240
ALDOLASES
[38] D e t e c t i o n
[38]
and Isolation of Mammalian
Fructose-diphosphate
Aldolases
B y EDWARD E. PENHOET and WILLIAM J. RUTTER
Three distinct class I fructose-diphosphate aldolases have been purifled in vertebrate tissues. 1 These aldolases, A, B, and C, have been purifled from vertebrate skeletal muscle, liver, and brain, respectively. 2 Although functionally homologous, they have different catalytic and physical properties and different amino acid sequences, indicating t h a t they are the products of three different genes. 3 Each of the enzymes contains 4 subunits, and hybrid tetrameric enzymes composed of the subunits of the parental types are found in m a n y vertebrate tissues. 4 The kind and amount of aldolase present in a given tissue are strictly regulated, and change during embryological development. 5 These developmental transitions result in patterns of aldolase in the adult which are characteristic for specific organs and tissues. 4,6 In m a n y cases, neoplasia in an adult tissue results in a reverse transition of aldolase complement to the fetal p a t t e r n J Thus analysis of multiple aldolase forms can be useful both for studying the regulation of aldolases themselves in differentiation and as a m a r k e r for differentiation or dedifferentiation in other studies of ontogeny or oncogeny. A full definition of the aldolase present in a given tissue sample must include an analysis of the total catalytic activity of aldolase and an analysis of the enzyme forms present which are responsible for this activity. Methods for performing these determinations are presented below.
Assay Method Preparation of Tissue Extracts. Dissect the organ or tissue to be analyzed from a freshly killed animal and drop it into a beaker containing
1E. E. Penhoet, T. V. Rajkumar, and W. J. Rutter, Proc. Nat. Acad. Sei. U.S. 56, 1275 (1966). 2E. E. Penhoet, M. Kochman, and W. J. Rutter, Biochemistry 8, 4391 (1969). W. J. Rutter, T. V. Rajkumar, E. E. Penhoet, and M. Kochman, Ann. N.Y. Acad. Sci. 151, 102 (1968). 4 H. G. Lebherz and W. J. Rutter, Biochemistry 8, 109 (1969). W. J. Rutter, and C. S. Weber, in "Developmental and Metabolic Control Meehanisms in Neoplasia" (D. N. Ward, ed.), pp. 193-218. Williams & Wilkins, Baltimore, Maryland, 1965. C. J. Masters, Biockim. Biophys. Acta 167, 161 (1968). Y. Nordmann and F. Schapira, Eur. J. Cancer 3, 247 (1967).
[38]
MAMMALIAN FRIJCTOSE-DIPHOSPHATE
ALDOLASES
241
]0 m M Tris C1, 1 m M E D T A pH at 0°C. After the sample has cooled for a few minutes, remove and weigh it. Mince it into small pieces with dissecting scissors and suspend the pieces in 2 volumes (v/w) of the above buffer at 0°C. Pour this suspension into a glass homogenizer with a Teflon plunger and homogenize for about 10 strokes. Pour the resulting homoggenate into appropriate-sized ultracentrifuged tubes and spin in an ul0
4-
I
Muscle Heart Spleen A4
Liver
Ji
B 4, B:--3A, BzA 2 , BA 3, A 4 ,
Kidney
Brain / / I \ \ A4, AsC, A2C2, AC5, C4, :FIG. 1. Patterns of aldolase activity of adult rabbit tissues after cellulose acetate electrophoresis. Electrophoresis and activity staining for aldolase were performed as indicated by W. A. Susor, E. Penhoet, and W. J. Rutter, this series, Vol. 41 [15].
242
ALDOLASES
[38l
tracentrifuge for 60 min at 100,000 g at 0-4 °. After the centrifugation is completed, carefully remove the clear supernatant with a Pasteur pipette and keep at 0 ° for short-term storage (0-8 hr) or freeze at --20 ° for longer-term storage. Assay. The type of aldolase present in a tissue extract can be qualitatively determined by electrophoresis of the extract followed by activity staining, as described in the accompanying paper. 8 Typical results of such analyses are presented in Fig. 1. A quantitative estimate of the total aldolase activity present in a tissue extract obtained as indicated above is determined by measuring the rate of cleavage of either fructose 1,6dip hosphate or fructose 1-phosphate according to the following reactions. Fructose 1,6-diphosphate •
• glyceraldehyde 3-phosphate + dihydroxyacetone phosphate Fructose 1-phosphate ~-----~glyceraldehyde + dihydroxyacetone phosphate
Details of the assay are presented by Lebherz and Rutter. s
Purification o] Aldolases A, B, and C For studies of aldolase enzymology it is necessary to obtain the various enzyme forms in a pure state. The methodologies presented below allow purification of these enzymes very rapidly and easily by affinity chromatography using phosphocellulose chromatographic columns and seelective elution with the substrate F D P , a modification of the method originally utilized by Pogell. 9
General Methodology All operations in the purification procedures are performed at 4 ° unless otherwise noted. Contact of aldolase preparations with the substrate is an integral part of the purification procedures, but the length of time of exposure to substrate should be minimized since it has been demonstrated that the enzymes are subject to slow inactivation in the presence of substrate. TM Concentrations of purified aldolases are determined spectrophotometrically using extinction coefficients of 0.91, 0.89, and 0.88 optical density unit per milligram per milliliter, in a 1-cm light path for aldolases A, B, and C, respectively. Purification o] Aldolase A. A summary of the purification procedure is presented in Table I. A detailed description of the methods follows. 8H. 'G. Lebherz and W. J. Rutter, this volume [39]. 9B. M. Pogell, Biochem. Biophys. Res. Commun. 7, 225 (1962). ~°B. M. Woodfin, Biochem. Biophys. Res. Commun. 29, 288 (1968).
[38]
MAMMALIAN F R U C T O S E - D I P H O S P H A T E ALDOLASES
243
TABLE I PURIFICATION OF ALDOLASE A FROM RABBIT MUSCLE
Purification steps 1. 14~000 g supernatant of crude extract. 2. 45-60% (NH4)2SO4precipitate 3. Phosphocellulosechromatography 4. Crystallization
Total activity
Total protein (g)
Specific actiyity
78,700
60.26
0.13
100
55,900 54,400 46,500
11.8 3.2 3.0
4.8-5.2 17 15-16
71 69 59
Yield (%)
Step 1. Crude Extract. Anesthetize a young (6 months or so) rabbit by injecting 30 mg of a Nembutal solution per kilogram. Remove the skin of the rabbit, quickly remove the skeletal muscles, and cool in an ice bath. Grind the meat in a meat grinder and weigh the ground nmscle. Suspend the ground muscle in 3 volumes (v/w) of 50 mM Tris C1, 5 mM EDTA, 4 mM 2-mercaptoethanol, pH 7.5. Homogenize this suspension in a commercial blender at low speed for 60 sec, and centrifuge at 14,000 g for 30 rain. Decant the supernatant solution for use in the following steps. Step 2. Ammonium Sulfate Fractionation. Bring the opalescent supernatant from above to 45% saturation ammonium sulfate by adding 278 g of ammonium sulfate per liter of extract over a l-hr period while maintaining the solution at approximately 2% Let the suspension stand for 1 hr and then centrifuge at 14,000 g for 1 hour. Remove the supernatant from this spin and discard the precipitate. Bring the supernatant to 60% saturation of ammonium sulfate by adding an additional 9 8 g per liter of supernatant. Adjust the pH of the suspension to 7.5 by adding an appropriate amount of 6 N NH40H and allow the solution to stand at least 2 hr before centrifuging at 14,000 g for 1 hr. Discard the supernatant from this spin and dissolve the precipitate in 10 mM Tris C1, 1 mM EDTA, pH 7.5. Adjust the protein concentration to approximately 40 mg/ml, and desalt the solution by passing it over a Sephadex G-25 column equilibrated with 10 mM Tris C1, 1 mM EDTA, pH 7.5. Step 8. Phosphocellulose Ch,romatography. Apply the desalted 45-60% ammonium sulfate fraction to a 3.7 X 100 cm phosphocellulose column equilibrated with 10 mM Tris C1, 1 mM EDTA, pH 7.5. After applying the sample wash the column with 1-2 liters of 50 mM Tris CI, 5 mM EDTA, pH 7.5 until the narrow red band visible on the column is eluted and the optical density of the effluent at 280 nm drops below 0.1. Proceed to elute the aldolase from the column with its substrate, fructose diphos-
244
[38]
ALDOLASES 30-
I I I
Buffer + FDP
20-
-SOOl
Buffer
~,
,~
-
I0r,
300 ~ ~' 200
!
1',
oo i
,. .....
6
io
40
..---.
6o
.....
so
,.....
,00
Fraction
._.--,:..__ ....
J20
,4o
._..,
160
iho 2oo
2:;o
number
Fro. 2. Phosphocellulose chromatography of rabbit muscle aldolase. The 45--60% a m m o n i u m sulfate fraction obtained as described in the text, and equilibrated with 10 m M Tris C1, 1 m M E D T A , p H 7.5 was applied to a 3.7 × 100 cm phosphocellulose column equilibrated with the same buffer. After the sample was applied, the column was washed with 50 m M Tris Cl, 5 m M E D T A , p H 7.5 until the red hemoglobin band was removed and the absorbance of the effluent at 280 nm fell below 0.1. The aldolase was then eluted with the same buffer to which 2.5 m M F D P had been added. The volume of individual fractions collected was 10 ml.
phate, by washing the column with approximately 1 liter of 2.5 mM fructose diphosphate in 50 mM Tris C1, 5 mM EDTA, pH 7.5. This substrate wash elutes the aldolase A sharply with a maximum specific activity of 14-18 t~moles of FDP cleaved per minute per milligram of protein as shown in Fig. 2. Step ~. Crystallization. Combine the peak fractions obtained from phosphocellulose chromatography above and dialyze against a solution which is 10 mM Tris C1, 1 mM EDTA, 50% saturated ammonium sulfate, pH 7.5 (4 ° saturated, diluted 1.1 to achieve 90%). Crystals will form in the dialysis tubing after a period of 12-20 hr. Collect the crystals by centrifugation at 35,000 g for 30 min. Resuspend the aldolase crystals in a small volume of the 50% saturated ammonium sulfate solution and store at 2-4 ° as a crystalline suspension for up to 6 months.
Purification of Aldolase A summary of the isolation of aldolase B is presented in Table II. With the following exceptions it is purified in the same manner as described above for aldolase A.
[38]
245
MAMMALIAN FRUCTOSE-DIPHOSPHATE ALDOLASES
~v
Z
e-,
0
o
©
©
II
G
:g 0
C~3
~.~ ~.~ ~..~ 1~ I~
o0
II
"~o~.
O
E~
Z
II
©
.
02
5 o
a. a h E O
o E
3
:1., b
20
40
6O FRACTION
80
I00
120
NUMBER
FIO. 3. D E A E A-50 Sephadex chromatography of the A-C aldolase set isolated from rabbit brain. Aldolases A-C eluted from phosphocellulose were equilibrated with 50 m M Tris C1, 4 m M EDTA, 200 m M sucrose, 5 m M fl-mercaptoethanol, p i t 8.0. This elution was then applied to a 0.8 × 50 cm DEAE-Sephadex A-50 column equilibrated with the same buffer. Elution was carried out with a linear sodium chloride gradient (0 to 0.4 M in the same buffer); 2-ml fractions were collected. Aldolase A was present in the breakthrough peak, and the order of elution beyond this was A3C, A=C.~, AC3, C~.
Step 2. DEAE-Sephadex Chromatog~'aphy. Combine the aldolase containing fractions from the phosphocellulose column above, and concentrate to 10-20 mg/ml using ultrafiltration. Then pass them over a Sephadex G-25 column equilibrated with 50 mM Tris C1, 3 mM EDTA, 200 mM sucrose, and 5 mM fl-mercaptoethanol, pH 8.0. Apply the desalted solution to a DEAE-Sephadex A-50 column (2.5 X 50 cm) equilibrated with the same buffer and elute the aldolases with a linear sodium chloride gradient (0 ~ 0.35 M NaC1) in 50 mM Tris C1, 3 mM EDTA, 200 mM sucrose, and 5 mM fl-mercaptoethanol, pH 8. As shown in Fig. 3, the various members of the AC hybrid set are resolved from each other by this procedure and elute from the column in the order A4, A2C2, AC3, and C4. Combine the fractions in each peak and concentrate to approximately 10 mgl/ml by ultrafiltration and then precipitate by dialyzing against 55% saturated ammonium sulfate containing 1 mM EDTA, pH 7.5.
248
ALDOLASES
1.2
a A!
[38]
.b
.........
4~
c~
.4
2
20
40 Fraction
60 Number
80
FIo. 4. Isoelectric resolution of crystalline rabbit muscle aldolase A of specific activity 16, electrofocused on an ampholine gradient (pH 7 to 9).
Purity of the Aldolase Preparations To determine purity of the aldolase preparations the samples may be subjected to polyacrylamide disc gel electrophoresis or analyzed by other traditional methods of protein chemistry such as ultracentrifugation, etc. To determine the efficacy of the resolution of the various members of the A-C set from each other in the purification from brain tissue perform the qualitative aldolase assay using cellulose acetate electrophoresis and activity staining which was presented in an earlier section of this manuscript. Purified, crystalline aldolase A preparations give rather diffuse bands in native polyacrylamide gels. This phenomenon is apparently due to an inherent heterodispersity of this protein, as shown in the studies of Susor, Kochman, and Rutter, 1°" who demonstrated that crystalline aldolase A preparations could be resolved into 5 peaks by electrofocusing (Fig. 4). These peaks behaved as a 5-membered hybrid set formed by combination of two different subunits in tetramerie molecules. This possibility was confirmed by the isolation of the two subunits and recombination to form the 5-membered set. Peptide maps confirmed a minor difference in the primary structure (probably the alteration of a single amino acid). This suggested the possibility of two separate aldolase A alleles in rabbits, or the degradation, for example by deamination in situ, or during the isolation procedure. The results of subsequent studies by Horeeker and colleagues support the latter alternative. 1~ loa W. A. Susor, M. Kochman, and W. J. Rutter, Science 165, 1260 (1969). 1, C. Y. Lai, C. Chen, and B. L. Horecker, Biochem. Biophys. Res. Commun. 40, 461 (1970).
[39]
CLASS I FRUCTOSE-DIPHOSPHATE ALDOLASE
249
This heterogeneity has little if any effect on general catalytic properties of the molecules. However, detailed studies of catalysis or chemistry should p r o b a b l y be carried out with an enzyme composed of a single subunit. The " n a t i v e " aldolase A is probably the species of p I 8.2.
[39] The Class I (Schiff Base) Fructose-diphosphate Aldolase of Peptococcus aerogenes B y HERBERT G. LEBHERZ and WILLIAM J. RuvrER
Fructose 1,6-diphosphate -~ dihydroxyacetone phosphate -I- n-glyceraldehyde 3-phosphate Fructose 1-phosphate ~ dihydroxyacetone phosphate -[- n-glyceraldehyde The fructose-l,6-diphosphate ( F D P ) aldolases found in biological systems can be assigned to one of two main classes. 1 Class I aldolases, as exemplified by the m a m m a l i a n muscle enzyme, catalyze the reversible cleavage of F D P and fructose 1-phosphate ( F - l - P ) via the formation of a Schiff base intermediate between substrate and a lysyl residue at the active site of the enzyme. ~ In contrast, class I I aldolases, as exemplified by the yeast enzyme, do not cleave F - 1 - P nor do they participate in a Schiff base complex with the substrate ( F D P ) . Rather, these aldolases are metalloenzymes; they require divalent cations, usually Zn 2÷ or Fe ~÷, for activity. 3,4 The two aldolase types can further be distinguished on the basis of their catalytic and molecular properties2 -7 Until recently the class I aldolases were thought to be restricted to "higher organisms"; namely, animals, plants, protozoa, and algae. Class I I aldolases were found in fungi and all prokaryotic cells tested. 1,6 E u g l e n a 6 and C h l a m y d o m o n a s ~ contain both aldolase types. We have recently shown that the aldolase isolated from the bacterium P e p t o c o c c u s aerogenes 9 A T C C No. 14963 (also called M i c r o c o c c u s a e r o 1W. J. Rutter, Fed. Amer. Soc. Exp. Biol. 23, 1248 (1964). B. L. Horecker, P. T. Rowley, E. Grazi, T. Cheng, and O. Tehola, Biochem. Z. 338, 36 (1-963). a R. D. Kobes, R. T. Simpson, B. L. Valtee, and W. J. Rutter, Biochemistry 8, 585 (1969). ' W. J. Rutter and K. H. Ling, Biochim. Biophys. Acta 30, 71 (1958). s 0. C. Richards, and W. J. Rutter, J. Biol. Chem. 236, 3177 (1961). W. J. Rutter and W. E. Groves, in "Taxonomic Biochemistry, Physiology and Serology" (C. A. Leone, ed.), p. 417. Ronald Press, New York, 1964. W. E. Groves, Ph.D. Thesis, University of Illinois, Urbana, Illinois, 1962. s G. K. Russell, and M. Gibks, Biochim. Biophys. Acta 132, 145 (1967). 9 R. S. Breed, W. G. D. Murray, and N. R. Smith, "Bergey's Manual of Determinative Bacteriology," 7th Ed. Williams & Wilkins, Baltimore, Maryland, 1957.
ALDOLASES
[38] D e t e c t i o n
[38]
and Isolation of Mammalian
Fructose-diphosphate
Aldolases
B y EDWARD E. PENHOET and WILLIAM J. RUTTER
Three distinct class I fructose-diphosphate aldolases have been purifled in vertebrate tissues. 1 These aldolases, A, B, and C, have been purifled from vertebrate skeletal muscle, liver, and brain, respectively. 2 Although functionally homologous, they have different catalytic and physical properties and different amino acid sequences, indicating t h a t they are the products of three different genes. 3 Each of the enzymes contains 4 subunits, and hybrid tetrameric enzymes composed of the subunits of the parental types are found in m a n y vertebrate tissues. 4 The kind and amount of aldolase present in a given tissue are strictly regulated, and change during embryological development. 5 These developmental transitions result in patterns of aldolase in the adult which are characteristic for specific organs and tissues. 4,6 In m a n y cases, neoplasia in an adult tissue results in a reverse transition of aldolase complement to the fetal p a t t e r n J Thus analysis of multiple aldolase forms can be useful both for studying the regulation of aldolases themselves in differentiation and as a m a r k e r for differentiation or dedifferentiation in other studies of ontogeny or oncogeny. A full definition of the aldolase present in a given tissue sample must include an analysis of the total catalytic activity of aldolase and an analysis of the enzyme forms present which are responsible for this activity. Methods for performing these determinations are presented below.
Assay Method Preparation of Tissue Extracts. Dissect the organ or tissue to be analyzed from a freshly killed animal and drop it into a beaker containing
1E. E. Penhoet, T. V. Rajkumar, and W. J. Rutter, Proc. Nat. Acad. Sei. U.S. 56, 1275 (1966). 2E. E. Penhoet, M. Kochman, and W. J. Rutter, Biochemistry 8, 4391 (1969). W. J. Rutter, T. V. Rajkumar, E. E. Penhoet, and M. Kochman, Ann. N.Y. Acad. Sci. 151, 102 (1968). 4 H. G. Lebherz and W. J. Rutter, Biochemistry 8, 109 (1969). W. J. Rutter, and C. S. Weber, in "Developmental and Metabolic Control Meehanisms in Neoplasia" (D. N. Ward, ed.), pp. 193-218. Williams & Wilkins, Baltimore, Maryland, 1965. C. J. Masters, Biockim. Biophys. Acta 167, 161 (1968). Y. Nordmann and F. Schapira, Eur. J. Cancer 3, 247 (1967).
[38]
MAMMALIAN FRIJCTOSE-DIPHOSPHATE
ALDOLASES
241
]0 m M Tris C1, 1 m M E D T A pH at 0°C. After the sample has cooled for a few minutes, remove and weigh it. Mince it into small pieces with dissecting scissors and suspend the pieces in 2 volumes (v/w) of the above buffer at 0°C. Pour this suspension into a glass homogenizer with a Teflon plunger and homogenize for about 10 strokes. Pour the resulting homoggenate into appropriate-sized ultracentrifuged tubes and spin in an ul0
4-
I
Muscle Heart Spleen A4
Liver
Ji
B 4, B:--3A, BzA 2 , BA 3, A 4 ,
Kidney
Brain / / I \ \ A4, AsC, A2C2, AC5, C4, :FIG. 1. Patterns of aldolase activity of adult rabbit tissues after cellulose acetate electrophoresis. Electrophoresis and activity staining for aldolase were performed as indicated by W. A. Susor, E. Penhoet, and W. J. Rutter, this series, Vol. 41 [15].
242
ALDOLASES
[38l
tracentrifuge for 60 min at 100,000 g at 0-4 °. After the centrifugation is completed, carefully remove the clear supernatant with a Pasteur pipette and keep at 0 ° for short-term storage (0-8 hr) or freeze at --20 ° for longer-term storage. Assay. The type of aldolase present in a tissue extract can be qualitatively determined by electrophoresis of the extract followed by activity staining, as described in the accompanying paper. 8 Typical results of such analyses are presented in Fig. 1. A quantitative estimate of the total aldolase activity present in a tissue extract obtained as indicated above is determined by measuring the rate of cleavage of either fructose 1,6dip hosphate or fructose 1-phosphate according to the following reactions. Fructose 1,6-diphosphate •
• glyceraldehyde 3-phosphate + dihydroxyacetone phosphate Fructose 1-phosphate ~-----~glyceraldehyde + dihydroxyacetone phosphate
Details of the assay are presented by Lebherz and Rutter. s
Purification o] Aldolases A, B, and C For studies of aldolase enzymology it is necessary to obtain the various enzyme forms in a pure state. The methodologies presented below allow purification of these enzymes very rapidly and easily by affinity chromatography using phosphocellulose chromatographic columns and seelective elution with the substrate F D P , a modification of the method originally utilized by Pogell. 9
General Methodology All operations in the purification procedures are performed at 4 ° unless otherwise noted. Contact of aldolase preparations with the substrate is an integral part of the purification procedures, but the length of time of exposure to substrate should be minimized since it has been demonstrated that the enzymes are subject to slow inactivation in the presence of substrate. TM Concentrations of purified aldolases are determined spectrophotometrically using extinction coefficients of 0.91, 0.89, and 0.88 optical density unit per milligram per milliliter, in a 1-cm light path for aldolases A, B, and C, respectively. Purification o] Aldolase A. A summary of the purification procedure is presented in Table I. A detailed description of the methods follows. 8H. 'G. Lebherz and W. J. Rutter, this volume [39]. 9B. M. Pogell, Biochem. Biophys. Res. Commun. 7, 225 (1962). ~°B. M. Woodfin, Biochem. Biophys. Res. Commun. 29, 288 (1968).
[38]
MAMMALIAN F R U C T O S E - D I P H O S P H A T E ALDOLASES
243
TABLE I PURIFICATION OF ALDOLASE A FROM RABBIT MUSCLE
Purification steps 1. 14~000 g supernatant of crude extract. 2. 45-60% (NH4)2SO4precipitate 3. Phosphocellulosechromatography 4. Crystallization
Total activity
Total protein (g)
Specific actiyity
78,700
60.26
0.13
100
55,900 54,400 46,500
11.8 3.2 3.0
4.8-5.2 17 15-16
71 69 59
Yield (%)
Step 1. Crude Extract. Anesthetize a young (6 months or so) rabbit by injecting 30 mg of a Nembutal solution per kilogram. Remove the skin of the rabbit, quickly remove the skeletal muscles, and cool in an ice bath. Grind the meat in a meat grinder and weigh the ground nmscle. Suspend the ground muscle in 3 volumes (v/w) of 50 mM Tris C1, 5 mM EDTA, 4 mM 2-mercaptoethanol, pH 7.5. Homogenize this suspension in a commercial blender at low speed for 60 sec, and centrifuge at 14,000 g for 30 rain. Decant the supernatant solution for use in the following steps. Step 2. Ammonium Sulfate Fractionation. Bring the opalescent supernatant from above to 45% saturation ammonium sulfate by adding 278 g of ammonium sulfate per liter of extract over a l-hr period while maintaining the solution at approximately 2% Let the suspension stand for 1 hr and then centrifuge at 14,000 g for 1 hour. Remove the supernatant from this spin and discard the precipitate. Bring the supernatant to 60% saturation of ammonium sulfate by adding an additional 9 8 g per liter of supernatant. Adjust the pH of the suspension to 7.5 by adding an appropriate amount of 6 N NH40H and allow the solution to stand at least 2 hr before centrifuging at 14,000 g for 1 hr. Discard the supernatant from this spin and dissolve the precipitate in 10 mM Tris C1, 1 mM EDTA, pH 7.5. Adjust the protein concentration to approximately 40 mg/ml, and desalt the solution by passing it over a Sephadex G-25 column equilibrated with 10 mM Tris C1, 1 mM EDTA, pH 7.5. Step 8. Phosphocellulose Ch,romatography. Apply the desalted 45-60% ammonium sulfate fraction to a 3.7 X 100 cm phosphocellulose column equilibrated with 10 mM Tris C1, 1 mM EDTA, pH 7.5. After applying the sample wash the column with 1-2 liters of 50 mM Tris CI, 5 mM EDTA, pH 7.5 until the narrow red band visible on the column is eluted and the optical density of the effluent at 280 nm drops below 0.1. Proceed to elute the aldolase from the column with its substrate, fructose diphos-
244
[38]
ALDOLASES 30-
I I I
Buffer + FDP
20-
-SOOl
Buffer
~,
,~
-
I0r,
300 ~ ~' 200
!
1',
oo i
,. .....
6
io
40
..---.
6o
.....
so
,.....
,00
Fraction
._.--,:..__ ....
J20
,4o
._..,
160
iho 2oo
2:;o
number
Fro. 2. Phosphocellulose chromatography of rabbit muscle aldolase. The 45--60% a m m o n i u m sulfate fraction obtained as described in the text, and equilibrated with 10 m M Tris C1, 1 m M E D T A , p H 7.5 was applied to a 3.7 × 100 cm phosphocellulose column equilibrated with the same buffer. After the sample was applied, the column was washed with 50 m M Tris Cl, 5 m M E D T A , p H 7.5 until the red hemoglobin band was removed and the absorbance of the effluent at 280 nm fell below 0.1. The aldolase was then eluted with the same buffer to which 2.5 m M F D P had been added. The volume of individual fractions collected was 10 ml.
phate, by washing the column with approximately 1 liter of 2.5 mM fructose diphosphate in 50 mM Tris C1, 5 mM EDTA, pH 7.5. This substrate wash elutes the aldolase A sharply with a maximum specific activity of 14-18 t~moles of FDP cleaved per minute per milligram of protein as shown in Fig. 2. Step ~. Crystallization. Combine the peak fractions obtained from phosphocellulose chromatography above and dialyze against a solution which is 10 mM Tris C1, 1 mM EDTA, 50% saturated ammonium sulfate, pH 7.5 (4 ° saturated, diluted 1.1 to achieve 90%). Crystals will form in the dialysis tubing after a period of 12-20 hr. Collect the crystals by centrifugation at 35,000 g for 30 min. Resuspend the aldolase crystals in a small volume of the 50% saturated ammonium sulfate solution and store at 2-4 ° as a crystalline suspension for up to 6 months.
Purification of Aldolase A summary of the isolation of aldolase B is presented in Table II. With the following exceptions it is purified in the same manner as described above for aldolase A.
[38]
245
MAMMALIAN FRUCTOSE-DIPHOSPHATE ALDOLASES
~v
Z
e-,
0
o
©
©
II
G
:g 0
C~3
~.~ ~.~ ~..~ 1~ I~
o0
II
"~o~.
O
E~
Z
II
©
.
02
5 o
a. a h E O
o E
3
:1., b
20
40
6O FRACTION
80
I00
120
NUMBER
FIO. 3. D E A E A-50 Sephadex chromatography of the A-C aldolase set isolated from rabbit brain. Aldolases A-C eluted from phosphocellulose were equilibrated with 50 m M Tris C1, 4 m M EDTA, 200 m M sucrose, 5 m M fl-mercaptoethanol, p i t 8.0. This elution was then applied to a 0.8 × 50 cm DEAE-Sephadex A-50 column equilibrated with the same buffer. Elution was carried out with a linear sodium chloride gradient (0 to 0.4 M in the same buffer); 2-ml fractions were collected. Aldolase A was present in the breakthrough peak, and the order of elution beyond this was A3C, A=C.~, AC3, C~.
Step 2. DEAE-Sephadex Chromatog~'aphy. Combine the aldolase containing fractions from the phosphocellulose column above, and concentrate to 10-20 mg/ml using ultrafiltration. Then pass them over a Sephadex G-25 column equilibrated with 50 mM Tris C1, 3 mM EDTA, 200 mM sucrose, and 5 mM fl-mercaptoethanol, pH 8.0. Apply the desalted solution to a DEAE-Sephadex A-50 column (2.5 X 50 cm) equilibrated with the same buffer and elute the aldolases with a linear sodium chloride gradient (0 ~ 0.35 M NaC1) in 50 mM Tris C1, 3 mM EDTA, 200 mM sucrose, and 5 mM fl-mercaptoethanol, pH 8. As shown in Fig. 3, the various members of the AC hybrid set are resolved from each other by this procedure and elute from the column in the order A4, A2C2, AC3, and C4. Combine the fractions in each peak and concentrate to approximately 10 mgl/ml by ultrafiltration and then precipitate by dialyzing against 55% saturated ammonium sulfate containing 1 mM EDTA, pH 7.5.
248
ALDOLASES
1.2
a A!
[38]
.b
.........
4~
c~
.4
2
20
40 Fraction
60 Number
80
FIo. 4. Isoelectric resolution of crystalline rabbit muscle aldolase A of specific activity 16, electrofocused on an ampholine gradient (pH 7 to 9).
Purity of the Aldolase Preparations To determine purity of the aldolase preparations the samples may be subjected to polyacrylamide disc gel electrophoresis or analyzed by other traditional methods of protein chemistry such as ultracentrifugation, etc. To determine the efficacy of the resolution of the various members of the A-C set from each other in the purification from brain tissue perform the qualitative aldolase assay using cellulose acetate electrophoresis and activity staining which was presented in an earlier section of this manuscript. Purified, crystalline aldolase A preparations give rather diffuse bands in native polyacrylamide gels. This phenomenon is apparently due to an inherent heterodispersity of this protein, as shown in the studies of Susor, Kochman, and Rutter, 1°" who demonstrated that crystalline aldolase A preparations could be resolved into 5 peaks by electrofocusing (Fig. 4). These peaks behaved as a 5-membered hybrid set formed by combination of two different subunits in tetramerie molecules. This possibility was confirmed by the isolation of the two subunits and recombination to form the 5-membered set. Peptide maps confirmed a minor difference in the primary structure (probably the alteration of a single amino acid). This suggested the possibility of two separate aldolase A alleles in rabbits, or the degradation, for example by deamination in situ, or during the isolation procedure. The results of subsequent studies by Horeeker and colleagues support the latter alternative. 1~ loa W. A. Susor, M. Kochman, and W. J. Rutter, Science 165, 1260 (1969). 1, C. Y. Lai, C. Chen, and B. L. Horecker, Biochem. Biophys. Res. Commun. 40, 461 (1970).
[39]
CLASS I FRUCTOSE-DIPHOSPHATE ALDOLASE
249
This heterogeneity has little if any effect on general catalytic properties of the molecules. However, detailed studies of catalysis or chemistry should p r o b a b l y be carried out with an enzyme composed of a single subunit. The " n a t i v e " aldolase A is probably the species of p I 8.2.
[39] The Class I (Schiff Base) Fructose-diphosphate Aldolase of Peptococcus aerogenes B y HERBERT G. LEBHERZ and WILLIAM J. RuvrER
Fructose 1,6-diphosphate -~ dihydroxyacetone phosphate -I- n-glyceraldehyde 3-phosphate Fructose 1-phosphate ~ dihydroxyacetone phosphate -[- n-glyceraldehyde The fructose-l,6-diphosphate ( F D P ) aldolases found in biological systems can be assigned to one of two main classes. 1 Class I aldolases, as exemplified by the m a m m a l i a n muscle enzyme, catalyze the reversible cleavage of F D P and fructose 1-phosphate ( F - l - P ) via the formation of a Schiff base intermediate between substrate and a lysyl residue at the active site of the enzyme. ~ In contrast, class I I aldolases, as exemplified by the yeast enzyme, do not cleave F - 1 - P nor do they participate in a Schiff base complex with the substrate ( F D P ) . Rather, these aldolases are metalloenzymes; they require divalent cations, usually Zn 2÷ or Fe ~÷, for activity. 3,4 The two aldolase types can further be distinguished on the basis of their catalytic and molecular properties2 -7 Until recently the class I aldolases were thought to be restricted to "higher organisms"; namely, animals, plants, protozoa, and algae. Class I I aldolases were found in fungi and all prokaryotic cells tested. 1,6 E u g l e n a 6 and C h l a m y d o m o n a s ~ contain both aldolase types. We have recently shown that the aldolase isolated from the bacterium P e p t o c o c c u s aerogenes 9 A T C C No. 14963 (also called M i c r o c o c c u s a e r o 1W. J. Rutter, Fed. Amer. Soc. Exp. Biol. 23, 1248 (1964). B. L. Horecker, P. T. Rowley, E. Grazi, T. Cheng, and O. Tehola, Biochem. Z. 338, 36 (1-963). a R. D. Kobes, R. T. Simpson, B. L. Valtee, and W. J. Rutter, Biochemistry 8, 585 (1969). ' W. J. Rutter and K. H. Ling, Biochim. Biophys. Acta 30, 71 (1958). s 0. C. Richards, and W. J. Rutter, J. Biol. Chem. 236, 3177 (1961). W. J. Rutter and W. E. Groves, in "Taxonomic Biochemistry, Physiology and Serology" (C. A. Leone, ed.), p. 417. Ronald Press, New York, 1964. W. E. Groves, Ph.D. Thesis, University of Illinois, Urbana, Illinois, 1962. s G. K. Russell, and M. Gibks, Biochim. Biophys. Acta 132, 145 (1967). 9 R. S. Breed, W. G. D. Murray, and N. R. Smith, "Bergey's Manual of Determinative Bacteriology," 7th Ed. Williams & Wilkins, Baltimore, Maryland, 1957.
