PURIFICATION AND CHARACTERIZATION OF ASCARIS SUUM ALDOLASE: AN INITIAL PHYLOGENETIC STUDY OF ALDOLASES
APPROVED:
Major Professor
uL/ // •IS- Minor Professor
*) xKiDt i. Chairmanofthe Department of Biology
Dean Uf the Graduate School " i?/i/
Dedman, John R., Purification and Characterization of
Ascaris suum Aldolase: An Initial Phvlogenetic Study of
Aldolases. Master of Science (Biology), August, 1972, 74 pp., 10 tables, 12 figures, 101 titles.
An efficient purification procedure of Ascaris suum muscle utilizing ion exchange column chromatography has been developed. Homogeneity of the enzyme preparation was con- firmed by rechromatography, ultracentrifugation, and disc gel electrophoresis. The molecular and catalytic properties of the enzyme are comparable to other muscle aldolases. The molecular weight, determined by ultracentrifugation and gel filtration, is 160,000 composed of four similar subunits.
The sedimentation coefficient, diffusion coefficient, fric- tional ratio, Stokes' radius, amino acid composition, and extinction coefficient have been determined. The Michaelis constants for fructose-1,6-diphosphate (FDP) and fructose-1- phosphate (F1P) are 6 x 10"" ^ M and 3 x 10~2 M, respectively.
The FDP/F1P ratio is 42.
Tryptic peptide fingerprints and amino acid analysis
Ascaris and rabbit muscle aldolases also exhibited a high degree of homology. It appears that muscle aldolases have been very conservative in maintaining their primary structure.
A proposal of a phylogenetic study of aldolases utilizing a quantitative method of comparing proteins from their amino acid compositions is discussed. PURIFICATION AND CHARACTERIZATION OF
ASCARIS SUUM ALDOLASE: AN INITIAL
PHYLOGENETIC STUDY OF ALDOLASES
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
By
John R. Dedman, B. S
Denton, Texas
August, 1972 "It is quite conceivable that every species tends to produce varieties of a limited number and kind, and that the effect of natural selection is to favour the development of some of these, while it opposes the development of others along their predetermined lines of modification."
Thomas H. Huxley Evolution in Biology. 1878 INTRODUCTION
Aldolase (E. C. 4.1.2.13 f ructose-1,6-diphosphate-
D-glyceraldehyde-3-phosphate lyase) provides an essential role in the glycolytic pathway of all organi&ms, with the exception of some heterofermative bacteria (Gunsalus, 1952).
It catalyzes the cleavage of fructose-lj6-diphosphate (FDP) to the trioses, dihydroxyacetone phosphate (DHAP) and glycer- aldehyde-3-phosphate (G-3-P) (reaction l).
HS-0-P03 c=o H HOCH HC-O-PO, HC=0 I I , I HC0H - Aldolase . C=0 + HCOH i I i HCOH HCOH HC-0-P0o I H H 3 HC-O-PO- H 3
FDP DHAP G3P
Although the equilibrium constant is in favor of FDP formation, the reaction is readily reversible; when there is sufficient removal of the triose products during glycolysis.
Aldolase has an absolute specifity for DHAP (Morse and Horecker,
1968), although a wide variety of aldehydes and their ketoses
(phosphorylated or non-phosphorylated) including L-glycer- aldehyde, D and L-erythrose, D and L-threose, formaldehyde acetaldehyde, glycolaldehyde, and propionaldehyde may constitute the remaining carbon requirement (Morse and Horecker, 1968}
Rutter, I960: Horecker, 1959)•
Aldolase activity was first demonstrated by Meyerhof and
Lohmann (1934)> who believed the enzyme to also contain the
catalytic function of triose phosphate isomerase. This
activity was found to be a contaminant when extensively puri-
fied by Herbert (1940). Warburg and Christian (1943) recorded
the first crystallization of the enzyme from rat muscle and
yeast.
Two distinct forms of aldolase have been reported in
biological systems. Rutter (1964) classified these unique
forms as Class I and Class II, each possessing strikingly
diverse physical and catalytic properties. Class II aldolase
is restricted to bacteria, fungi, yeast, and blue-green algae.
Its most distinguishing characteristics are a requirement
of a divalent metal ion and a molecular weight of 80,000
(Rutter, 1964j Harris et al., 1969; Kobes et al., 1969;
Mildvan et al., 1971).
Class I aldolases are present in animal and higher plant tissues. They exist as tetramers of molecular weight 160,000. I The exception is spinach leaf aldolase, which has a molecular weight of 120,000 (Fluri et al., 1967). Class I aldolases are not affected by metal chelators. They form a Schiff base intermediate between DHAP and the £-amino group of a lysine residue in the active site (Grazi et al., 1962; Morse and Horecker, 1968: Rutter, 1964; Horecker, 1970). The comparative properties of Class I and Class II aldolases are summarized in Table I. Euglena and Chlamydomonas (Rutter, 1964) possess both forms of aldolase, Class II during heterotrophic growth, and Class I during autotrophic phases.
The animal aldolases have shown variformity in tissue localization (Penhoet et al., 1966; Masters, 1967; Rutter et al., 1968; Masters, 1967} 1968; Lebherz and Rutter, 1969J
Kochman et al., 1971? Penhoet et al., 1969a). There are three types, A, B, arid C, characteristic of muscle, liver, and brain aldolase, respectively (see Table II). The three types are similar in physical properties, including molecular weight, subunit structure, and catalytic mechanism forming a Schiff- base intermediate (Morse and Horecker, 1968a; Penhoet et al..
1969b: Schachman et aj.., 1966; Penhoet et al., 1967; Sia et al.. 1968; Kawahara et al., 1966; Gracy et al., 1969).
However other parameters exhibit significant differences.
Distinctions may be seen in immunological cross-reactions
(Penhoet et al., 1966; Penhoet et al., 1969; Penhoet and
Rutter, 1971; Marquart, 1971; Baron et al., 1969), amino-acid compositions (Gracy et al., 1970), tryptic fingerprints (Gracy I St J 1970; Penhoet et al., 1969b; Rutter, 1964), sequences of amino-acids in the active sites (Morse and
Horecker, 1968b), and end-group analysis (Ting et al., 1971;
Lai and Oshima, 1969). These properties are summarized in
Table III. The major catalytic distinctions between the Class I TABLE I
PROPERTIES OF ALDOLASE CLASSES
Class I Class II
Animals, plants, green Bacteria, yeast, fungi, blue- algae, euglena and green algae, euglena &1 chlamy- chlamydomonas (Autotrophic) domonas (Heterotrophic)
Schiff-base intermediate Divalent metal ion requirement ((-amino lysine-DHAP) K+ activation
Functional carboxyterminal Functional SH groups tyrosine
Broad pH profile Sharp pH profile (7»0-7.5)
MW 120,000-160,000 MW 70-80,000 s 20,» 6.5-8.0 S20,w 5-5-6'.0 S 4 subunits 2 subunits
FDP/FIP ratio 50,1,10 FDP/F1P ratio 2000-2500
Specific Activity 8r25 Specific Activity 80-100
From: Rutter, 1964* Rutter et al., 1968. TABLE II
TISSUE DISTRIBUTION OF ALDOLASE ISOZYMES
Tissue Isozyme
Muscle A
Heart A
Spleen A Kidney cortex A-B medulla A
Brain A-C
Liver A-B
^-Intestine A, B
*Lens C From: Lebherz and Rutter, 1969*
•frKochman et al., 1971. TABLE III
PROPERTIES OF CLASS I ALDOLASE VARIANTS (RABBIT)
Properties A B C
^Molecular Weight 160,000 160,000 160,000 #Schiff-base Intermediate + + + °Immunological cross-reactivity Anti-A + Anti-B + Anti-C + ^Residual act- ivity after carboxypep- tidase A 5% 55% 1056 w^max (FDP Cleavage) 2,900 250 1,000 (FDP Synthesis) 10,000 2600 4,900 *Km 6 (FDP) 3 X 10~ 1 x 10~9 3 x 10-6 ( F IP ) 1 x 10""2 3 x 10-4 4 x 10"3 (DHAP) 2 x 10~3 4 x 10-4 3 x 10-4 (G3P) 1 x 10-3 3 x 10-4 8 x 10"4 *FDP/F1P Ratio 50 1 10 #Rutter et al., 1968. " " °Penhoet, Kochman, and Rutter, I969, *Penhoet and Rutter, 1971. aldolases are the maximum velocities of cleavage of the two
substrates (ie, FDP/F1P ratio). Mammalian muscle (type A),
liver (type B), and brain (type C) possess FDP/F1P ratios of
50, 1, 10, respectively (Penhoet et al., 1966; Penhoet et al..
1969b). The physiological function of type A and B aldolases
have been correlated with these catalytic properties. The
muscle form functions primarily in glycolysis, and the muscle
does not utilize F1P as a substrate. On the other hand, the
liver can convert fructose into F1P by a specific fructokinase
which in turn is cleaved by liver aldolase. The glyceraldehyde
formed from this reaction can be phosphorylated by a specific
triose-kinase. Likewise, ATP is a competitive inhibitor of
the muscle type and the liver enzyme is strongly inhibited
by AMP (Spolter et al., 1965; Gracy et al., 1970). Both types,
A and B, fulfill physiological function in the specific tissues
(Rutter et al., 1963; Horecker, 1967) • The specialized func-
tion of brain aldolase has yet to be elucidated (Herskovits
et al., 1967).
The ontogeny of aldolase has been studied by examining
the isozyme patterns in specific tissues (Rutter et al., 1963;
Weber and Rutter, 1964; Sheedy and Masters, 1971; Masters,
1968). In most tissues, mixed hybrid forms are originally
present, and not until the tissue develops and differentiates do the parental forms become predominant (Masters, I968)
(Table II). Adult muscle maintains type A and liver is pre- dominately type B, while kidney and brain possess the five 8 respective A-B and A-C hybrids (Penhoet et al., 1966; Rutter et al., 1968; Kochman et al., 1971; Lebherz and Rutter, 1969). Although ABC and BC hybridization has been produced in vitro, such combinations have yet to be found in biological systems
(Penhoet et al,, 1966; Penhoet et ajL., 19
Bn-Cn mixtures possessed proportional FDP/F1P ratios indi- cating that, catalytically, the subunits act independently. This has also been verified by the hybridization experiments using succinic anhydride inactivated subunits (Meighen and
Schachman, 1970). Anti-sera against A does not react with B or C and vice versa. But anti-sera from A, B, or C inhibit activity of any combination of its hybrids.
Because of its omnipresence in biological systems and relative ease of purification, aldolase has been extensively studied and amply characterized. This has created an area of extensive information and interest in comparative bio- chemistry of living organisms. Until now, aldolase has been purified and studied in a large variety of vertebrates
(Anderson et al., 1969; Caban and Hass, 1971; Ikehara et al.. 1969), spinach (Fluri et al.., 1967; Rapaport et al., I969), and microorganisms (Kobes et al., 1969; Mildran et al., 1971). Although this study encompasses a wide variety of organisms, there is a large void of information in the invertebrate classes, the lobster (Buha et al., 1971) being the lone species studied. This has resulted in a strong need for a
thorough investigation of the invertebrates.
The present study of Ascaris aldolase is twofold; first,
from a comparative biochemical standpoint, and secondly from
a clinical aspect. There is a need for more efficient anthel-
mintic drugs. From recent studies, many drugs have been found
to inhibit specific Ascaris enzymes (Saz and Bueding, 1966;
Bueding, 1969; Saz and Lescure, 1968; Van den Bossche and
Jansen, 1969). The greater our knowledge of the differences
between the parasite's and host's metabolic pathway^ the more
confident we can be of a vermifuge's effectiveness. There
are marked differences in the metabolic pathways of the worm
and host (Saz and Lescure, 1969; Saz, 1971; 1971b). Although
helminth enzymology is in its infancy, a few reports of the.
properties of purified helminth enzymes have recently appeared
(Burke et al., 1972; Li et al., 1972; Fodge et al., 1972; Hill
et al., 1971). In addition, partial purification of nematode
aldolases has been reported (Mishra et al., 1970; Srivastava
et al., 1971).
The present pcper describes the purification and properties
of the enzyme aldolase from the muscle tissue of the pig intes-
tinal roundworm, Ascaris suum. In addition, the properties of the ascarid enzyme have been correlated with muscle aldolases of other organisms in an attempt to evaluate the evolutionary development of the enzyme. MATERIALS AND METHODS
Materials — 1 Ion exchanger, cellulose phosphate (0.91 meq g ), the
sodium salts of NAD, NADH, fructose-1,6-diphosphate (FDP);
2-mercaptoethanol, ammonium sulfate (enzyme grade), crystalline bovine serum albumin, ^-glycerol phosphate dehydrogenase,
triose phosphate isomerase, ethylenediaminetetracetic acid
(EDTA), phenazine methosulfate, MTT tetrazolium 3(4,5-
dimethylthiazolyl-2-)-2-5-diphenyl tetrazolium bromide,
triethanolamine, and the barium salt of fructose-l-phosphate
(F1P) were obtained from Sigma Chemical Company. Guanidinium chloride, sodium dodecyl sulfate (SDS), and ninhydrin were
1Sequanal grade' and purchased from Pierce Chemical Company.
Sephadex G-200, aldolase, chymotrypsin, albumin, and ovalbumin were acquired from Pharmacia Fine Chemicals. Aldolase and glyceraldehyde-3-phosphate dehydrogenase, crystallized from rabbit muscle, and TPCK-treated trypsin were prepared by
Worthington Chemicals. Iodoacetic acid (Eastman Organic
Chemicals) and urea (Mallinckrodt) were recrystallized prior to use. The barium salt of F1P was converted to the sodium salt with Dowex50 (Mesh 200-400). All other chemicals were of reagent grade and all solutions were prepared in distilled water.
10 11
Enzyme Assay
Aldolase was assayed according to the coupled enzyme procedure of Racker (1947). Assays were performed in a temperature—rfegulated Beckman Model DB—GT Spectrophotometer attached to a Beckman Ten-inch Laboratory Potentiometric
Recorder. Initial velocities were determined by monitoring the linear decrease in the absorbance of oxidized NADH at
340 nm at 25°C. The routine reaction solution consisted of
130 yumoles triethanolamine buffer (pH 7»5)> 0.75 xunoles
NADH, 3.0 jumoles FDP, and 10 xig each of triose phosphate isomerase and ©^-glycerophosphate dehydrogenase in a 3 ml final volume. One unit of activity is defined as the amount of enzyme catalyzing the conversion of 1>umole of FDP per minute at 25°C, Specific activities are expressed as units per mg protein. During kinetic studies, precise measurements were made by observing the optical change from 0.0-0.1 0D with a chart speed of 10 inches per minute. Substrate con-
centrations were determined spectrophotometrically.
Protein Determination
The protein concentration of crude homogenate was deter- mined by the Biuret method (Bailey, 1967) using a standard
curve of 1-5 mg ml""*- crystalline bovine serum albumin. All
other protein estimations were determined from absorption at
280 nm using the molar absorbancy index of 1.06. 12
Ion-Exchange Chromatography
Cellulose phosphate was washed repeatedly with 0.5 N
HC1 and 0.5 N NaOH until the filtrate remained colorless
(Peterson and Sober, 1962). The exchanger was then equil- ibrated in 10 inM triethanolamine, 1 mM EDTA, and 10 mM
2-mercaptoethanol at the desired pH and stored at 0-5°C.
A suspension of approximately 1 gm per 15 ml buffer was allowed to de-aerate under vacuum with vigorous stirring. Columns were packed under hydrostatic pressure and washed with buffer (50 ml hr-l) at 5°C for 24 hours. The eluant was tested for the desired pH before the sample was applied. Constant flow rates
(50 ml hr ) were maintained with a piston minipump. Fractions of 10 ml were collected. Protein was monitored by the absor- bance at 280 nm.
Gel Filtration
Sephadex G-200 (particle size 40-120 AJL) was equilibrated in 5 mM phosphate buffer suggested by Pharmacia (1970) and packed under 10 cm hydrostatic pressure. The 1.6 x 120 cm column was washed with the buffer at 5°C until stabilization of bed height and a constant flow rate of 6 ml hr""l were achieved. Samples in 20% sucrose (w/v) were layered onto the top of the column bed. Dimensions of the column, void volume
(VQ), internal volume (V^), and elution volumes (Ve) were determined with blue dextran, tryptophan, and standard proteins
(Pharmacia). The elution volumes were correlated with molec- ular weights (Andrews, 1964j Determann and Michel, 1966) or 13
Stokes' radii (Andrews, 1970).
Polyacryamide Gel Electrophoresis
Disc gel electrophoresis was conducted according to the
procedure of Davis (1964)- Gels were prepared with a 7*5%
monomer concentration in Tris-glycine buffer (pH 8.9) and
run on a Canalco Model 200 apparatus. Sodium dddecyl sulfate
gel electrophoresis was performed following the procedure of
Weber and Osbox-n (1969), using 10$ gel in 0.1% SDS and phosphate
buffer (pH 7.0). The mobilities of the protein subunits
were plotted against their molecular weights. Protein was
stained with 0.25$ (w/v) Coomassie Brilliant Blue in methanol:
acetic acidsI^O (5:5:90). Activity bands were developed via
the method of Lebherz and Rutter (1969), coupling glyceralde—
hyde-3-phosphate dehydrogenase reduction of NAD with phenozine
methosulfate and MTT tetrazolium.
Ultracentrifugation
Sedimentation velocity and sedimentation equilibrium
experiments were conducted in a Beckman-Spinco model E ana-
lytical ultracentrifuge equipped with RTIC temperature and
electronic speed control. Schlieren patterns and Rayleigh
interference fringes were measured with a Nikon model 60 microcomparator with digital encoders. High-speed sedimen- tation equilibrium experiments were carried out as described by Yphantis (1964) and Van Holde (1967). Densities and vis- cosities of all buffers were calculated as described by Rozacky 14
et al. (1971) and Kawahara and Tanford (1966). The partial
specific volume was estimated from the amino-acid composition.
Tryptic Fingerprints
Samples of purified aldolase (10-20 mg) were S-carbox-
ymethyiated in 10 ml of 8M urea, 0.2% EDTA, 0.1 ml 2-mercap-
toethanol, and 0.268 g iodoacetic acid (Scoffone and Fontana,
1970). A 1/50 weight ratio of TPCK treated trypsin was used
to digest the aldolases at pH 8.0 in 2.5% trimethanolamine.
After dialysis in y% formic acid and lyophilization, the
samples were diluted to a concentration of 10 mg ml-"'". Approx-
imately 200 jag of protein was carefully spotted near the
corner of a 20 x 20 cm celluose-coated (160 microns) thin-
layer plate (Eastman Organic Chemicals). Best results were
obtained when electrophoresis was carried out first at 300
V for 2 hrs under varsol in pyridine:acetic acid:H20 (100: 30:3000) pH 5.5 at 5°C. The plate was then chromatographed
in the second dimension in n-butanol:pyridine : acetic acid:
^2® (150:100:30:120) to 1 cm of the upper edge. After the dried chromatogram was sprayed with 2% ethanolic ninhydrin with 10$ collidine, it was allowed to develop for 30 min in a 75°C oven (Tarui et al., 1972). The colored spots were outlined and the plates stored at 4°C with little loss in intensity for several weeks.
Amino Acid Analysis
One milligram samples were hydrolyzed for 24 and 48 hrs 15 in sealed evacuated tubes containing 2.0 ml of 6N HC1 at
110°C (Moore and Stein, 1963). Norleucine was used as an internal standard. Analyses were performed on a Beckman model 120C automatic amino acid analyzer (Spackman et al.,
1958). Cysteine was determined from the S-carboxymethyl derivative (Scoffone and Fontana, 1970) and tryptophan was estimated spectrophotometrically (Edelhoch, I967). RESULTS
Purification
Female Ascaris were obtained from a local abattoir.
Muscle tissue was dissected free from reproductive and di- gestive organs then, washed in cold buffer. The muscle was then homogenized in 3 volumes of 20 mM triethanolamine, 0.1
M NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol (pH 7.5) for three
1-min intervals with 5—min intermittent cooling periods. The mixture was centrifuged for 1 hr at 12,000 x g in a refrigerated centrifuge at 0-5°C. The resulting pellet was rehomogenized for 1 min in 1 volume of buffer and recentrifuged as above.
The supernatant solutions were pooled and again centrifuged at 90,000 x g for 1 hr (Beckman model L 3-40) and strained through glass wool to remove any residual lipid. The resul- tant pale-pink solution ('Extract') was then allowed to dialyze against 4 liters 10 mM triethanolamine, 1 mM EDTA, 10 mM 2- mercaptoethanol buffer (pH 7.1) for 24 hrs with three changes of buffer.
The dialized fraction was applied to a column (2.5 x 45 cm) of phosphocellulose and washed with the respective buffer until the eluant was protein-free. Subsequently, a linear salt gradient (0.0-0.5M NaCl; 500 ml to 500 ml) was begun (Fig. l),
Fractions containing aldolase activity (Fig. 1) were combined
('Salt Elutant') and dialyzed 20 hrs against the same buffer
16 17
Fig. 1. Column chromatography of Ascaris aldolase on phosphocellulose. 'EXTRACT' (480 ml) was applied to a phos- phocellulose column (2.5 x 60 cm). Buffer (10 mM trieth- anolamine 10 mM 2-mercaptoethanol, ImM EDTA, pH 7*1) was pumped through until the eluate was free of protein. A linear salt-gradient was employed starting with buffer (500 ml) in the mixing chamber and 500 ml of 0.5M NaCl buffer solution in the reservoir. Arrow designates the initiation of the gradient. The major peak of activity was pooled ('SALT ELUTANT') and dialyzed in the same buffer at pH 7»4« lp (t_|iu 6ui)Ni3xoad
?Osoonoio^ P)N»-
J %UJ 3 —J o>
CO CN i-j oodo •—• (.[UI^UIUipOADaQ dad S®|OW9-0t 19 at pH 7.4. The sample was applied to a second phospho- cellulose column (1.5 x 90 cm) equilibrated in the appropriate buffer. Again the non-binding protein was washed free from the column. A 2.5 mM fructose-1,6-diphosphate solution (in the same buffer) was applied eluting all of aldolase activ- ity in a single, sharp peak in which the activity and protein fractions coincided ('FDP Elutant', Fig. 2). The purified enzyme solution was immediately dialyzed for 15 hrs in satur- ated ammonium sulfate to avoid loss of activity (Woodfin and Temer, 1967)• The precipitated protein was collected by cen- trifugation at 25,000 x g. The total purification of Ascaris muscle aldolase (Table IV) is 157-fold, a value consistent to that reported for other muscle aldolases. A final yield of 11.2 mg enzyme represented # 67% recovery of activity from an original 130 gm of Ascaris muscle. A specific activity of 10.2 can be compared with other type A aldolases, frog, 11.5 (Ting et al., 1971), shark, 25.5 (Caban and Hass, 1971), tuna, 7.5 (Kwon and Olcott, 1968), lobster, 14.8 (Guha et al., 1971), chicken, 24 (Marquart, I969), codfish, 8 (Lai and Chen, 1971), and rabbit, 12 (Schwartz and Horecker, 1966). Comparable results were obtained with several other preparations, and the precipitated enzyme was pooled and stored at 4°C in 70% ammonium sulfate. The absorbance of a 0.1% solution of Ascaris aldolase at 280 nm is 1.06, This is consistent with the greater number of tryptophan residues in the ascarid enzyme (vide infra). 20
Fig. 2, Substrate elution of Ascaris aldolase from phosphocellulose. Partially purified enzyme ('SALT ELUTANT'j 300 ml, 310 mg, specific activity = 0.4) was applied to a phosphocellulose column (1.5 x 90 cm) and washed with buffer until fractions were protein free. A 2.5 mM FDP solution (in same buffer) was then applied (arrow A), specifically eluting aldolase. The purified enzyme solution was immediately dia- lyzed against saturated ammonium suffate. Salt solution (1.0M: B) was applied to elute the remaining protein which was devoid of aldolase activity. 2,1
10~6 Moles FDP Cleaved min"1 ml"1 •—• __i ro w •o o• •
O< r— c £ m
o o o
o% o o i t O Ui PROTEIN (mg ml""1) 22
TABLE IV
PURIFICATION OF ASCARIS MUSCLE ALDOLASE
Procedure Vol. Protein Units Spec- Yield Fold (ml) (mg) ific % Purif. Act.
'Extract' 480 2620 170 0.065
'Salt Elutant' 300 310 123 0.4 72 6.2
'FDP Elutant' 45 H.2 114 10.2 67 157.0 23
Protein concentration of the solution was determined by the method of Lowry et al. (1951)•
Gel Filtration
The molecular size of Ascaris muscle aldolase was also
determined from quantitative gel filtration of a calibrated
Sephadex G-200 column (1.5 x 120 cm; see Figure 3). Chro-
matography of Ascaris and rabbit muscle aldolases on Sephadex
(Fig. 3 and 4) results in coinciding elution peaks, demon-
strating the striking similarity of molecular size of the two muscle aldolases. The apparent molecular weight of Ascaris muscle aldolase from gel filtration is 158,000 + 3000.
The Stokes' radius was determined from the gel filtration
data according to the procedure of Ackers (1968). The Stokes'
radius of Ascaris aldolase is 46.4 ^ (Fig. 5). The diffusion
constant was estimated from the relationship D^Q w — KT/6Trtytt, ,
(Siegel and Monty, 1965), where 1 is the Stokes' (46.4 A), K
is the Boltzman constant, and is the viscosity of water at
20°C (0.01005 P). Ascaris aldolase has a diffusion constant n O of 4.43 x 10 cm /sec. Further, a frictional ratio (f/fQ) of 1.29 for Ascaris aldolase is indicative of an essentially spherical molecule. These are essentially the same values as rabbit muscle, since it was used as a standard.
Folyacrylamide Gel Electrophoresis
Disc gel electrophoresis of Ascaris muscle aldolase re- sulted in a single band of protein which corresponded to the 24
Fig. 3. Determination of molecular weight of Ascaris aldolase by gel filtration. The logarithm of the molecular weight is plotted against the ratio of protein elution volume (Ve) to column void volume (VQ) . A Sephadex G-200 column (1.5 x 120 cm) was calibrated with (A) chymotrypsin A, MW 25,000, (B) ovalbumin, MW 45>000, and (C) rabbit muscle aldolase, MW 158,000. The open circle (0) represents Ascaris aldolase. Fractions of 40 drops each were collected (flow rate of 6.0 ml per hour). n
2.75 —
\a
S>S«B >° 2.25 - >"
\|C 1.75 mm
1 1 1 1 30 50 100 150 MOLECULAR WEIGHT (xlO3) 26
Fig. 4. Comparative elutions of Ascaris and rabbit muscle aldolase on Sephadex G-200 column(1.5 x 120 cm). Samples were chromatographed on separate runs. 27 RABBIT MUSCLE ALDOLASE OD280
io o
7>3 O n -H o a z O z c £ CO rn CO 79 o
o o
ASCARIS ALDOLASE ACTIVITY (units ml"1) •—• 28
Fig. 5. Determination of molecular radius of Ascaris aldolase from gel filtration data. values (Ve-V0/V.)are plotted against the Stokes' gadii of CA) chymotrypsin A, 20.9 A, (B) ovalbumin, 27.3 A, and rabbit muscle aldolase, 45 a. The open circle (0) designates Ascaris aldolase. 2$
K.
0.4-
20 SO 40 MOLECULAR RADIUS (A) 30
single band when stained for aldolase activity {Fig, 6), thus providing another criterion of homogeneity. Electrophoresis in the presense of sodium dodecyl sulfate (SDS) yielded a single band. Ascaris aldolase, disassociated in SDS, exhibits a subunit molecular weight of 42,000 + 3000(Fig, 7).
Ultracentrifugation Studies
As shown in Figure 8,the sedimenting enzyme formed a single, symmetrical boundary, indicating a homogeneous pre- paration of the enzyme. Values of the sedimentation coeffi- cient were calculated from three protein concentrations (2.87,
1.49. 0.74 mg ml~^) and the s2o w was °btained by extrapolation to zero protein concentration. The value of 7.98 S (Fig. 9) is in the same order as reported for Class I aldolases (Penhoet
al., 1969b; Gracy et al. 1969; Ting et al, 1971; Lai and
Chen, 1971; Guha et al., 1971). The S2o,w may be calculated from the relationship S2QjW =® 7.98 (l~0.008c), where c is the protein concentration in milligrams ml""**'.
Sedimentation equilibrium studies yielded a molecular weight of 156,000 + 3000. When disassociated in 6M guanidine hydrochloride the molecular weight is 40,800 + 1000, indicating a tetrameric structure of similar subunits with virtially equal mass. Plots of (In y vs x2) resulted in straight lines, suggesting a monodisperse system (Fig. 10).
Catalytic Properties
The FDP/F1P ratio for Ascaris muscle aldolase is 42 from 31
Fig. 6. Polyacrylamide gel electrophoresis of Ascaris aldolase. Purified enzyme (50 micrograms with specific activity = 10.2) was applied to a 7.5% standard 0.6 x 7.0 cm polyacrylamide gel. Electrophoresis was carried out at 5° in Tris-glycine buffer (pH 8.9). The gel shown at left was stained for protein,while the gel on the right was devel- oped in a stain specific for aldolase activity ('Methods'). 32 33
Fig. 7. Determination of subunit molecular weight on sodium dodecyl sulfate gel electrophoresis. Ascaris aldolase (0.5 mg) and standard proteins were prepared in 1% SDS solution and subjected to electrophoresis on separate gels (10% monomer concentration, 8 ma, 5 hours). The mobility of each protein was plotted against the logarithm of its molecular weight. Letters designate standard proteins, (A) albumin, MW 68,000, (B) ovalbumin, MW 43,000, (C) pepsin, MW 35,000, and (D) trypsin, MW 23,3000. 34
(£0l*)
J.HOI3M avmDaiow 35
Fig. 8. Sedimentation-velocity centrifugation of Ascaris aldolase. Purified enzyme (2.87 mg ml"1) was centrifuged in a 12 mm double sector cell at 60,000 rpm at 20°C. The solute sector and reference sector contained 0.40 ml of 10 mM Tris-Cl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 M NaCl, pH 7.5. 36 37
Fig. 9. Determination of sedimentation coefficient of Ascaris aldolase. Purified Ascaris aldolase in concentrations of 2.87* 1«43* and 0.72 mg ml" were subjected to sedimentation va ue velocity experiments at 60,000 rpm. The §20 w l (7*985) was obtained by extrapolating to zero protein concentration. 38
* CON CO
1.0 2.0 3.0 PROTEIN (mg ml4) 39
Fig. 10. Fringe displacement obtained from sedimentation equilibrium of native Ascaris aldolase. The protein (0.5 mg ml ) was dialyzed against 50 mM Tris-Cl, 0.1 M NaCl, 10 mM 2-mercaptoethanol, pH 8.0. Sedimentation was at 16,000 rpm in the An-D rotor in a 12-mm double sector cell with sapphire windows for 24 hours at 20°C. The protein concentration was calculated from the fringe displacement (y"i-y0) • The abscissa represents the square of the distance (cm) from the center of rotation. 40
1.0
£ ~ 0.5 w C
0.0
50.8 50.9 51.0 51.1 51.2
v2 41
comparisons of the Vmax values. The Michaelis constant for FDP is 6 x 10~^M and for F1P is 3 x 10~^M at 25°C. The con- centration range for FDP was 5.18 x 10~^M to 3.11 x 10~^M and that for FIP was 5.75 x 10~2M to 1.01 x 10-1M. Values were calculated using a computer program with weighted regression analysis (Cleland, I963). When dialyzed against 0.1 M EDTA, there was no loss in activity, and divalent or potassium ions displayed no augmentation in activity. These results are in accordance with those reported for all other muscles aldolases except lobster (Guha et al., 1971), which has a catalytic ratio of 200. Properties of Ascaris muscle aldolase are summarized on Table V*.
Peptide Maps Composite maps were drawn from at least five chromatograms each for rabbit and Ascaris muscle aldolase (Fig. 11). Locali- zation of the peptides and comparisons were made by overlaying a 2-cm grid on the maps. The total number of peptides (35) from both enzymes was consistent with the predicted number of arginines and lysines present in the amino-acid composition and the presence of four identical subunits. Surprising sim- ilarities were discovered from the fingerprints. Most of the peptides from each muscle type had nearly identical migrations. Small differences are present in region 2-3, F-G (Fig. 11), in which Ascaris aldolase has a few additional peptides. Other relatively small differences in location can be accounted for 42 p"i HI i-"I Hi r-4 rH I t I I I t a> a a a •H •rl IH J* >> m U X> P> •p > •p •H d \ O •P JC CM Fig. 11. Tryptic peptide fingerprints of rabbit (i) an<* Ascaris (II) muscle aldolases. S-carboxymethylation, tryptic digestion, electrophoresis, and chromatography were performed as described in 'Methods'. The origin is desig- nated by (*). Each drawing is a composite from five chromatograms (cellulose thin-layer plate). 44 M o #v 00 N J°0 0 : O T H tr» r V» - CJ | 4k c%. ; K> j £ o o _ I i i i i i i i i i n —« A C G D H B E F I O "O X i i i • i i r i 1 1 O m pa 9 m 8 is* CA 7 6 0 # 5 »«;• o o in . 1 e> 0 a xfS 0 (2) CHROMATOGRAPHY 45 by minor intra-peptide changes in composition. This would result in slight alterations in homologous peptides. Amino-Acid Analysis The amino acid composition of Ascaris muscle aldolase was calculated on a basis of a molecular weight of 160,000. In comparing Ascaris aldolase to the amino acid compositions of rabbit, the mammalian muscle prototype (Ting et al(, 1971a), and the pig (Anderson et al^, 1969), significant differences were found (Table VX ). Table VII lists the major differences of 10% or greater. Also as shown in Table VI , the number of hydrophilic amino acids (lys, arg, his, asp, glu) varies by just 6%. Further, the hydrophobic amino acids (val, ile, leu, phe) differ by a mere . The greatest change seems to be a very conservative substitution of glycine. Ascaris has 29 and 21 more residues than pig and rabbit, respectively. It is evident that Ascaris muscle aldolase differs only slightly overall from both rabbit and pig muscle aldolase. 46 TABLE VI AMINO-ACID ANALYSIS RESIDUES PER MOLE Ascaris Piga Rabbit'3 Muscle Muscle Muscle Lysine 112 110 104 Histidine 46 39 44 Arginine 52 56 56 Aspartic Acid 112 116 116 Threonine 82 81 88 Serine 94 79 84 Glutamic Acid 180 162 164 Proline 85 74 80 Glycine 145 116 124 Alanine 162 156 .. 172 HaIf-cystine 30 28 32 Valine 75 84 80 Methionine 18 15 12 Isoleucine 73 77 76 Leucine 118 138 140 Tyrosine 47 45 48 Phenyalanine 39 30 28 Tryptophan 20 12 12 Total 1490 1418 1460 Corrected to 160,000 MW. aValues from Anderson et al,(1969). b Values from Ting et al. (1971a), # 47 TABLE VII COMPARISON OF AMINO-ACID COMPOSITIONS Pig Rabbit Amino Acid Residues % Residues % Different Difference Different Difference Histidine +7 15 +2 4 Serine +15 16 + 10 11 Glutamic acid +18 10 +16 9 Glycine +29 20 +21 .14 Proline +11 13 +5 6 Valine -11 11 -5 1 Methionine +3 17 +6 33 Leucine -20 14 -22 16 Phenylalanine +9 23 +11 28 -^Tryptophan +8 40 +8 40 *Not included in deviation function calculation. DISCUSSION Fructose diphosphate aldolase was purified 157-fold from the muscle of the roundworm, Ascaris suum. The pur- ified enzyme exhibited a specific activity of 10.2 units per mg of protein. The preparation was shown to be homoge- neous by the criteria of analytical ultracentrifugation, zone electrophoresis, and rechromatography. Ascaris aldolase is composed of 4 identical subunits of 40,000 daltons each. Aldolase was partially purified from the nematodes Ascaris suum (Mishra, 1970) and Ascaridia galli (Strivastava et al.. 1971). Mishra (1970) reported that Ascaris aldolase exhibited a FDP/F1P ratio of 82. In addition, ATP, ADP, and AMP exhibited positive allosteric effects of the enzyme, which is in contrast with other studies of muscle aldolase (Spolter et al., 1965; Lacko et al., 1970; Kawabe et al.. I969). The allosteric effects reported by Mishra may be due to contamination of his preparation with other enzymes such as phosphofructokinase. Srivastava et al. (1971) purified Ascaridia galli aldolase 44-fold and recorded a Km value for FDP of 4»5 x 10~3 M,100-fold higher than any other muscle aldolase. Due to the inconsistency of these workers' results, their heterogeneous preparations, and their methods of assaying the enzyme, their resulting data must be considered tenuous at this time. 48 49 The present study involved a comparison between the primi- tive invertebrate, Ascaris suum. and mammalian (rabbit) muscle aldolase. The molecular weights, sedimentation coefficients, Stokes' radii, tryptic fingerprints, and amino-acid compositions are surprisingly similar. Catalytic studies also supported these similarities. Further, other studies from this labora- tory (Dedman et al., 1972) suggested that Ascaris aldolase, like rabbit muscle aldolase, is inhibited by glyceraldehyde- 3-phosphate and also responds to reversible, dark inactivation by pyridoxal phosphate. The enzyme is also desensitized by pyridoxal phosphate in the presence of light. Likewise, Ascaris aldolase is inactivated in the presence of sodium borohydride and FDP. Finally, Dedman et al. (1972) demon- strated identical migration of Ascaris and rabbit muscle al- dolases during cellulose-acetate eletrophoresis. There are several obvious functional components that are maintained through the phylogeny of muscle aldolase. The act- ive site peptide retains essentially all of its primary struc- ture (vida infra). Every muscle aldolase has an amino-terminal proline and alanyltyrosine-terminating carboxyl end (Caban and Hass, 1971)^ and the tyrosine is essential in the cleavage of FDP (surprisingly, the removal of a single C-terminal tyrosine decreases the Kffl by 100-fold (Gracy et al., 1970). Furthermore, the amazing similarity of the tryptic fingerprints Ascaris and rabbit muscle aldolase stresses the conservatism in the structure of the enzyme. Many of the alterations in 50 proteins (especially enzymes) do not occur randomly. Folding, interaction between the subunits and most important, pre- servation of the active center must be maintained. Radical changes (hydrophobic for hydrophilic) or even conservative replacements might alter the catalytic properties of the enzyme. In observing the amino-acid compositions of animal al- dolases, there is a high degree of homology. Major variations from average y^lues exist in lobster, having 136 • lysines (25 higher), 132 alanines (25 fewer), and in Ascaris. with 180 glu- tamic acids (20 higher) and glycines (25 higher). The half- cystine residues have been maintained at approximately 30 residues per mole, with the exception of codfish (16) and lobster (12). The most variant amino-acid is methionine, ranging from 4 (frog) through 32 (codfish) residues per mole. There is no suggestion^of a trend in the number of residues of a single amino-acid and the complexity of the animal, as hypothesized for methionine (Gracy et a_l., 1970). Several investigators have attempted to relate homologies in the structure of aldolases through a variety of methods (Rutter, 1964; 1965; Rutter e_t al., 1963; Rutter et al., 1963; Penhoet et al., 1967; Rutter et al., 1968; Anderson et al., 1969; Idehara et al., 1969; Koida et al., 1969). Molecular weight, effect of chelators, specificity of substrates (FDP/ F1P ratio), electrophoretic mobilities, terminal amino acids, and tryptic and cyanogen bromide peptide patterns, all have very little quantitative significance from a phylogenetic 51 standpoint. Other homologies between different aldolases have been attempted through the use of amino-acid sequences of the active site peptide (Guha et al., 1971; Lai and Oshima, 1971), but the results have yielded limited evolutionary in- formation (vida infra). Until the complete primary sequence of amino-acid in proteins, including aldolase, are completed, other means of quantitative comparisons must be sought. Slobin (1970) com- pared the Metzger difference index (Metzger et al., 1968) to the number of sequence changes in various cytochromes c. He reported that a value of less than 7 or less than 9 resulted in sequence likenesses of 90$ and 80%, respectively. Metzger's data (I968) indicates that unrelated proteins seldom yield a value of less than 15. This study has utilized three statistical procedures, the correlation coefficient (Steele and Torrie, 1962), the deviation function (Harris et al., 1969), and the difference index (Metzger et al. . 1968), to compare amino-acid compositions of numerous aldolases (Appendix A). TableVHC displays the amino-acid composition of Ascaris aldolase with aldolases from 16 other sources, using the three comparative procedures. The correlation between the independent comparisons is very high. The deviation function (DF) and difference index (Dl) have a correlation coefficient (CC) of O.98, while the correlation value for DF-CC and DI-CC comparisons are greater than 0.95. DE and DI can be interconverted from the relationship DI=1.65 (DF). 52 TABLE VIII COMPARISONS OF DEVIATION FUNCTION, DIFFERENCE INDEX, AND CORRELATION COEFFICIENT OF ALDOLASE AMINO-ACID COMPOSITIONS Ascaris Compared With: DF DI CC H Ox 3.02 4.50 .976 Human 4.33 7.35 .942 Pig 2.97 4.42 .973 Rat 2.85 4.43 .965 Rabbit A 2.82 4.45 .976 Rabbit B 4.18 6.92 .951 Rabbit C 4.90 8.30 .933 Chicken 3 i 28 5.07 . 966 Snake 5.18 8.73 .923 Frog 3.63 6.12 .960 Codfish 3.37 6 .12 .966 Sturgeon 4.00 7.03 .950 Shark 4.67 7.28 .944 Lobster 4.92 8.82 .926 Spinach 4.46 7.38 .953 Yeast 5.25 8.97 .911 53 Extending this study, 136 pairs of aldolases from 17 sources were compared, employing the deviation function (Table IX). The average DF values between mammals were used as a standard value (1.32). The DF values of the other classes of animals were then compared to this standard value. By using this method it is readily seen that as the phylogenetic scale is descended from mammals the DF values gradually in- crease. This is evidence of increasing dissimilarities in the amino-acid compositions. These DF differences were then plotted against the geologic age in which the classes evolved (except for mammals, where the modern orders of evolution were used) (Fig. 12). There is a direct correlation between the increasing DF and increasing evolutionary time. The animal classes fall in exceptionally close order to the classical phylogenetic sequence. To confirm this relationship, the sequence changes in cytochromes c were also plotted against time. The evolutionary pathway of this respiratory protein has been quantitatively established (Dickerson et al., 1971; Margoliash et al., I965). Thus, cytochrome c is an excellent, reference to compare the findings of aldolase phylogeny. Correlation between the DF of aldolase and sequence changes in cytochrome c was exceptionally high, a value greater than O.98 (Table X). There is little correlation (O.63) between the interclass comparisons, ie., birds-fish, fish-reptiles, etc. Homologous aldolase isozymes from muscle tissue of various species have more similarity than heterologous isozymes (muscle, . 54, CO 525 CT* o xo H ro H c > rH H a ) CO c > ro O 1 PH r- C"1 rH & ir J CN V ^iqqea 1 C-" O 1 ro If ) c\ 1 0 p- r- r- H q-tqqey Q (T1 0 00 0 CN 00 X! W r- CN a r- CO CN 00 O a>teus H CO cn < r~ 00 ro •St\ m tP CN 3.8 4 ro M 00 00 CN 00 {JH rH CD CD VJC 00 cn 00 MD 00 rH O <£> o> v£» a\ ** uoafian^s CN 00 00 ro 1 00 CN 3.4 0 CN| If} Fig. 12, Plot of DF of aldolases and changes in cyto- chromes c against the time of evolution of each Class (except for mammals). 56 • • SEQUENCE CHANGES IN CYTO C 10 15 20 25 600 500 ARTHROPODS ac 400 PISCES •© < LU >~ Q O oLi— AMPHIBIANS co 300 ASCARIS REPTILES 200 Q© AVES MAMMALS 100 PRESENT 1 DEVIATION FUNCTION ©—© OF ALDOLASE 57 TABLE X CORRELATION AND COMPARISON OF DF OF ALDOLASE WITH SEQUENCE CHANGES IN CYTOCHROMES C Comparison DF Cyto. c Changes Mammals - Mammals 1-32 4.75 Chicken 2.12 9.9 Snake 3.99 20.3 Frog 2 .66 12.1 Codfish 2.961i 17.7 (tuna) CC=>0.98 Sturgeon 3.18 ' 3.40 Shark 4.07, Ascaris 2.88 Lobster 4.29 24.6 (insect) 58. liver, brain) from the same animal (rabbit) as previously reported (Marquart, 1971). Furthermore, muscle and liver isozymes are more closely related than muscle and brain or liver and brain. These results may be compared with reports from the laboratory of Horecker (Guha et al., 1971), who has compared the active site amino-acid sequences of rabbit, frog, codfish, sturgeon, and lobster. The number of sequence alterations per 28 amino-acid-peptide is as follows: Comparison Sequence Changes Rabbit-frog 4 -codfish 5 -sturgeon 4 -lobster 5 -liver 5 Frog-codfish 4 -sturgeon 2 -lobster 4 Codfish-sturgeon 4 -lobster 6 Sturgeon-lobster 6 Using this method, it is difficult to reveal any descent through the classes studied. Applying the studies of Ingram (1961) and Zuckerkandl and Pauling (1962) to the relationship of myglobin-hemoglobin series, these substitutions give the appearance that lobster muscle aldolase is more closely re- lated to rabbit than to either of the lower vertebrates, codfish or sturgeon. 59 The active sites do show that muscle aldolases are homo- logous and exhibit extreme conservativism. For instance, the sequence around the Schiff-base forming lysine (15), residues 9-22, is identical for all aldolases determined. Likewise, cysteine is always located at residue 25 and methionine at position 18, even in the extreme case when just one methionine is present in the total peptide chain (Ting et al., 1971)• Thus, the active site is not likely to be indicative of the sequence changes that are capable of occurring in less crit- ical regions of the proteins. Molecular paleontology can be a very valuable tool. Data obtained from hemoglobins (Ingram, 196l) and cytochromes c (Dickerson et al. 1971) have greatly contributed in substan- tiation of previously established geological records, and have also provided data during periods in which fossil records are absent or incomplete. This is readily seen in the present study. From the DF of aldolases it appears that Ascaris evolved approximately 300 million years ago (Fig. 12), with the advent of terrestrial vertebrates. This is in contrast with the presently accepted time of 600 million years for free-living roundworms. It is conceivable that Ascaris evolved later than the free-living roundworms since this parasite has "adapted" to become totally dependent upon its host. Also, Ascaris has been protected from selection pres- sures by living in the well-regulated environment of the intestine. It would be interesting to see if the cytochrome 60 c sequence supports this theory, since this protein has re- cently been purified from Ascaris (Hill et al., 1971). Further, fossil records of Ascaris are absent since it is soft-bodied and is found within the body of its host. Cau- tion and discretion must be used to prevent overextending the information gained thus far. More animals and more proteins must be examined before a molecular phylogenetic tree can be confirmed. From this quantitative study of aldolase the question arose pertaining to the relationship of Class I and Class II aldolases. Because of the diverse catalytic and molec- ular differences, Rutter (1964; 1965) proposed that the two classes were analogous, evolving from unique genes. These 2 classes of aldolase then are considered as examples of convergent evolution. At the time of these proposals (1964)* Class I aldolases were reported to be trimers with subunit molecular weights of 50,000 (Deal et al., 1963; Kowalsky and Boyer, I960; Stellwagen and Schachman, 1962) and Class II aldolases dimers, subunit molecular weight, 35*000 (Rutter, 1964; 1965). New developments have since taken place. The subunit molecular weights are essentially identical, 40,000 (Harris et al., 1969; Kawahara and Tanford, I966). The deviation values (Table IX) for yeast and animals fall be- tween 5.14 and 6.91. This displays a relative degree of similarity, since the DF for unrelated proteins is seldom less 61 than 8.8 (Metzger et al., 1968)'. Harris et al. (1969) show DF values for rabbit A-yeast comparisons to be 6.25, rabbit A~cytochrome c, 13.6, and rabbit A-chymotrypsin, 12.1. By employing Rutter's original model of aldolase evolu- tion, the apparent 'homology' of Class I and II aldolases may be explained. Class II Metal Requirement Properties Ancestral Gene Conserved Mutations Retained Gene T Dupli- (Class I cation Useless Non-functional Gene > More Free Purposeful Mutations Gene 'Creative Event' Most organisms possess a great deal of gene redundancy. When duplicity is present, metabolically meaningful variants tend to develop (Rutter, 1964). A duplicate gene thus may undergo unconserved mutations, free from selective pressures. This "useless" gene may develop into a functional genome. Finally this "created" genome may then express itself and aid in the physiological needs of the organism. Thus, from a common ancestral gene, becoming progressively more divergent, •5K Value obtained by converting DI to DF. 62 Class I aldolases may have mutated significantly to develop a more purposeful catalytic mechanism. The length of the genome (MW of the subunits) and amino-acid composition (DF) have been conserved. This study, as well as previous ones, foims the basis for a comparative study of aldolase. There is a vast number of invertebrates yet unstudied that would provide pertinent and valuable information. A great deal of insight might be gained from examining all of the pre-adult stages of Ascaris. Aldolases from several animals in the same class would con- firm a clear picture of aldolase phylogeny. Such studies would yield a confirmed molecular phylogenetic tree in a relatively short period of time (in comparison to sequencing). It is cleaij then, that this study of aldolase may just be a springboard for a wide variety of further comparisons. SUMMARY 1. Fructose-1,6-diphosphate aldolase has been purified 157-fold from "the intestinal roundworm, Ascaris suum. with ' a final specific activity of 10.2 units per min per mg. 2. The enzyme was adjudged to be homogeneous by the criteria of analytical ultracentrifugation, zone electro- phoresis, and rechromatography. 3. Concomitant with the homogeneity studies, the follow- ing physical parameters were established: S^Q w = 7.98 x 2 10 ^ sec; D2Q = 4.43 x 10 ? cm per sec; Stokes' radius o =46.4 A; f/fQ = 1.29. 4. Sedimentation velocity, sedimentation equilibrium, and analytical gel filtration yield a molecular weight of 158,000 + 4000 for the native enzyme. The enzyme dissociates in 6 M guanidinium-Cl and SDS to four identical subunits of molecular weight 41,000. 5. The apparent Michaelis constants determined for FDP and F1P were J x 10"5 and 3 x 10~2, respectively. The catalytic ratio (FDP/F1P) was 42. The enzyme was unaffected by EDTA or potassium ions. 6. Comparison of the tryptic peptide maps indicate a high degree of homology between the mammalian (rabbit muscle) and Ascaris, aldolases. This indicates that the enzyme has retained its structural properties during its evolution. 63 64 7. The aldolase amino acid compositions from 17 sources have been compared by the deviation function, difference index, and correlation coefficient. A proposal of a phyloge- netic study of aldolases utilizing these quantitative methods of comparing proteins from their amino acid compositions is discussed. 65 APPENDIX A Description of Procedures Used for Comparisons of Amino-Acid Compositions Deviation Function (DF) = L ~ ^n^-}2 * Difference Index (Dl) = £|"^n ~ ^n| * ^0 Correlation Coefficient (CC) = £(XY) - (£x) (^Y)/n AI^X)2 - iix)2/n-(W2-<.(^/n All amino-acid compositions were normalized to 100,000 MW. X and Y represent mole fractions of corresponding amino- acids from the pair of compositions compared. Tryptophan was not included in calulations. REFERENCES Ackers, G. 1968. A new calibration procedure for gel fil- tration columns. J. Biol, Chem. 242; 3237-3228. Anderson, P. J., Gibbons, I., and Perham, R. N. 1969. A comparative study of the structure of muscle fructose-1, 6~diphosphate aldolases. European J. Biochem. 11: 503-509. Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91: 222-233. Andrews, P. 1970. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of Biochemical Analysis (edited by Glick, D.) 18: 1-53. Interscience Publishers, New York. Bailey, J. L. 1967. Techniques in Protein Chemistry. p. 341. Amsterdam, Netherlands. Baron, D. N., Maureen, B., and Foxwell, C. J. 1969. Multiple forms of aldolase in mammalian brain, gonads, fetal tissue, and muscle. Advances in Enz. Reg. 2.: 325-336. Blostein, R. and Rutter, W. J. 1963. Comparative studies of liver and muscle aldolase. II. Immunological and chromatographic differenciation. J. Biol. Chem. 238: 3280-3285. Bueding, E. 1969. Some biochemical effects of anthelmintic drugs. Biochem. Pharmacol. 18: 1541-1547. Burke, W. F., Gracy, R., and Harris, B. G. 1972. Studies on enzymes from parasitic helminths. III. Purification and properties of lactate dehydrogenase from Hymenolepis diminuta. Comp. Biochem. Physiol. (in pressT. Caban, C. E., and Hass, L. F. 1971. Studies on the structure and function of muscle aldolase molecular characterization of the enzyme from shark (Mustelus canis). J. Biol. Chem. 246: 6807-6813. Cleland, W. W. 1963. Computer programmes for processing enzyme kinetic data. Nature. 198: 463-465. 67 Davis, B. J. 1964- Disc electrophoresis - II. Methods and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. Dedman, J., Lycan, A., Gracy, R., and Harris, B. 1972. Studies on enzymes from parasitic helminths. IV. Puri- fication and properties of aldolase from Ascaris suum. (In preparation). Determann, H. and Michel, W. 1966. The correlation between molecular weight and elution behavior in the gel chro- matography of proteins. J. Chromatog. 2 5: 303-313- Dickerson, R. E. 1971. The structure of cytochrome c and the rates of molecular evolution. J. of Molecular Evolution. 1: 26-45« Dikow, A. L., Jeckel, D., and Pfleiderer, G. 1971. Isolierung und charakterisierung von aldolase A, B, and C aus mens- chlichen organen. Hoppe-Sevler1s Zeits. Phys. Chem. 352; 1151-1156. Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 6_: 1948-1954. Fluri, R., Ramasarma, T., and Horecker, B. L. I967. Puri- fication and properties of fructose diphosphate aldolase from spinach leaves. European J. Biochem. 1: 117-124. Fodge, D., Gracy, R., and Harris, B. 1972. Studies on enzymes from parasitic helminths. I. Purification and physical properties of malic enzyme from the muscle tissue of Ascaris suum. Biochim. Biophys. Acta. (in press). Gibbons, I., and Perham, R. N. 1970. Comparison of aldolases extracted from rabbit muscle at low and high ionic strenght. Biochem. J. 117: 415-146. Gracy, R. W., Lacko, A., and Horecker, B. 1969. Subunit structure and chemical properties of rabbit liver aldolase. J. Biol. Chem. 244; 3913-3919. Gracy, R. W. , Lacko, A., Brox, L., Adelman, R., and Horecker, B. 1970. Structural relations in aldolases purified from rat liver and muscle and Novikoff hepatoma. Arch. Bio- chem. Biophys. 136: 480-490. Grazi, E., Cheng, T., and Horecker, B. 1962. The formation of a stable aldolase-dihydroxyacetone phosphate complex. Biochem. Biophys . Res . Commun. J.' 250-253. Guha, A., Lai, C., and Horecker, B. 1971- Lobster muscle aldolase: Isolation, properties and primary structure at the substrate-binding site. Arch. Biochem. Biophvs. 147: 692-706. 68 Gunsalus, I., and Gibbs, M. 1952. The heterolactic fermen- tation. J. Biol. Chem. 194: 871-876. Harris, C., Kobes, R., Teller, D., and Rutter, W. I969. The molecular characteristics of yeast aldolase. Biochemistry, 8: 2442-2454. Herbert, D., Gordon, H., Subrahamyan, V., and Green, D. 1940• Zymohexase. Biochemistry J. 34: 1108-1123. Herskovits, J., Masters, C., Wassagrman, P., and Kaplan, N. 1967. On the tissue specificity and biological signi- ficance of aldolase C in the chicken. Biochem. Biophys. Res. Commun. 26; 24-29. Hill, G., Perkowski, C., and Mathewson, N. 1971. Purifi- cation and properties of cytochrome Cpr0 from Ascaris lumbricoides variety suum. Biochem. Biophys. Acta. 236: 242-246. Horecker, B. L. 1959. Aldol and ketol condensations. J. Cell. Comp. Physiol. 54: Suppl. 1, 90-107. Horecker, B. L. 1967- Glucose-6-phosphate dehydrogenase, the pentose phosphate cycle, and its place in carbo- hydrate metabolism. Am. J. Clin. Path. 47: 271-281. Horecker, B. L. 1970. Active site of aldolases. FEBS Symposium 19: 181-189• In Metabolic Regulation and Enzyme Action (ed. A. Solo and S. Grisolia) Acad. Press, N. Y. Ikehara, Y., Yanagi, S., and Endo, Hideya. 1969. Comparative studies on muscle aldolases purified from rat, rabbit, human, pigeon, and hen. J. of Biochem. 66: 493-501. Kawahara, Kazuo and Tanford, C. 1966. The number of polypep- tide chains in rabbit muscle aldolase. Biochemistry. i.: 1578-1584. Kochman, M., Krzywda, U., Kwiatkowska, D., and Baranowski, T. 1971. Multiple molecular forms of fructose-1,6- diphosphate aldolase in ontogeny and phylogeny. Int. J- Biochem. 2: 221-231. Kobes, R., Simpson, R., Vallee, B., and Rutter, W. 1969- A functional role of metal ions in a Class II aldolase. Biochemistry. 8,: 585-588. Koida, M., Lai, C., and Horecker, B. 1969• Subunit structure of rabbit muscle aldolase: extent of homology of the and subunits and age-dependent changes in their ratio. 69 Arch. Biochem. Biophys. 134: 623-631. Kwon, T., and Olcott, H. 1965. Tuna muscle aldolase: I, Purification and Properties. Comp. Biochem. Physiol. 1£: 7-16. Lacko, A., Brbx, L., Gracy, R., and Horecker, B. 1970. The carboxy-terminal structure of rabbit liver aldolase (aldolase B). J. Biol. Chem. 24 5: 2140-2141. Lai, C. and Chen, C. 1971. Codfish muscle aldolase: Puri- fication, properties, and primary structure around the substrate-binding site. Arch. Biochem. Biophys. 144: 467-475. 1968. Studies of the structure of rabbit muscle aldolase I. Cleaving with cyanogenbromide. Arch. Biochem. Biophys. 128: 202-207. and Oshima, T. 1971. Studies on the structure of rabbit muscle aldolase. Ill, Primary structure of the BrCN peptide containing the active site. Arch. Biochem. Biophys. 144: 363-374. Tchola, 0., Cheng, T., and Horecker, B. 1965a. The mechanism of action of aldolases. VIII. The number of combining sites in fructose diphosphate aldolase. J. Biol. Chem. 240: 1347-1350. ——, Hoffee, P., and Horecker, B. 1965b. Mechanism of action of aldolases. XII. Primary structure around the substrate binding site of rabbit muscle aldolase. Arch. Biochem. Biophys. 112: 567-579. Lebherz, H., and Rutter, W. 1969. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry. 8_: 109-121. Li, T., Gracy, R., and Harris, B. 1972. Studies on enzymes from parasitic helminths. II. Purification and properties of malic enzyme from Hymenolepis diminuta. (in press). Lowry, H., Rosenbrough, N., Farr, A., and Randall, R. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. ~" Margoliash, E., and Smith, E. I965. Structural and functional aspects of cytochrome c in relationship to evolution. In Evolving Genes and Proteins (eds. Bryson, V. and Vogel H.) pp 221-242. Academic Press, N. Y. ' 70 Marquardt, R. 1969a- Multiple molecular forms of avian aldolases. I. Crystallization and physical properties of chicken (Gallus domesticus) breast muscle aldolase. Can. J. Biochem. 47: 517-526. 1969b. Multiple molecular forms of avian aldolases. II. Enzyftiatic properties and amino acid composition of chicken (Gallus domesticus) breast muscle aldolase. Can. J. Biochem. 42» 527-534. 197la, Multiple molecular forms of avian al- dolases. V. Purification and molecular properties of chicken (Gallus domesticus) liver aldolase. Can. J. Biochem. 49: 647-657. . 1971 b. Multiple molecular forms.of avian al- dolases. VI. Enzymatic properties and amino acid com- position of chicken liver aldolase and comparative immunological properties. Can. J. Biochem. 49: 658- 667. Masters, C. 1967. Characteristics of aldolase variformity. Biochem. Biophys. Res. Commun. 28: 978-984. Masters, J. 1968. The ontogeny of mammalian fructose-1,6- diphosphate aldolase. Biochem. Biophvs. Acta. 167: 161-171. Metzger, H. Assessment of compositional relatedness between proteins. Nature. 219: 1166-1168. Meighen, E., and Schachman, H. 1970. Hybridization of native and chemically modified enzymes. I. Development of a general method and its application to the study of the subunit structure of aldolase. Biochemistrv. Q: 11 AT- 1176. "*• Meyerhof, 0. and Lohmann, K. 1934. Uber die enzymatische gleichgewichtsreaktion zwischen hexosediphosphorasaure und dihydroxyacetonphosphorsaure. Biochem. Z. 217: 89-110. - —L •• 7'.T • !936 . Uber die aldolase, ein kohlenstoff-ver- knupfendes ferment. Biochem. Z. .286: 319-335. Mildvan, A., Kobes, R., and Rutter, W. 1971. Magnetic re- sonance studies of the role of the divalent cation in the mechanism of yeast aldolase. Biochemistry. 10: 1191-1204. Mishra, N. K. 1970. Aldolases of Ascaris suum: An immunologic and biochemical study. Ph. D, dissertation. Univ. of Neb. 71 Moore, S. and Stein, W. 1963. In Methods in Enzymology (S. P. Colowick and N. 0. Kaplan, eds.X"Vol. VI, pp. 819-831- Academic Press, N. Y, Morse, D. and Horecker, B. 1968a. In Advances in Enzy- mology (ed. F. F. Nord) j£l: pp. 126-181. John Wiley and Sons. New York. Morse, D. and Horecker, B. 1968b. Rabbit liver aldolase: Determination of the primary structure at the active site. Arch. Biochem. Biophys. 125.: 942-963. Penhoet, E., Kochman, M., Valentine, R., and Rutter, ¥. 1967» The subunit structure of mammalian fructose diphosphate aldolase. Biochemistry.> 6: 2940-2949. — ? Rajkumar, T., and Rutter, W. I966. Multiple forms of fructose diphosphate aldolase in mammalian tissues. Proc. Nat. Acad. Sci. 56: 1275-1282. Kochman, M., and Rutter, W. 1969s* Isolation of fructose diphosphate aldolases A, B, and C. Biochemistry 8: 4391-4395. ~~ JL' f Kochman, M., and Rutter, ¥. 1969b. Molecular and catalytic properties of aldolase C. Biochemistry 8: 4396-4402. . _ ^ and Rutter, W. 1971. Catalytic and immunochemical properties of homomeric and heteromeric combinations of aldolase subunits. J. Biol. Chem. 246: 318-323. Peterson, E. and Sober, H. 1962. In Methods in Enzymoloev (eds. S. P. Colowick and N. 0. Kaplan) Vol. V pp. 3-27 Academic Press, N. Y. Pharmacia Fine Chemicals. 1970. Sephadex gel filtration in theory and practice. Pharmacia Fine Chemicals Inc. Appelbergs Boktryckeri, Sweden. Racker, E. 1947. Spectrophotometry measurement of hexo- 843^854and phosphokinase activity. J. Biol. Chem. 167: Rapaport G Da^is, L. and Horecker, B. 1969. The subunit structure of fructose diphosphate aldolase from spinach • Arch » Bxochem* Biophvs . 132 : 286-293, Rozacky E., Sawyer, T., Barton, R., and Gracy, R. 1971. Studies on human triosephosphate isomerase I. Isolation the enzyme from erythrocytes. Arch. 72 Rutter, W. , Rajkumar, T., Penhoet, E., and Kochman, M. 1968. Aldolase variants: structure and physiological sig- nificance. Ann. N. Y. Acad. Sci. 151: 102-117. Rutter, W. 1964* Evolution of aldolase. Fedn. Proc. Fedn. Am. Socs. Exp. Biol. 2£: 1248-1257. Rutter, W., Blostein, R., Woodfin, B., and Weber, C. 1963* Enzyme variants and metabolic diversification. Advances Enz• Regulation. 1: 39-53* Rutter, W. J. 1965. Enzymatic Homology and analogy in phylogeny. In Evolving Genes and Proteins (eds. Bryson, V. and Vogel, H.) pp 279-291. Academic Press, N. Y. Sai, C. and Horecker, B. 1968. The molecular weight of rabbit muscle aldolase and the properties of the sub- units in acid solution. Arch. Biochem. Biophys. 123 i 186-194- Saz, H., and Bueding, E. 1966. Realtionships between ant- helmintic effects and biochemical and physiological mechanisms. Pharmac. Rev. 18: 871-894. and Lescure, 0. 1968. Effects of anticestodal agents on mitochondria from the nematode, Ascaris lumbricoides. Molecular Pharmacol. 407-410. —, and Lescure, 0. 1969. The function of PEPCK and malic enzyme in the anerobic formation of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30: 49-60. 1971a. Anaerobic phosphorylation in Ascaris mito- chondria and the effects of anthelmintics. Comp. Biochem. Physiol. 39: 627-637. 1971b, Falcultative anaerobisois in the inverte- brates: pathways and control systems. Am. Zoologist. 11: 125-135. Schwartz, E. and Horecker, B. I966. Purification and pro- perties of fructose diphosphate aldolase from Boa constrictor constrictor. Arch. Biochem. Biophvs. 115: 407-412. Scoffone, E. and Fontana, A. 1970. In Protein Sequence Determination {ed. S. B. Needleman) pp. 202-203. Springer-Verlag. Berlin, Heidelberg. Sheedy, R. and Masters, C. 1971. On the ontogeny of amphib- ian aldolase. Int. J. Biochem. 2: 173-176. 73 Siegel, L, and Monty, K. 1965. Determination of molecular weights and functional ratios of macromolecules in impure systems: aggregation of urease. Biochem. Biophys. Res• Commun. 19; 494-498. Spackman, D., Moore, S., and Stein, W. 1958. Automatic recording apparatus for using in the chromatography of amino acids. Anal. Chem. 30: 1190-1206. Spotter, P., Adelman, R., and Weinhouse, J. 1965. Distinc- tive properties of native and carboxypepidase-treated aldolases of rabbit muscle and liver. J. Biol. Chem. 240: 1327-1337. Srivastava, V., Ghatak, S., and Krishanamurti, C. 1971. Partial purification of Ascaridia galli aldolase. Labdev. £: 27-30. Steel, R. and Torrie, J. i960. In Principles and Procedures of Statistics. p. I83. McGraw-Hill Book Company Inc. N. Y. Ting, S., Sai, C., Lax, C., and Horecker, B. 1971a. Frog muscle aldolase: purification of the enzyme and struc- ture of the active site. Arch. Biochem. Biophys. 144: 485-490. Ting, S., Lai, C., and Horecker, B. 1971b. Primary struc- ture of the active sites of beef and rabbit liver aldolases. Arch. Biochem, Biophys. 144: 476-484. Tarui, S., Kono, N., and Uyeda, K. 1972. Purification and properties of rabbit erythrocyte phosphofructokinase. J- Biol. Chem. 247: 1138-1145. van den Bossche, H. and Jansen, P. 1969. The biochemical mechanism of action of the antinematodal drug tetramisole, Biochem. Pharmacol. 18: 35-42. Van Holde, K. 1967. Sedimentation equilibrium in Fractions. No. 1, Beckman Instruments, Inc. Spinco Division, Palo Alto, California. Warburg, 0., and Christain^ W. 1943. Isolierung und Kristallisation des garungsferments zymohexase. Biochem. Z. 2M- 149-164. Weber, K. and Osborn, M. 1969. The realibility of molecular weight determination with dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412. 74 Woodfin, R. 1967- Substrate induced dissociation of rabbit muscle aldolase into active subunits. Biochem. Biophys. Res. Commun. 29: 288-293- Yphantis, D. 1964. Equilbrium ultracentrifugation of dilute solutions. Biochemistry. 3.: 297-317. Zuckerkandl, E. and Pauling, L. 1962. In Horizons in Bio- chemistry (M. Kasha and B. Pullman, eds.) p. 189» Academic Press, New York.