228
ALDOLASES
[36]
phate. I t is interesting to note that the loss of activity toward fructose 1,6-diphosphate does not parallel the removal of tyrosine residue. When 80% of this activity is lost, less than half of the tyrosine have been released. On the other hand, loss of activity toward fructose 1-phosphate is observed only during release of the last 2 tyrosine residues. This suggests a high degree of interaction between subunits of lobster muscle aldolase t h a t has not been observed for rabbit muscle aldolase. -~
[35] F r u c t o s e - d i p h o s p h a t e
Aldolase from Blue-Green
A l g a e 1,2
B y JAMES M. WILLARD and .~,~ARTIN GIBBS Me~+-bRSH
Fructose 1,6-diphosphate •
~ dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate
Rutter 3 proposed two broad classes of fructose-l,6-diphosphate ( F D P ) aldolases: T y p e I aldolases not requiring a divalent metal, strongly inhibited by mercurials, and unaffected by chelating agents and K÷; t y p e I I aldolases requiring a divalent metal, slightly inhibited by mercurials, strongly inhibited by chelating agents, and stimulated by K ÷. Variants within each aldolase type have been proposed. 4,5 The F D P aldolase of blue-green algae is a type I I aldolase2 ,5-7 Unlike most other type I I aldolases, the enzyme from the blue-green algae, Anacystis nidulans, is unaffected by K +, has an absolute requirement for cysteine, and Fe 2+ stimulates the rate obtained with cysteine alone, s
Assay Methods Principle. The assay of aldolase is based on the estimation of triose phosphate formed with F D P cleavage. Two methods of assay can be employed depending upon the specific assay conditions and both yield com-
1Fructose-l,6-biphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13. 2 This work was assisted by grants from the National Science Foundation and the United States Atomic Energy Commission. a W. J. Rutter, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 23, 1248 (1964). 4 W. J. Rutter, B. M. Woodfin, and R. E. Blostein, Acta Chem. Seand. 17, Suppl. 1, 226 (1963). 5j. M. Willard and M. Gibbs, Plant Physiol. 43, 793 (1968). 6 C. Van Baalen, Nature (London) 206, 193 (1965). 'J. M. Willard, M. Schulman, and M. Gibbs, Nature (London) 206, 195 (1965). s j. M. Willard and M. Gibbs, Bioehim. Biophgs. Acta 151,438 (1968).
[36]
ALDOLASE FROM BLUE-GREEN ALGAE
229
parable results: the colorimetric method of Sibley and Lehninger9 and the spectrophotometric method of Wu and Racker. TM Specifically, the colorimetric method should be used whenever there is appreciable NADH oxidase activity or when additions result in UV-absorbing complexes. The spectrophotometric assay can be used in the presence of cysteine and iron only if both are preincubated at least 5 rain prior to initiation of the reaction with FDP. 1~ The rate with either assay is linear with time and proportional with enzyme.
Reagents ]or Assay Tris(hydroxymethyl)aminomethane.HC1 (Tris.HCl) (Sigma), 0.2 M, pH 7.6 FDP-Na, 50 mM (Sigma) Hydrazine sulfate (Eastman Chemicals), 0.56 M, pH 7.5 Cysteine-HC1.H~O (Pfanstiehl Laboratories), 40 mM pH 7.5, (prepared just before use) Fe(NH~)~(SO,)~.6 H..,O (Mallinckrodt Chemicals), 20 mM Trichloroacetic acid, 10% (w/v) (Fisher Chemicals) 2,4-Dinitrophenylhydrazine (Eastman Chemicals) (0.1% in 2 N
HCI) NADH (Sigma), 10 mM Triosephosphate isomerase and a-glycerolphosphate dehydrogenase (Boehringer and Soehne).
Colorimetrie Assay. The assay is performed at 37 ° in 2.5-ml reaction mixtures and contains 0.5 ml of Tris.HC1, 0.25 ml hydrazine sulfate, 0.2 ml FDP, 0.5 ml cysteine-HC1, 0.125 ml Fe (NH,).~(S04)2 and aldolase. The aldolase is incubated 10 min to achieve maximum activity after which FDP is added to initiate the reaction. Incubations are terminated at the end of 20 rain by the addition of 2.0 ml cold trichloroacetic acid. Blanks consist of adding FDP after the acid and controls with no enzyme present are run in all cases where cofactors or inhibitors are employed. Following chromagen development9 the absorption at 540 nm is determined in 1-cm cuvettes with a Beckman DU spectrophotometer. Spectrophotometric Assay. This is the method of choice and may be 0j. A. Sibley and A. L. Lehninger, J. Biol. Chem. 177, 859 (1949). 10IR. Wu and E. Racker, J. Biol. Chem. 234, 1029 (1959). 1~The intense red-violet color strongly absorbing at 340 nm slowly disappears as cysteine stoichometrically reduces ferric ions to the ferrous state. A. E. Martell and M. Calvin, in "Chemistry of the Chelate Compounds," p. 384. Prentice-Hall, Englewood Cliffs, New Jersey, 1956.
230
ALDOLASES
[36]
employed provided (a) the N A D H oxidase activity is low and (b) a 5-min preincubation occurs when using cysteine and iron. Assays are performed at either 26 ° or 37 ° employing the coupling system of triosephosphate isomerase and a-glycerolphosphate dehydrogenase. In a final volume of 1.0 ml the reaction mixture contains 0.2 ml of Tris.HC1, 9.6 E U of triosephosphate isomerase, 0.24 E U of a-glycerolphosphate dehydrogenase, 0.02 ml of N A D H , 0.1 ml of F D P , 0.2 ml of cysteine HC1, 0.05 ml of F e ( N H ~ ) 2 ( S Q ) 2 , and aldolase. Reference cu. vettes lack N A D H while control cuvettes lack F D P or aldolase. The reaction is initiated by addition of F D P or aldolase. N A D H oxidation at 340 nm is followed in 1-cm cuvettes with a Beckmann D U spectrophotometer equipped with a Gilford Model 2000 multiple absorbance recorder. A molecular extinction coefficient for N A D H of 6.22 X 106 cm2/mole is employed22 The t e m p e r a t u r e is controlled by use of a H a a k e Model F circulator. Units. Units are expressed as micromoles of F D P cleaved per minute at a specified temperature of 26 ° or 37 °, and specific activities are expressed in units per milligram of protein. Protein is measured spectrophotometrically 13 in purified preparations and colorimetrically 1~ at 750 nm in crude preparations using crystalline bovine serum albumin as standard. The two methods yield similar results provided the 280/260 ratio is greater t h a n 0.9. Purification of Anacystis Aldolase s A typical purification obtained in the different steps is summarized in the table. All procedures are carried out at 4 ° unless otherwise stated. Source of E n z y m e . Anacystis nidulans (Richt.) was obtained from Dr. J. M e y e r at the University of Texas, Austin. Ten-liter cultures were grown at 30 ° in 12-1 Florence flasks in a medium ~5 containing, per liter, sodium citrate-2H20, 0.165 g; MgSO4.TH~O, 0.122 g; KN03, 1 g; K~HP04, 1.0 g; Ca (NO.~) ..) 4H,.,O, 0.025 g, 1 ml of 1% (w/v) FeSO4-EDTA solution 16 and 1 ml of Hoagland and Arnon A5 solution. ~ Cells were 12B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). '~0. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); see this series, Vol. 3 [73]. 140. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 15W. A. Kratz and J. Myers, Amer. J. Botany 42, 282 (1955). 16L. Jacobson, Plant Physiol. 20, 411 (1951). "D. R. ttoagland and D. I. Arnon, Calif. Agr. Exp. Sta. Circ. 347 (1938). 1 ml of solution contains 2.86 mg of H.~BO~; 1.81 mg of MnCI:.H20; 0.11 mg of ZnCl.; 0.079 mg of CuSO~'SH20; 0.03 mg of Na2MoO4.2H20.
[35]
ALDOLASE FROM :BLUE-GREEN ALGAE SUMMARY OF PURIFICATION PROCEDURE FOR
Fractionation step Cell-free extracts: sum of 3 lots 35-75% (NH4)2SO4:sum of 3 lots Stored and pooled 35-75% (NH4)~SO4 Calcium phosphate gel and 35-80% (NH4)2SO4 eluate Combined Sephadex G-25 fractions Combined DEAE-cellulose fractions 80% (NH4):SO4 fraction off DEAEcellulose
Total activity" (units) 64 38 38 33 31 20 11
231
Anacystis ALDOLASE Specific activity (units/mg protein) 0.13 0.58 0.58 2.1 2.3 -128
Recovery (%) 100 60 60 51 48 30 17
Aldolase activity was determined by the colorimetric assay method at 37° with 8 mM cysteine and 1 mM Fe ~+ present. grown under continuous fluorescent illumination of 500 foot-candles with a constant supply of 1% C0._,/99% air. Five-day-old cultures yield 0.5 to 1.0 g wet weight of cells per liter. Step 1. Preparation of Cell-Free Extracts. Two grams wet weight of Anacystis cells were suspended in a final volume of 15 ml of 50 m M Tris.HC1 (pH 7.6}. After deposition in a 25-ml Rosette cell the suspension was deaerated 4 min with N.,, then sonicated under N2 at full power on a Branson Model S-75 sonifier. After centrifugation at 10,000 g the supernatant fraction constituted the cell-free extract. The aldolase of this step is extremely unstable (80% loss in 10 hrs at 4 °, 50% loss in 2 days at --15 °) and the following step 2 must be performed immediately for maximal yield. Step 2. Fractionation with Ammonium Sulfate. Freshly prepared cellfree extract (29 ml) is brought to 35% saturation with 7.2 g solid (NH4)2SO~ and centrifuged at 10,000 g. The resultant pellet is discarded and the supernatant is brought to 75% saturation with a further 8.2 g (NH4)=,SQ. After centrifugation the 75% supernatant is discarded and the pellet is suspended in 4 ml of deaerated (by N._,) 50 m M Tris.HC1 (pH 7.6). This 35-75% (NH4)~SO~ fraction is stable for several months when stored under N~ at --15 °. This remarkable stability in (NH4)2S04 allows for the accumulation of sufficient material for subsequent purification.
Step 3. Calcium Phosphate Treatment and Ammonium Sulfate Elution. Calcium phosphate gel suspension, 61 ml in 50 m M Tris.HC1 (pH 7.6, containing 16.5 mg of gel dry weight per milliliter) is added to 10
232
ALDOLASES
[36]
ml of three pooled 35-75% (NH4)_~S04 fractions (66.25 mg protein). After 10 min of magnetic mixing, the suspension is centrifuged and the supernarant is discarded. The calcium phosphate gel pellets are extracted twice with 30 ml each of 35% saturated (248.5 g/liter) (NH~)._,SO~. This (NH4)2SO4 solution is then raised to 80% saturation with 13.6 g of solid (NH4)2SO4 and centrifuged; the resultant precipitate is suspended in 4.5 ml of 50 mM Tris. HC1 (pH 7.6) and recentrifuged. Step 4. Chromatography on DEAE-CelIulose. This step completely separates aldolase from the molecularly similar biliprotein, phycocyanin, the latter constituting 40% of the total soluble protein, having an isoelectric point of 4.5-5.0 and a molecular weight of 138,000.1~ The 35-80% (NH~)~SO4 fraction (5.3 ml) is passed through a 1.5 X 15 cm Sephadex G-25 column previously equilibrated with 50 mM Tris (pH 7.6). Fractions of 2.3 ml are collected at 0.8 ml/minute. Fractions 6 through 11 are pooled, and 13.5 ml are applied directly to a 1.5 }( 23 cm DEAE-cellulose column equilibrated with 50 mM Tris-HC1 (pH 7.6). Fractions of 4.6 ml are then collected at 1.9 ml/min. Stepwise gradient elution with NaC1 solutions of 50, 100, 150, 200, and 350 mM concentrations is performed and aldolase and phycocyanin (reflected by its absorption at 615 nm) located. The aldolase in fractions 43 through 57 (67 ml) is concentrated by addition of 37.8 g solid (NH4).,SO4 to yield 80% saturation. The resultant precipitate after centrifugation is suspended in a final 3.0 ml of 50 mM Tris-HC1 (pH 7.6). The final preparation has a specific activity of 128 representing a 980-fold purification.
Properties Appcbrent Molecular Weight. When determined by the sucrose density gradient method, 19 the Anacystiz aldolase migrates with muscle lactic acid dehydrogenase~° and therefore has an apparent molecular weight of 137,000. This molecular weight was obtained with both catalytically inactive and activated samples. Purity. The purified aldolase appears homogeneous when subjected to polyacrylamide disc gel electrophoresisY 1 Aldolase activity corresponds exactly to the single protein band obtainedY 2 The pul'ified enzyme contained no NAD- or NADP-linked glyceraldehyde-3 phosphate dehydrogenase activity and, on a unit of activity basis, contained about 0.1% triosephosphate isomerase.
~C. O'hEocha, in "Physiology and Biochemistry of Algae" (R. A. Lewin, ed.). p. 421. Academic Press, New York, 1962. 1,R. G. Martin and B. N. Ames,J. Biol. Chem. 236, 1372 (1961). soGenerously provided by Dr. N. O. Kaplan. 21B. J. Davis, Ann. N. Y. Acad. Sci. 121, 32:1 (1964). 22j. M. Willard, Ph.D. Thesis, Cornell Univ., Ithaca, New York, 1967.
[35]
ALDOLASE FROM BLUE-GREEN ALGAE
233
pH Optimum. Like other type II aldolases, the Anacystis enzyme exhibits a sharp pH optimum at pH 7.62 No difference is seen when either activating and assaying at the same pH or activating at pH 7.6, then assaying at varying pH. Co]actor Requirements. The Anacystis aldolase exhibits maximal activity only when both 8 mM cysteine and 0.1 mM Fe 2+ is present. Use of either alone elicits no activity. A 10-rain preincubation is required for maximum activity. Unlike other type II aldolases, K + is without effect. When assayed in the presence of cysteine, Fe 3+ and Mn 2÷ could replace Fe -~÷.In the presence of Fe 2÷, reduced glutathione and thiolycolate yield rates 20% and 58%, respectively, those obtained with cysteine. /!t-Mercaptoethanol and BAL were uneffective. Evidence ]or Metal in Anacystis Aldolase. When excess aldolase is assayed in the presence of cysteine alone, the rate obtained is 3% that obtained with both cysteine and Fe 2+ present. This rate with cysteine alone is totally inhibited by o-phenanthroline and 2,2'-bipyridine suggesting the presence of a tightly bound metal, possibly iron. Specificity and Kinetics. The Anacystis enzyme exhibits a high degree of specificity for FDP (Kin = 0.16 raM). The enzyme cleaved 4 mM sedoheptulose-l,7-P (Kin = 10 mM) at a rate 59% of that obtained with FDP. No activity was noted with fructose-l-P, ribulose 1,5-diphosphate, 2-keto,3-deoxyphosphogluconate, sorbose 1-phosphate, sorbose 1,6-diphosphate, rhamnulose 1-phosphate, or fuculose 1-phosphate, each at 4 mM concentration. The turnover number for the Anacystis enzyme is 5200 compared to 6900 obtained for the yeast enzyme. 23 It appears that type II, metal-requiring aldolases have 10-fold lower affinities for FDP than do type I, nonmetal-requiring, aldolases, but their turnover numbers are twice those of the type I aldolases. Distribution. Type II aldolase activity for which there is an absolute requirement for cysteine with Fe 2÷ stimulating such activity has been demonstrated in cell-free extracts of the following blue-green algae: Anabaena variabilis, Plectonema sp., Anabaenopsis sp., and Nostoc ?~tU8corum. 5
A type II aldolase with cofactor requirements identical to those of the yeast enzyme, i.e., maximal activity results with cysteine, Zn 2, and K ÷ present, T M has been obtained from the flexibacterium, Saprospira
thermalis2 Russell and Gibbs 24,2~ have demonstrated that autotrophically grown
Chlamydomonas mundana possesses a type I aldolase whereas heterotro:20. C. Richards and W. J. Rutter, J. Biol. Chem. 236, 3177 (1961). 24G. K. Russell and M. Gibbs, Biochim. Biophys. Acta 132, 145 (1967). :5 G. K. Russell and M. Gibbs, Plant Physiol. 41, 885 (1966).
234
ALDOLASES
[37]
phically grown cells possess only a type I I aldolase? 4,~5 Similarly, autotrophic Euglena gracilis and Chlorella pyrenoidosa possess type I aldolases, and heterotropic, d a r k grown cells, type I I aldolases. 5 The type I I aldolase of such dark-grown Euglena has been shown to be of cytoplasmic and not chloroplastic origin. 2G I t appears t h a t in the leaves of higher plant and green algae the type I aldolase participates in both photosynthesis and cellular respiration. In those cells which lose their ability to photosynthesize, a t y p e I I enzyme functions in cellular respiration. The red alga Chond.rus crispus and the golden brown algae Ochromonas danica possess type I and I I aldolases. 5 Seeds and the etiolated leaves and cotyledons of plants appear to possess a v a r i a n t of the type I aldolase, i.e., they lack mercurial inhibition. Subsequent illumination results in normal type I aldolase formation. ~ 2ey. Mo, B. G. Harris, and R. W. Gracy, Arch. Biochem. Biophys. 157, 580 (1973).
[37] F r u c t o s e
Bisphosphate
By
Aldolase from Spinach
B. L. HORECKER
Fructose 1,6-bisphosphate ~- dihydroxyaeetone phosphate + D-glyceraldehyde 3-phosphate Fructose-l,6-bisphosphate (Fru-P2) aldolase in higher plants has been shown to form a Schiff-base intermediate with the substrate and therefore belongs to the class I aldolases. 1 The enzyme has been purified from peas, 2-4 cactus, 5 and spinach chloroplasts 6,7 and whole spinach leaves, s T h e procedure described is t h a t of Fluri et al2 as modified by Davis. 9 There have been some suggestions t h a t plant cells m a y contain two forms of Fru-P2 aldolase. The green alga Chlamydamo,nas has been reported to contain a class I I aldolase as well as a class I aldolase, the latter probably playing a role in photosynthetic carbon dioxide fixation. 1° 1See this series, Vol. 9 [87]. P. K. Stumpf, J. Biol. Chem. 176, 233 (1948). s C. Hatz and F. Leuthardt, Hoppe Seyler's Z. Physiol. Chem. 348, 354 (1967). 4j. M. Willard, Thesis dissertation, Cornell University, Ithaca, New York, 1967. 5 G. G. Sanwal and P. S. Krishnan, Enzymologia 23, 249 (1961). K. Brooks and R. S. Criddle, Arch. Biochem. Biophys. 117, 650 (1966). 7G. Jacobi, Z. Pflanzen Physiol. 56, 262 (1967). 8 R. Fluri, T. Ramasarma, and B. L. Horecker, Eur. J. Biochem. 1, 117 (1967). J. C. Davis, Thesis dissertation, Albert Einstein College of Medicine, New York, N.Y., 1970. 10G. K. Russell and M. Gibbs, Biochim. Biophys. Acta 132, 145 (1967).
ALDOLASES
[36]
phate. I t is interesting to note that the loss of activity toward fructose 1,6-diphosphate does not parallel the removal of tyrosine residue. When 80% of this activity is lost, less than half of the tyrosine have been released. On the other hand, loss of activity toward fructose 1-phosphate is observed only during release of the last 2 tyrosine residues. This suggests a high degree of interaction between subunits of lobster muscle aldolase t h a t has not been observed for rabbit muscle aldolase. -~
[35] F r u c t o s e - d i p h o s p h a t e
Aldolase from Blue-Green
A l g a e 1,2
B y JAMES M. WILLARD and .~,~ARTIN GIBBS Me~+-bRSH
Fructose 1,6-diphosphate •
~ dihydroxyacetone phosphate + D-glyceraldehyde 3-phosphate
Rutter 3 proposed two broad classes of fructose-l,6-diphosphate ( F D P ) aldolases: T y p e I aldolases not requiring a divalent metal, strongly inhibited by mercurials, and unaffected by chelating agents and K÷; t y p e I I aldolases requiring a divalent metal, slightly inhibited by mercurials, strongly inhibited by chelating agents, and stimulated by K ÷. Variants within each aldolase type have been proposed. 4,5 The F D P aldolase of blue-green algae is a type I I aldolase2 ,5-7 Unlike most other type I I aldolases, the enzyme from the blue-green algae, Anacystis nidulans, is unaffected by K +, has an absolute requirement for cysteine, and Fe 2+ stimulates the rate obtained with cysteine alone, s
Assay Methods Principle. The assay of aldolase is based on the estimation of triose phosphate formed with F D P cleavage. Two methods of assay can be employed depending upon the specific assay conditions and both yield com-
1Fructose-l,6-biphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13. 2 This work was assisted by grants from the National Science Foundation and the United States Atomic Energy Commission. a W. J. Rutter, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 23, 1248 (1964). 4 W. J. Rutter, B. M. Woodfin, and R. E. Blostein, Acta Chem. Seand. 17, Suppl. 1, 226 (1963). 5j. M. Willard and M. Gibbs, Plant Physiol. 43, 793 (1968). 6 C. Van Baalen, Nature (London) 206, 193 (1965). 'J. M. Willard, M. Schulman, and M. Gibbs, Nature (London) 206, 195 (1965). s j. M. Willard and M. Gibbs, Bioehim. Biophgs. Acta 151,438 (1968).
[36]
ALDOLASE FROM BLUE-GREEN ALGAE
229
parable results: the colorimetric method of Sibley and Lehninger9 and the spectrophotometric method of Wu and Racker. TM Specifically, the colorimetric method should be used whenever there is appreciable NADH oxidase activity or when additions result in UV-absorbing complexes. The spectrophotometric assay can be used in the presence of cysteine and iron only if both are preincubated at least 5 rain prior to initiation of the reaction with FDP. 1~ The rate with either assay is linear with time and proportional with enzyme.
Reagents ]or Assay Tris(hydroxymethyl)aminomethane.HC1 (Tris.HCl) (Sigma), 0.2 M, pH 7.6 FDP-Na, 50 mM (Sigma) Hydrazine sulfate (Eastman Chemicals), 0.56 M, pH 7.5 Cysteine-HC1.H~O (Pfanstiehl Laboratories), 40 mM pH 7.5, (prepared just before use) Fe(NH~)~(SO,)~.6 H..,O (Mallinckrodt Chemicals), 20 mM Trichloroacetic acid, 10% (w/v) (Fisher Chemicals) 2,4-Dinitrophenylhydrazine (Eastman Chemicals) (0.1% in 2 N
HCI) NADH (Sigma), 10 mM Triosephosphate isomerase and a-glycerolphosphate dehydrogenase (Boehringer and Soehne).
Colorimetrie Assay. The assay is performed at 37 ° in 2.5-ml reaction mixtures and contains 0.5 ml of Tris.HC1, 0.25 ml hydrazine sulfate, 0.2 ml FDP, 0.5 ml cysteine-HC1, 0.125 ml Fe (NH,).~(S04)2 and aldolase. The aldolase is incubated 10 min to achieve maximum activity after which FDP is added to initiate the reaction. Incubations are terminated at the end of 20 rain by the addition of 2.0 ml cold trichloroacetic acid. Blanks consist of adding FDP after the acid and controls with no enzyme present are run in all cases where cofactors or inhibitors are employed. Following chromagen development9 the absorption at 540 nm is determined in 1-cm cuvettes with a Beckman DU spectrophotometer. Spectrophotometric Assay. This is the method of choice and may be 0j. A. Sibley and A. L. Lehninger, J. Biol. Chem. 177, 859 (1949). 10IR. Wu and E. Racker, J. Biol. Chem. 234, 1029 (1959). 1~The intense red-violet color strongly absorbing at 340 nm slowly disappears as cysteine stoichometrically reduces ferric ions to the ferrous state. A. E. Martell and M. Calvin, in "Chemistry of the Chelate Compounds," p. 384. Prentice-Hall, Englewood Cliffs, New Jersey, 1956.
230
ALDOLASES
[36]
employed provided (a) the N A D H oxidase activity is low and (b) a 5-min preincubation occurs when using cysteine and iron. Assays are performed at either 26 ° or 37 ° employing the coupling system of triosephosphate isomerase and a-glycerolphosphate dehydrogenase. In a final volume of 1.0 ml the reaction mixture contains 0.2 ml of Tris.HC1, 9.6 E U of triosephosphate isomerase, 0.24 E U of a-glycerolphosphate dehydrogenase, 0.02 ml of N A D H , 0.1 ml of F D P , 0.2 ml of cysteine HC1, 0.05 ml of F e ( N H ~ ) 2 ( S Q ) 2 , and aldolase. Reference cu. vettes lack N A D H while control cuvettes lack F D P or aldolase. The reaction is initiated by addition of F D P or aldolase. N A D H oxidation at 340 nm is followed in 1-cm cuvettes with a Beckmann D U spectrophotometer equipped with a Gilford Model 2000 multiple absorbance recorder. A molecular extinction coefficient for N A D H of 6.22 X 106 cm2/mole is employed22 The t e m p e r a t u r e is controlled by use of a H a a k e Model F circulator. Units. Units are expressed as micromoles of F D P cleaved per minute at a specified temperature of 26 ° or 37 °, and specific activities are expressed in units per milligram of protein. Protein is measured spectrophotometrically 13 in purified preparations and colorimetrically 1~ at 750 nm in crude preparations using crystalline bovine serum albumin as standard. The two methods yield similar results provided the 280/260 ratio is greater t h a n 0.9. Purification of Anacystis Aldolase s A typical purification obtained in the different steps is summarized in the table. All procedures are carried out at 4 ° unless otherwise stated. Source of E n z y m e . Anacystis nidulans (Richt.) was obtained from Dr. J. M e y e r at the University of Texas, Austin. Ten-liter cultures were grown at 30 ° in 12-1 Florence flasks in a medium ~5 containing, per liter, sodium citrate-2H20, 0.165 g; MgSO4.TH~O, 0.122 g; KN03, 1 g; K~HP04, 1.0 g; Ca (NO.~) ..) 4H,.,O, 0.025 g, 1 ml of 1% (w/v) FeSO4-EDTA solution 16 and 1 ml of Hoagland and Arnon A5 solution. ~ Cells were 12B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). '~0. Warburg and W. Christian, Biochem. Z. 310, 384 (1941); see this series, Vol. 3 [73]. 140. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 15W. A. Kratz and J. Myers, Amer. J. Botany 42, 282 (1955). 16L. Jacobson, Plant Physiol. 20, 411 (1951). "D. R. ttoagland and D. I. Arnon, Calif. Agr. Exp. Sta. Circ. 347 (1938). 1 ml of solution contains 2.86 mg of H.~BO~; 1.81 mg of MnCI:.H20; 0.11 mg of ZnCl.; 0.079 mg of CuSO~'SH20; 0.03 mg of Na2MoO4.2H20.
[35]
ALDOLASE FROM :BLUE-GREEN ALGAE SUMMARY OF PURIFICATION PROCEDURE FOR
Fractionation step Cell-free extracts: sum of 3 lots 35-75% (NH4)2SO4:sum of 3 lots Stored and pooled 35-75% (NH4)~SO4 Calcium phosphate gel and 35-80% (NH4)2SO4 eluate Combined Sephadex G-25 fractions Combined DEAE-cellulose fractions 80% (NH4):SO4 fraction off DEAEcellulose
Total activity" (units) 64 38 38 33 31 20 11
231
Anacystis ALDOLASE Specific activity (units/mg protein) 0.13 0.58 0.58 2.1 2.3 -128
Recovery (%) 100 60 60 51 48 30 17
Aldolase activity was determined by the colorimetric assay method at 37° with 8 mM cysteine and 1 mM Fe ~+ present. grown under continuous fluorescent illumination of 500 foot-candles with a constant supply of 1% C0._,/99% air. Five-day-old cultures yield 0.5 to 1.0 g wet weight of cells per liter. Step 1. Preparation of Cell-Free Extracts. Two grams wet weight of Anacystis cells were suspended in a final volume of 15 ml of 50 m M Tris.HC1 (pH 7.6}. After deposition in a 25-ml Rosette cell the suspension was deaerated 4 min with N.,, then sonicated under N2 at full power on a Branson Model S-75 sonifier. After centrifugation at 10,000 g the supernatant fraction constituted the cell-free extract. The aldolase of this step is extremely unstable (80% loss in 10 hrs at 4 °, 50% loss in 2 days at --15 °) and the following step 2 must be performed immediately for maximal yield. Step 2. Fractionation with Ammonium Sulfate. Freshly prepared cellfree extract (29 ml) is brought to 35% saturation with 7.2 g solid (NH4)2SO~ and centrifuged at 10,000 g. The resultant pellet is discarded and the supernatant is brought to 75% saturation with a further 8.2 g (NH4)=,SQ. After centrifugation the 75% supernatant is discarded and the pellet is suspended in 4 ml of deaerated (by N._,) 50 m M Tris.HC1 (pH 7.6). This 35-75% (NH4)~SO~ fraction is stable for several months when stored under N~ at --15 °. This remarkable stability in (NH4)2S04 allows for the accumulation of sufficient material for subsequent purification.
Step 3. Calcium Phosphate Treatment and Ammonium Sulfate Elution. Calcium phosphate gel suspension, 61 ml in 50 m M Tris.HC1 (pH 7.6, containing 16.5 mg of gel dry weight per milliliter) is added to 10
232
ALDOLASES
[36]
ml of three pooled 35-75% (NH4)_~S04 fractions (66.25 mg protein). After 10 min of magnetic mixing, the suspension is centrifuged and the supernarant is discarded. The calcium phosphate gel pellets are extracted twice with 30 ml each of 35% saturated (248.5 g/liter) (NH~)._,SO~. This (NH4)2SO4 solution is then raised to 80% saturation with 13.6 g of solid (NH4)2SO4 and centrifuged; the resultant precipitate is suspended in 4.5 ml of 50 mM Tris. HC1 (pH 7.6) and recentrifuged. Step 4. Chromatography on DEAE-CelIulose. This step completely separates aldolase from the molecularly similar biliprotein, phycocyanin, the latter constituting 40% of the total soluble protein, having an isoelectric point of 4.5-5.0 and a molecular weight of 138,000.1~ The 35-80% (NH~)~SO4 fraction (5.3 ml) is passed through a 1.5 X 15 cm Sephadex G-25 column previously equilibrated with 50 mM Tris (pH 7.6). Fractions of 2.3 ml are collected at 0.8 ml/minute. Fractions 6 through 11 are pooled, and 13.5 ml are applied directly to a 1.5 }( 23 cm DEAE-cellulose column equilibrated with 50 mM Tris-HC1 (pH 7.6). Fractions of 4.6 ml are then collected at 1.9 ml/min. Stepwise gradient elution with NaC1 solutions of 50, 100, 150, 200, and 350 mM concentrations is performed and aldolase and phycocyanin (reflected by its absorption at 615 nm) located. The aldolase in fractions 43 through 57 (67 ml) is concentrated by addition of 37.8 g solid (NH4).,SO4 to yield 80% saturation. The resultant precipitate after centrifugation is suspended in a final 3.0 ml of 50 mM Tris-HC1 (pH 7.6). The final preparation has a specific activity of 128 representing a 980-fold purification.
Properties Appcbrent Molecular Weight. When determined by the sucrose density gradient method, 19 the Anacystiz aldolase migrates with muscle lactic acid dehydrogenase~° and therefore has an apparent molecular weight of 137,000. This molecular weight was obtained with both catalytically inactive and activated samples. Purity. The purified aldolase appears homogeneous when subjected to polyacrylamide disc gel electrophoresisY 1 Aldolase activity corresponds exactly to the single protein band obtainedY 2 The pul'ified enzyme contained no NAD- or NADP-linked glyceraldehyde-3 phosphate dehydrogenase activity and, on a unit of activity basis, contained about 0.1% triosephosphate isomerase.
~C. O'hEocha, in "Physiology and Biochemistry of Algae" (R. A. Lewin, ed.). p. 421. Academic Press, New York, 1962. 1,R. G. Martin and B. N. Ames,J. Biol. Chem. 236, 1372 (1961). soGenerously provided by Dr. N. O. Kaplan. 21B. J. Davis, Ann. N. Y. Acad. Sci. 121, 32:1 (1964). 22j. M. Willard, Ph.D. Thesis, Cornell Univ., Ithaca, New York, 1967.
[35]
ALDOLASE FROM BLUE-GREEN ALGAE
233
pH Optimum. Like other type II aldolases, the Anacystis enzyme exhibits a sharp pH optimum at pH 7.62 No difference is seen when either activating and assaying at the same pH or activating at pH 7.6, then assaying at varying pH. Co]actor Requirements. The Anacystis aldolase exhibits maximal activity only when both 8 mM cysteine and 0.1 mM Fe 2+ is present. Use of either alone elicits no activity. A 10-rain preincubation is required for maximum activity. Unlike other type II aldolases, K + is without effect. When assayed in the presence of cysteine, Fe 3+ and Mn 2÷ could replace Fe -~÷.In the presence of Fe 2÷, reduced glutathione and thiolycolate yield rates 20% and 58%, respectively, those obtained with cysteine. /!t-Mercaptoethanol and BAL were uneffective. Evidence ]or Metal in Anacystis Aldolase. When excess aldolase is assayed in the presence of cysteine alone, the rate obtained is 3% that obtained with both cysteine and Fe 2+ present. This rate with cysteine alone is totally inhibited by o-phenanthroline and 2,2'-bipyridine suggesting the presence of a tightly bound metal, possibly iron. Specificity and Kinetics. The Anacystis enzyme exhibits a high degree of specificity for FDP (Kin = 0.16 raM). The enzyme cleaved 4 mM sedoheptulose-l,7-P (Kin = 10 mM) at a rate 59% of that obtained with FDP. No activity was noted with fructose-l-P, ribulose 1,5-diphosphate, 2-keto,3-deoxyphosphogluconate, sorbose 1-phosphate, sorbose 1,6-diphosphate, rhamnulose 1-phosphate, or fuculose 1-phosphate, each at 4 mM concentration. The turnover number for the Anacystis enzyme is 5200 compared to 6900 obtained for the yeast enzyme. 23 It appears that type II, metal-requiring aldolases have 10-fold lower affinities for FDP than do type I, nonmetal-requiring, aldolases, but their turnover numbers are twice those of the type I aldolases. Distribution. Type II aldolase activity for which there is an absolute requirement for cysteine with Fe 2÷ stimulating such activity has been demonstrated in cell-free extracts of the following blue-green algae: Anabaena variabilis, Plectonema sp., Anabaenopsis sp., and Nostoc ?~tU8corum. 5
A type II aldolase with cofactor requirements identical to those of the yeast enzyme, i.e., maximal activity results with cysteine, Zn 2, and K ÷ present, T M has been obtained from the flexibacterium, Saprospira
thermalis2 Russell and Gibbs 24,2~ have demonstrated that autotrophically grown
Chlamydomonas mundana possesses a type I aldolase whereas heterotro:20. C. Richards and W. J. Rutter, J. Biol. Chem. 236, 3177 (1961). 24G. K. Russell and M. Gibbs, Biochim. Biophys. Acta 132, 145 (1967). :5 G. K. Russell and M. Gibbs, Plant Physiol. 41, 885 (1966).
234
ALDOLASES
[37]
phically grown cells possess only a type I I aldolase? 4,~5 Similarly, autotrophic Euglena gracilis and Chlorella pyrenoidosa possess type I aldolases, and heterotropic, d a r k grown cells, type I I aldolases. 5 The type I I aldolase of such dark-grown Euglena has been shown to be of cytoplasmic and not chloroplastic origin. 2G I t appears t h a t in the leaves of higher plant and green algae the type I aldolase participates in both photosynthesis and cellular respiration. In those cells which lose their ability to photosynthesize, a t y p e I I enzyme functions in cellular respiration. The red alga Chond.rus crispus and the golden brown algae Ochromonas danica possess type I and I I aldolases. 5 Seeds and the etiolated leaves and cotyledons of plants appear to possess a v a r i a n t of the type I aldolase, i.e., they lack mercurial inhibition. Subsequent illumination results in normal type I aldolase formation. ~ 2ey. Mo, B. G. Harris, and R. W. Gracy, Arch. Biochem. Biophys. 157, 580 (1973).
[37] F r u c t o s e
Bisphosphate
By
Aldolase from Spinach
B. L. HORECKER
Fructose 1,6-bisphosphate ~- dihydroxyaeetone phosphate + D-glyceraldehyde 3-phosphate Fructose-l,6-bisphosphate (Fru-P2) aldolase in higher plants has been shown to form a Schiff-base intermediate with the substrate and therefore belongs to the class I aldolases. 1 The enzyme has been purified from peas, 2-4 cactus, 5 and spinach chloroplasts 6,7 and whole spinach leaves, s T h e procedure described is t h a t of Fluri et al2 as modified by Davis. 9 There have been some suggestions t h a t plant cells m a y contain two forms of Fru-P2 aldolase. The green alga Chlamydamo,nas has been reported to contain a class I I aldolase as well as a class I aldolase, the latter probably playing a role in photosynthetic carbon dioxide fixation. 1° 1See this series, Vol. 9 [87]. P. K. Stumpf, J. Biol. Chem. 176, 233 (1948). s C. Hatz and F. Leuthardt, Hoppe Seyler's Z. Physiol. Chem. 348, 354 (1967). 4j. M. Willard, Thesis dissertation, Cornell University, Ithaca, New York, 1967. 5 G. G. Sanwal and P. S. Krishnan, Enzymologia 23, 249 (1961). K. Brooks and R. S. Criddle, Arch. Biochem. Biophys. 117, 650 (1966). 7G. Jacobi, Z. Pflanzen Physiol. 56, 262 (1967). 8 R. Fluri, T. Ramasarma, and B. L. Horecker, Eur. J. Biochem. 1, 117 (1967). J. C. Davis, Thesis dissertation, Albert Einstein College of Medicine, New York, N.Y., 1970. 10G. K. Russell and M. Gibbs, Biochim. Biophys. Acta 132, 145 (1967).
