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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 H C 0 H - 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., 1 9 6 9 a ) . 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., 1 9 6 9 ) .
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, 1 9 6 4 ) ,
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 algae, euglena and chlamydomonas (Autotrophic)
Schiff-base intermediate ((-amino lysine-DHAP)
Functional carboxyterminal tyrosine
Broad pH profile
MW 120,000-160,000
s 20 ,» 6 .5 -8 .0
4 subunits
FDP/FIP ratio 50,1,10
Specific Activity 8r25
Bacteria, yeast, fungi, blue-green algae, euglena &1 chlamy-domonas (Heterotrophic)
Divalent metal ion requirement K + activation
Functional SH groups
Sharp pH profile (7»0-7.5)
MW 70-80,000
S20,w 5-5-6'.0 S
2 subunits
FDP/F1P ratio 2000-2500
Specific Activity 80-100
From: Rutter, 1964* Rutter et al., 1968.
TABLE II
TISSUE DISTRIBUTION OF ALDOLASE ISOZYMES
Tissue Isozyme
Muscle
Heart
Spleen
Kidney cortex medulla
Brain
Liver
^-Intestine
*Lens
A
A
A
A-B A
A-C
A-B
A, B
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) (FDP Synthesis)
2,900 10,000
250 2600
1,000 4,900
*Km (FDP) 3 X 10~6
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..
1 9 6 9 b ) . 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., 1 9 6 7 ) .
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,
1 9 6 8 ) . In most tissues, mixed hybrid forms are originally
present, and not until the tissue develops and differentiates
do the parental forms become predominant (Masters, I 9 6 8 )
(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<?7). Recently,
Penhoet and Rutter ( 1971) examined the catalytic and immu-
nological properties of aldolase variants. An-Cn and
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, 1 9 7 0 ) . 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..
1 9 6 9 ) , spinach (Fluri et al.., 1967; Rapaport et al., I969),
and microorganisms (Kobes et al., 1969; Mildran et al., 1 9 7 1 ) .
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«
( t _ | i u 6 u i ) N i 3 x o a d
lp
? O s o o n o i o ^ P ) N » -
CO CN i- j o o d o
•—• ( . [ U I ^ U I U i p O A D a Q d a d S ® | O W 9 - 0 t
J UJ % 3 —J
o >
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 . 0 6 5
'Salt Elutant' 300 310 123 0 . 4 72 6 . 2
'FDP Elutant' 45 H . 2 114 1 0 . 2 67 1 5 7 . 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
>° 2.25 >"
1.75
—
\ a
-
S > S « B
mm \ | C
1 1 1 1 30 50 100 150
MOLECULAR WEIGHT
(x lO 3 )
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
O D280
io o
73 > n -H o z z c £ CO rn 79
O
a O
CO 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 4 0 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 w a s °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 m ay be calculated
from the relationship S 2Q j W =® 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 x 2) 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
( £ 0 l * )
J . H O I 3 M a v m D a i o w
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 velocity experiments at 60,000 rpm. The §20 w
v a l u e (7*985) was obtained by extrapolating to zero protein concentration.
38
* O CN 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
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41
comparisons of the V m a x 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
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43
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
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32
1
(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.
TABLE VI
AMINO-ACID ANALYSIS RESIDUES PER MOLE
46
Ascaris Muscle
Piga
Muscle Rabbit'3
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
Amino Acid Pig Rabbit
Amino Acid Residues Different
% Difference
Residues 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 K m 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).
TABLE VIII
COMPARISONS OF DEVIATION FUNCTION, DIFFERENCE INDEX, AND CORRELATION COEFFICIENT OF ALDOLASE AMINO-ACID COMPOSITIONS
52
Ascaris Compared With: DF DI CC
Ox
H
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,
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55
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
600
• • SEQUENCE CHANGES IN CYTO C
10 15 20 25
500
ac 400 < LU >~
Li— o co 300
200
100
ARTHROPODS
PISCES • ©
Q O AMPHIBIANS
ASCARIS REPTILES
PRESENT
Q© AVES
MAMMALS
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 2.961 i 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
Ancestral Gene
Gene
Dupli-cation
Metal Requirement Conserved Mutations
Properties Retained
T (Class I
Useless Non-functional Gene
> Free
Mutations
More Purposeful 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,
• 5 K 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
10 ^ sec; D 2 Q = 4.43 x 10 ? cm2per 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.
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