a change in absorblected on a Buchner funnel and washed ance at 340 In@of 0.01-0.03 per minute. One unit of act,ivity represents the quantity required to successively with distilled water, 0.5 N HCl, cleave 1 @mole of fructose 1,6-diphosphate per water, 0.5 N NaOH, and finally with disminute, when a molar absorptivity coefficient of tilled water until neutral. The gel was then 6200 is used for DPNH (25) and it is assumed that suspended at 4” in 50 mM potassium phos2 moles of DPNH are formed for each mole of phate buffer, pH 7.25, cont~ning 1.0 mM fructose 1,6-diphosphat,e cleaved. mer~aptoethanol and 0.1 mM fructose 1,6b Estimated from the experiments with 1 ml diphosphate. The supernatant solution was aliquots. decanted several times to remove the fine particles. A column (4 X 25 cm) was prekept for several days at 4” with little loss of pared and washed with 25 ml of 10 mM activity. The extract (250 ml) was adjusted EDTA, followed by 2 column volumes of to pH 6.8-7.0 and diluted with 100 ml of 50 m&Y phosphate buffer, containing merwater to bring the protein concentration to captoethanol and fructose 1,6-diphosphate 40-45 mg per milliliter. P-Mercaptoethanol as above. The EDTA wash was essential and fructose 1,6-diphosphate were added in order to obtain stable enzyme preparato final concentrations of 2 and 0.1 miV, tions after elution from the column. The respect~ively, and 525 ml of cold saturated G-25 eluate was placed on the column which ammonium sulfate solution, which had been was then washed with 50 ml of the same adjusted to pH 7.5 with concentrated XHB, mixture of buffer, mercaptoethanol, and was added slowly with &irring. The susfructose 1,6-diphosphate. El&on was begun pension was kept at 0” for 10 minutes and with the same buffer mixture containing the precipitate was removed by centrifugaLion. The supernatant solution (825 ml) was 0.1 M potassium chloride. When the first, protein peak was eluted and the absorbance treated with 55 gm of ammonium sulfate of the effluent at 280 rnp had decreased to added slowly with constant stirring. During about 0.100 (3 column volumes), the eluting these procedures care was taken to maintain the pH above 6.8 to avoid loss of enzyme mixture was replaced by a linear gradient M KC1 in 1300 ml of the buffer activity. The precipitate was again removed of 0.143 mixture (4 column volumes). Enzyme acby centrifugation and the supernatant tivity was eluted at, about 0.15 M KC3 as solution was treated with an additio~l PURIFICATION
OF ~~~d~dn
utiEis ALDOLASE
-1
KOWAL,
Cl~E~~O~A,
1i
i;
VOLUME
OF
ELUATE
hnl)
FIG. 1. Sephadex G-100 chromatography of C. utilis FDP aldolase after purification in DEAESephadex. Open circles, absorbance; closed circles, units/ml. (For details see text.)
the first of two slightly overlapping protein peaks. Fractions in the first half of the enzyme peak, wit,h specific activity greater than 70 units per milligram, were pooled (Fraction I, 140 ml). The pooled eluate was then concentrated by negative pressure dialysis in the cold until the protein concentration reached about 2-3 mg per milliliter (15-20 ml). The remaining fractions from the column containing FDP aldolase were pooled (Fraction II, 220 ml) and concentrated in the same way. Other procedures for concentrating the enzyme at this stage, including precipitation with aInmonium sulfate, resulted in large losses of activity. The dilute column effluent was stable for several days at 0” but was unstable to freezing and thawing. The ~oncel~~~rated enzyme was stable for several weeks at 0” in the presence of 1.0 mM mercaptoethanol. Some further purification was achieved by chromatography on Sephadex G-100. For this purpose l-ml aliquots containing 3 mg of enzyme were placed on Sephadex G-100 CoIumns (0.8 X 30 cm) that had been equilibrated with 10 mM tris-HCl buffer, pH 7.2, containing 1.0 mM mercaptoethanol. The protein was eluted with the same buffer mixture and fractions of about 1 ml volume
AND HORECKER,
were collected. The 4 fractions with highest specific activity were pooled (Fig. 1) (Sephadex G-100 fraction, 3 ml). Puriky and molecular weight. Disc electrophoresis of DEAE-Sephadex fraction I on a~ryl~~ide gel at pH 5.4 prior to Sephadex G-100 chromatography revealed a dark band and a minor component that migrated slightly more rapidly, and a faint band close t,o the origin (Fig. 2). These were partly removed by chromatography on Sephadex G-100. When unstained samples were cut into segments and eluted witah 50 mM glycylglycine buffer, it was found that the major dark band corresponded t,o the location of enzyme activity. When the procedure was repeated with the same G-100 effluent that had been stored at 2” for lo-15 days, it was found t,hat several additional components that migrated more slowly than the original enzyme band had appeared. Solutions of enzyme containing 0.2 and 0.35 mg per milliliter (specific activity, 79 units per l~lli~am), obtained aft,er Sephadex G-100 chromatography, were used for molecular weight determination by the equilibrium ~ont.~fugat,ion method of Yphantis (26). Figure 3 shows the data obtained from a typical solvent-solution pair at equilibORIGIN I)
t-1
____..
,/,, . ..._
(+I MAE-SEPHADEX FRACTION I
~ G-100 EFFLUENT
WOO EFFLUENT (AGED1
FIG. 2. Diagrammatic representation of the disc gel electrophoresia patterns with C. utilis aldolase. Electrophoresis of 75Mg C. ~tilis Jdolase in pH 8.4 buffer was carried out according to the procedure of Davis (24). The gel was stained with amido black. The aged effluent was stored for 10 days at 0” in ice.
FDP-ALDOI,AS~
OF CAiVDiD-4
The average molecular weight calculated from 3 determinations was 67,500 gm per mole. Stability of the purified enzyme preparations. The FDP aldolase of 6. utiZis was found to be sensitive to mild acid conditions. During the purification procedure exposure of the enzyme solutions to pH below 6.5 result,ed in progressive inactivation. The purified enz,yme preparations lost, more than 90% of their activity at, 0” after 10 minut.es at pH 5.0. -Piearly 90% of this activity was recovered IThen the pH was again adjusted to 7.0. Return of activity was not immediate and required incubation at 0” for about 10 minutes. The loss of activity at pH 5.0 became irreversible if the preparations were maintained for longer periods at t’his pH. Effect qf pH and monovalent cations on enzyme activity. The purified enzyme preparations from C. utilis showed maximum activity between pH 7.0 and 7.4 (Fig. 4). The nat,ure of the buffer was important; act>ivity was greatest in glycyIglycine buffer and somewhat less in tris-HCl or triethanolamine buffer. Histidine, malonate, maleate, phosphate, and citrate were found to be inhibitory at t,he concentrations employed; the inhibit,ion by maleate was not reversed by addition of tris buffer. Examination of
rium.
3.0
------l
49
50
51 X2 (cm2)
FIG. 3. Molecular weight determination of C. with t,he high speed equilibrium method (26). Meas~Irement~ were made of the net fringe diapla~ement of an 0.03y0 solution of C. z&is aldolase (specific activity, 79 unite/mg) in 50 m&f tris buffer at pH 7.2. The data are from a single solution-solvent pair run in a 12 mm doublewindowed cell after 17 hours at 24,630 rpm at 10”.
utilis aldolase
UTILIS
17
t,be kinetics of inhibit.ion by phosphate and citrate showed these anions to be competitive with substrate. The values of Kc ealculated from reciprocal plots of the data (27) were I..2 X 10W2M for phosphate and 3.5 X 1O-3 M for citrate. The most poLent anion inhibitor was pyrophosphate, which, at a concentration of 6 mM, reduced the enzyme activity to 40 % of the control value. This inhibition was reversed when pyrophosphat,~ was removed by passa,ge though Sephadex G-25 or by a ZO-fold ~Iutio~~. Inhibition of FDP aldolase from S. cerevisiae by histidine and by high concentrations of tris buffer has been reportsed by Richards and Rutter (6). Tris buffer at 0.1 M had no effect on the Candida enzyme. Like yeast aldolase (6), the Candida enzyme showed a requirement for K*(Fig. 5). At a concentration of lo-20 mM K+ the activity was one half of maximum, while at concentrations above 0.2 M, K+ was inhibitory. K+ could be replaced by N&+, which was active at similar concentrations, but not by Li+ or Na+; however, in the presence of these ions higher concentrations of K+ were required to produce maximal activity. Substrate! speci$city. The FDP aldolase of C. utilis was active with sedoheptulose 1,7-diphosphate as well as with fructose 1,6-diphosphate, although the relative activity with the former substrate was much less than with FDP aldolase from rabbit muscle (28). The value of K, for fructose 1,6-diphosphate was 0.8 X 10F4 M, and the extrapolated value for V,,, w&h the most active preparation was 80 units per milligram (Table II). There was no evidence of inhibition by excess substrate at* concenup to 5 trations of fructose 1,6-diphosphate mM. Sedoheptulose 1,7-diphosphate was cleaved at only 2-3% of the rate observed with fructose 1,6-diphosphate (Table II), compared with 45% for FDP aldolase from rabbit muscle. The muscle and Cundida enzymes showed comparable specific activities with sedohel3tulose 1,7-~phosphate, but the latter was much more active with fructose 1,6-~phosphate than was the enzyme from rabbit muscle. The K, of C. utilis aldolase for sedoheptulose 1,7-diphos-
18
KOWAL,
CRE~O~A,
AND IIORECKER
I
I 7.5
7.0
I
I
8.0
8.5
I
9.
PH
FIG. 4. Effect of pH and anions on enzyme activity. All assays were performed as described in the footnote to Table I, except that the glycylglycine buffer in the standard assay was replaced with other buffers as indicated in the figure. The pH of the reaction mixture was checked after each assay. The enzyme preparation was 0.07 pg of the DEAESephadex Fraction I. The results are expressed as a percentage of the activity observed with glycylglycine buffer at pH 7.2. Glyc-glycine and TEA refer to glycylglycine and triethanolamine, respectively.
phate (1.4 x lo-* M) was comparable to that for fructose 1,6-diphosphate. The enzyme from C. ~~~~~was also shown to be capable of catalyzing the synthesis of sedoheptulose 1,7-diphosphate from erythrose 4-phosphate and dihydroxyacetone phosphate. The rate of the synthetic reaction at maximum was similar for the muscle and Candida enzymes. The effect of K+ on the rate of sedoheptulose 1,7-diphos-
phate cleavage was found to be identical to that observed with fructose 1,6-diphosphate as substrate. Fructose 1-phosphate (1 n&f) was cleaved at a rate which was only 0.05% of the rate with fructose 1 ,6-diphosphate. No activity was detected with fructose 6-phosphate as substrate. The large differences in the rates of cleavage of fructose 1,6-~phosphate and
19
I .05 MOLARITY
I II 0.1 fl OF K+
I 0.2
I 0.3
I 0.4
FIG. 5. Effect of potassium acetate concentratjon on activity of C. utilis aldolase. The assays were performed as described in Table I, except that the coneentra~~onof potassium acetate was varied, as shown in the figure. The enzyme preparation was 0.04 pg of DEAESephadex Fraction I {specific activity, 70 units/mg). The ordinate represents mpmoles fructose 1,6-diphosphate cleaved per minute
sedoheptulose 1,7-diphosphate permitted a study of competitive effects of these substrates. Sedoheptulose 1,7-diphosphate was found to be a competitive inhibitor of fructose 1,6-diphosphate cleavage with a Ki value of 1.5 X lo-* .M (Fig. S}. E.~2~~~.~ ~~~i~it~o~ and reactivation. Addition of EDTA to the assay mixtures at concentrations of 0.5 mM severely inhibited catalytic at:tivit,v. This inhibition was progressive with time and was greater in the presence of mercaptoethanol (Fig. 7). In the presence of 10 mM mercaptoethanol, inhibition with 1 mM EDTA was complete after 15 minutes; 5 mM EDTA produced complete inhibition within 2 minutes. Concentrations of EDTA as low as 0.01 mM were inhibitory after longer incubation. ~~ercap~ethanol alone had little effect. o-PhenanthroIine and pyrophosphate (see above) were also potent inhi~tors of the FDP aldolase of 6. utilis; concentrations of o-phenanthroline as low as 0.5 mM produced
complete inhibition within 1 or 2 minutes. In this case mercaptoethanol (or cysteine) did not alter the rate and degree of inhibition. The enzyme, after treatment with EDTA and mercaptoethanol or with ophenanth~oline, was reactivated by dilution or by the addition of a small excess of a divalent cation such as Zn++ , Mg++, or Mn++ (Table III). The 3 cations tested were equally effective, suggesting that their action was related to their ability to bind EDTA. The results also suggested that inhibition was due to formation of dissociable enzyme-EDTA or enzyme-o-phenanthroline complexes, rather than to removal of metal from the protein. This was confirmed by determination of the Zn++ content of the protein before and after treatment with the chelating agent (see below). Zn* ~0~~~~ oj Candida FLIP a~oZ~e. Analysis of the enzyme prep~atio~ for Znf+ showed this metal to be present in amounts which were proportional to the
KOWAL,
20 TABLE
CREMONA,
AND HORECKER
II
C~MPA~I~O~OFSUBSTRATESFECI~ICITI~~OF ALDOUSE FROM C.utilis AND RABBIT MUSCLE ALDOLASE
Fructose diphosphatea 0.8 X 10-“1 80.0 Sedoheptulose diphos- 1.4 X l(r* 1.9 phates 0.05 Fructose 1-phosphatec
FDP
10.5 4.7 0.19
a For determination of specific activity the reaction mixtures contained 2 m&f fructose 1,6diphosphate, 0.1 m&f DPNH, and 10 pg of the mixture of a-glycerophosphate dehydrogenase and triosephosphate iaomerase in 1 ml of 50 mM glycylglycine buffer, pH 7.2. For the C. utilis FDP aldolase assays potassium acetate was added to 100 mM. For determination of K, for C. utilis aldolase the asssys were as above, except that the concentration of fructose 1 ,Gdiphosphate was varied between 0.02 and 0.4 mM. The concentration of enzyme in each assay was 0.05 &ml for C. u$iZis FDP aldolase, and 0.3 p&/ml for rabbit muscle aldolase. 6 For determination of specific activity the reaction mixtures were the same as above, except that sedoheptulose 1,7-diphosphate, 1 mM, was substituted for fructose 1,Gdiphosphate. For determination of K, the concentration of sedoheptulose 1,7-diphosphate varied between 0.02 and 0.4 m&f. The concentration of enzyme used wa,s 4.0 pg/ml for C. ~~tiZi~ FDP aldolase and 1.2 #g/ml for rabbit muscle aldolase. c The assays were the same as above (a) except that 1 mM fructose l-phosphate was substituted for fructose 1,6-diphosphate. The concentration of enzyme was 75 pg/ml for C. utilis aldolase and 2.0 pg/ml for rabbit muscle aldolase. d Calculated from reciprocal plots according to Lineweaver and Burk (27).
J
FIQ. 6. Effect of sedoheptulose 1,7-diphosphate on the rate of cleavage of fructose 1,6-diphosphate. Enzyme assays were carried out as described in the footnote to Table I, except that the concentration of fructose 1,6-diphosphate varied between 0.02 and 0.4 mM. Sedoheptulose 1,7diphosphate was added to the assays as indicated. The enzyme preparation was 0.04 pg of DEAESephadex Fraction I. Velocity was measured as absorbance change per minute. The data are plotted according to Lineweaver and Burk (28).
catalytic activity (Table IV). On the basis of the Zn++ content of the mo& purified enzyme preparations it was estimated that the protein contained one Zn atom per mole&x weight of 70,000. No change in Zn++ content was detected when the enzyme was treated with a solution containing EDTA and mercaptoethanol and then
atomic absorption spectroscopy.3 A preparation with specific activity equal to 78 units per milligram was found to contain 1.03 pg of Zn++ per milligram protein, corresponding to 1.10 atoms per 70,000 gm (Table V). Exa~nation of the ~rep~atio~ for iron, copper, magnesium, and manganese by atomic absorption spectroscopy showed these metals to be present in much smaller quantities. The reversible loss of activity when enzyme solutions were kept at pH 5.0, described in a previous section, could not be attributed to dissociation of Zn++ from the protein, since activity could be restored to the extent of 80 % by neutraliza-
passed through a Sephadex G-25 column. Analysis for Zn++ was also carried out by
aWe are indebted to Miss Sabina Sprague of the Perkin-Elmer Corporation for these analyses.
FDP-ALDOLASE
OF CANDIDA
U!i”ZLZS TABLE
REACTIVATION AFTER
21 III
OF CANDIDA OR INACTIVATION WITH O-P~ENA~THROLINE
FDP ALDOLASE EDTAo=
ictivity
Conditions
tWOk%/
for reactivation
min)
Dilution Addition
MINUTES
FIG. 7. rnhibition of C. z&&is FDP aldolase by EDTA and mercaptoethanol. Assays were carried out as described in Table I with 0.05 pg of DEAESephadex Fraction I. EDTA (2 X 10-a M) and ~-mercaptoethanol (1.5 X 10-S M) were added as indicated in the figure. The ordinate represents mMmoles of fructose 1,6-diphosphate cleaved per minute. tion after passage through a Sephadex G-25 column. No method has yet been found for the reversible dissociation of Zn++ from the enzyme. E$ect of treut~e~t of ~zy~~-~~~~tru~ mixtures with borohydride. The aldolase of C. utilis does not appear to bind dihydroxyacetone phosphate by a Schiff base mechanism. A number of attempts were made to inactivate the enzyme by reduction with borohydride in the presence of dihydroxyacetone phosphate or fructose 1 ,6-diphosphate. The enzyme was stable to treatment with borohydricie under these conditions, and virtually no loss of activity occurred either in the presence or absence of substrate at pH L&7.0. The t,reatment with borohydride was continued for 10 minutes at the lower pH and for 30-60 minutes at the higher pH values. Treatment at pH 6.0 and 7.5 with radioactive dihydroxyace~ne phosphate and borohydride resulted in the binding of less than 0.2 equivalent of substrate per mole of enzyme. When muscle FDP aldolase was treated under similar conditions, at pH 6.0, there was 90% inactivation with the uptake of about 2 equiva-
of Zn++, 1.0 mM 2.5 mM 3.5 mM of Mg++, 3.5 mM of Mn++, 3.5 mM
Addition Addition Dilution Addition of Zn++, 3.0 mM Addition of MS++, 3.5 m&f Addition of Mn*, 3.5 mM
-
3.0 0 1.1 2.2 2.1 2.1 4.9 4.3 4.3 4.5
91 0 37 75 70 72 100 90 90 94
a The incubation mixture for inactivation (0.2 ml) contained 0.22 mg of FDP aldolase (specific activity, 60 units/mg), 50 mM phosphate buffer, pH 7.2, 2.5 mM EDTA, and 10 mM mercaptoethanol. After 30 minutes at 25” aliquots (10~1 of a 1:2OOdilution) were added to the standard assay mixture. In the experiment with added cations the assay mixture also contained 2.6 mM EDTA and 10 m&f meroaptoethanol. With 2.5 mM EDTA in the assay mixture no activity was detected in the absence of the divalent cations. *The conditions were as in Experiment I, except, that 0.5 mM o-phenanthroline was present instead of EDTA and mercaptoethanol, and the content of enzyme in 0.2 ml was 0.35 mg. After 3-5 minutes, aliquots (1 ~1) were analyzed in the standard assay mixture. In the experiment with added cations the assay mixture also contained 0.5 mM o-phenanthroline.
lents of radioactive ~hydroxyacetoue phate per mole of enzyme (3,29).
phos-
DISCUSSION
Although the FDP aldolase of G. utitis is relatively unstable, particularly to acid conditions, we have obtained preparations of high specific activity that can be stored at low temperature for at least several weeks. Maintenance of activity during storage appears to require a sulfhydryl or other reducing reagent, but the purified enzyme does not require the addition of zinc or cysteine, or both, in the assay mixture, unlike preparations from S. cerevisiae.
22
KOWAL, TABLE
Assay
OF
IV
ALDOLA~EPREP~\RATIONS FORZN++ zn++
sp. act. (units/mg)
30 40 70 70
pmoles
0.37 0.460 0.800 0.740
FROMC.Z&~~
contenta
r&N
CREMONA,
Zn++/mg
moles Zn++/ 7a,ooo gm
0.530 0.705 1.230 1.140
0.390 0.495 0.860 0.800
a The preparations analyzed were obtained by purification through the DEAE-Sephadex step. They were dialyzed for 15 hours against 5 mM tris buffer, pH 7.2, prepared in Zn-free water containing 1 m&f mercapt.oethanol. The last entry in the table represents a preparation which had been treated with 2.5 mM EDTA and 10 mM mercaptoethanol and passed through a Sephadex G-25 column equilibrated with 5 mM tris buffer, pH 7.4. Each value represents the average of 3 determinations which agreed within 10%. Zn++ assays were carried out by a modification of the dithizone method of Malmstrom (15). TABLE
V
ATOMIC ABSORPTION SPECTROGRAPHIC ANALYSIS FORDI~~LENTMET~LSIN YEAST ALDOLASE
content ExperimenP
Cont.(ppm)
Metal
moles/ #dmx 70,000 gm
protein I II III
IV V
Zn++ cuff Fe++ Mn++ Mg++
-
0.635 1; --ii’0.120 0:193 0.106 0.161 None found 0
1.10 0.21 0.20 0 <0.06
5 C. utitis aldolase (specific activity, 79) was prepared for assay by passage through a small Chelex column after concentration by negative pressure dialysis. Traces of zinc, iron, and copper amounting to less than 0.1 ppm were found in the sample of buffer provided for analysis.
The results of our studies clearly place the FDP aldolase of C. ~~i~~ in the category of Class 2 aldolases. It is similar in many respects to the enzyme isolated from X. cercvisiue (4, 6). The molecular weight is approximately 70,000 and the enzyme contains 1 mole of Zn++ per mole of protein. It is strongly inhibited by chelating agents,
AND HORECKER
and evidence has been obtained that this i~hibitio~l is due to formation of an enzymechelator complex, rather than to removal of t,he metal. Inhibition by chelating agents is readily reversed by dilution, dialysis, or passage through Sephadex columns. Attempts to remove the tightly bound zinc with the formation of an active apoenzyme were unsuccessf~~1. Numerous efforts were made to obtain evidence for a Schiff base intermediate with this enzyme; these were uniformly unsuccessful. This result is in agreement with the findings of Rutter (7) that Class 2 aldolases do not form such intermediates. iEtut,ter has proposed a model in which Znf+ serves as a binding site for the substrate. The development of a satisfactory method for removing zinc in a reversible manner with the formation of a metal-free apoenzyme wouId be invaluable for further studies of the active site of this enzyme. unfortunately, no such met,hod is yet available. FDP aldolase from C. z&is, like the enzyme from rabbit muscle, catalyzes the cleavage of sedoheptulose 1,7-diphosphate and fructose l-phosphate, as well as of fructose 1,6-diphosphate. However, the activity with these substrates is low compared to that with fructose 1,6-diphosphate. Thus, the rate of cleavage of sedoheptulose 1,7-diphosphate was found to be less than 3 % of that with fructose 1,6-diphosphate; the low activity with fructose l-phosphate is similar to that reported for the enzyme from S. cerevisiae (30). The lack of significant activity with fructose l-phosphate is not surprising, since there is no evidence for a role of fructose l-phosphate in the intermediary met,aboIism of C. utilis. &md&&r utilis thus contains aldolases of two classes, since it has been demonstrated that transaldolase in this organism does form a Schiff base intermediate (8). The presence of both classes of aldolases in a single cell is of interest in relation to t.he evolutionary origin of these enzymes. REFERENCES 1. MEYEILHOF, O., ANDLOHMANN, K., Bi0chem.Z. 271, 89 (1934). 2. MEYERHOF,O.,B~&SOC.
1345 (1938).
Chim. Biol.20.1033,
FDP-ALI~OLAS~
OF C.4NDIL)B
3. HORECBER, B. L., ROWLEY, P. T., GRAZI, E., CHENG, T., AND TCHOLA, O., Biochem. Z. 338, 36 (1963). 4. WARBURG, O., AND CHRISTIAN, W., Biochem. 2. 314, 149 (1943). BARD, R. C., AND GUNSALUS, I. C., J. Bacteriol. 69, 387 (1950). RICHARDS, 0. C., AND RIJTTER, W. J., J. Biol. Chem. 236, 3177 (1961). Proc. 231, 1248 RUTTER, W. J., Federation (1964). HORECKER, B. L., PO~TREMOLI, S., RICCI, C., AND CHPNG, T., Proc. Xatr. Acad. Sci. U. S. 47, 1949 (1961). 9. HOFFEE, P., R.OSEN, O., AND HORECBER, B. L., J. BioE. Chem. 240, 1512 (1965). 10. MELOCHE, H. P., AND WOOD, W. A., J. Biol. Chem. 239, 3515 (1964). B. S., MEINHART, J. O., 11. VANDERHEIDEN, DODSON, R. G., AND KEEPS, E. G., J. Biol. Chem. 237, 2095 (1962). 12. TAYLOR, J. F., GREEN, A. A., AND CORI, G. T., J. Biol. Chem. 173, 591 (1948). 13. PONTREMOLI, S., PRANDINI, B. D., BONSIGNORE, A., AND HORECKER, B. L., Proc. Natl. Aead. Sci. U. S. 47, 1942 (lQ61). 14. BAXTER, J. N., PERLIN, A. S., AN’D SIMPSON, F. J., Can. J. Bioehem. Physiol. 37, 199
,,nZZZ?\ .
fIJr)J/
15. MALMSTR~M, B., ia “Methods of Biochemical Analysis” (D. Click, ed.), Vol. 3, p. 340. Wiley ([nterscience), New York (1956). 16. CREMONA, T., AND SINGER, T. P., J. Biol. Chem. 239, 1466 (1964).
17. FISKE,
UTILTS C. H.,
23 AND SUBBARO~,
Y., J. Biol.
Chem. 81, 629 (1929). 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 19. B~.!cHER, T., Biochim. Biophys. Acta 1, 292 (1947). 20. RACKER, E., AND SCHROEDER, E. A. R., Arch. Biochem. Biophys. 74, 326 (1958). 21. HORECKER, B. L., in. “Methods in Enzymology” (S. P. Colowiek and N. 0. Kaplan, eds.), Vol. III, p. 105. Academic Press, New York (1955). 22. COOPER, J., SRERE, P. A., TABACHNICK, M., AND RACKER, E., Arch. Biochem. Biophys. 74, 306 (1958). 23. DAVIS, B. J., Ann. N. Y. Acad. Sci. 212, 404 (1964) * 24. SMYRNIOTXS, P. Z., AND HORECKER, B. b., J. Biol. Chem. 218, 745 (1956). 25. HORECKER, B. L., AND KORNBERG, A., J. Biol. Chem. 176, 385 (1948). 26. YPHANTIS, D. A., Biochemistry 3, 297 (1964). 27. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66, 658 (1934). 28. HORECEER, B. L., S~YRNIO~S, P. Z., HEATH, H. H., AND MARKS, P. A., J. Biol. Chem. i@!& 827 (1955). 29. LAI, C. Y., TCHOLA, O., CHENG, T., AND HORECKER, B. L., J. Biot. Chem. 240, 1347 (1965). 30. RICHARDS, 0. C., AND RUTTER, W. J., J. Biol. Chem. 236, 3185 (1961).