ELECTORPHORETIC VARIATION IN ENZYMES OF SEBASTES PAUCISPINIS
AND SEBASTES GOODEI
Kami Andersoi
Biology 175
Advisors: D. Powe
and C. Baxter
ABSTRACT
Electrophoretic techniques were used to analyze the genetic variation of
Sebastes paucispinis and Sebastes goodei. Three types of variation were examined:
variation between the muscle and liver tissues of an individual, between individuals
of a species, and between species. Six enzyme systems were used:
phosphoglucomutase (PGM), glucosephosphate isomerase (GPI), esterase (EST),
isocitrate dehydrogenase (IDH), sorbital dehydrogenase (SDH), and malate
dehydrogenase (MDH). GPI, IDH, and MDH showed variation between tissues
within individual fish. Allelic variation was detected at GPI-B in S. paucispinis.
MDH proved to be the only distinguishing enzyme between the two species; all
other isozymes had identical electrophoretic mobilities in both species.
INTRODUCTION
An important aspect in understanding speciation is determining the degree of
genetic variability between and within a species. One method of examining genetic
variation is by comparing the electrophoretic mobility of enzymes using gel
electrophoresis. This method was used to examine the enzymes:
phosphoglucomutase (PGM), glucosephosphate isomerase (GPI), esterase (EST),
isocitrate dehydrogenase (IDH), sorbitol dehydrogenase (SDH), and malate
dehydrogenase (MDH) in Sebastes paucispinis and Sebastes goodei.
Gel electrophoresis is a technique used to separate molecules or proteins
based on charge and/or size. Charged molecules in solution migrate through a
porous medium (starch gel) in the presence of an electric field. Nonpigmented
molecules are made visible in the gel by an appropriate staining procedure (E.A.
Martin, 1983). Three types of electrophoretic variation of enzymes are possible: (1)
variation between tissues within an individual, (2) variation between individuals
within a species, and (3) variation between species.
The expression of different loci for the same enzyme in different tissues is an
example of variation within an individual. These variant forms of an enzyme,
produced by different loci, are known as isozymes and can be distinguished by.
electrophoresis. Isozymes vary in their electrophoretic mobility, not in the reactions
they catalyze within the cell. Alternatively, differential expression of loci in the
same tissue type among individuals within the same species is variation within a
species. For example, muscle tissue may contain four isozymes in the same species.
Finally, there is variation that exists between species, where one species shows a
different isozyme than another species. All three types of variation were detected in
this experiment.
The bands formed by the histochemical staining of the gel vary in thickness
and intensity. This corresponds to the level of enzyme activity within the cell, i.e.
the heavier the band the greater the concentration of the enzyme.
The two species of Sebastes studied in this experiment differ in their external
morphology. The S. paucispinis, commonly called the Bocaccio (B), is recognized
for its large mouth and projecting chin. The upper jaw usually extends beyond the
rear of the eye, and the profile is slightly concave between the snout and dorsal fin,
with a noticeable dip above eye. The S. goodei, commonly known as the chilipepper
(C), has a convex space between the eyes (curves outward) and the upper jaw ends
below midpoint of the eye. Also noteworthy in the Chilipepper is the white lateral
line in a clear red or pink zone, not present in the Bocaccio (Peterson, 1983). The
skin of both species of fish can be pinkish, reddish, or grayish in color and therefore
is not a useful distinguishing characteristic. Both species were found at depths of
300-500 feet in the Monterey area.
MATERIALAND METHODS
Chemicals
Hydrolyzed starch for gel electrophoresis and all reagents for the
histochemical staining of each system were obtained from Sigma Chemicals (St.
Louis, Missouri). All other chemicals were reagent grade.
Tissue Preparation
Muscle and liver samples were dissected from fish caught on local party
boats. A piece of each tissue was taken from S. paucispinis and S. goodei, and placed
in a labeled 17x10 mm polyethylene test tube; the tubes were then covered with ice.
The tissues were brought to the lab and frozen in liquid No or used fresh. No
discernable difference was noticed in the stained gels of the tissues stored in liquid
No, as consistent with the findings of Place and Powers (1977).
The fresh or frozen tissue (0.1-0.2 g) was minced with a razor blade and
placed into a labeled microfuge tube. Approximately 0.5 ml sonicating buffer
containing 20 mM Tris/citrate pH 7.5, .3 MNaSO4, lmM MnSO, and 10% (v/v)
glycerol was added before sonification. Cells were disrupted with five 5-sec bursts
from a Heat Systems Sonifier set at 30 W. All homogenates were kept on ice.
Lipid and solid particles were removed by centrifugation for 5-10 minutes in
a eppendorf centrifuge at approximately 12,000-15,000g.
Electrophoretic Procedure
PGM, GPI, and EST were resolved utilizing the lithium hydroxide buffer
system of Selander et al. (1969) and were stained according to procedures outlined
by Shaw and Prasad (1970).
SDH, IDH, and MDH were resolved using a tris-citrate buffer system, which
contained 78g starch, 60g sucrose, 2 drops B-mercaptoethanol, 2.5 ml Triton-X 100,
50 ml glycerol, 9 ml Tris-citrate buffer pH 6.9 (Place and Powers, 1978). The gels
were stained using the methods of Shaw and Prasad (1970).
High purity agarose (Difco Lab., Detroit, MI) was used for PGM, GPI, SDH,
IDH in the overlay stain since it provides the clearest background for photographic
documentation. Stained gels were fixed and stored in methanol-acetic acid-water
(S:1:5). This is also done for photographic reasons. The solution sharpens the color
difference between the bands and the white background.
Lithium hydroxide gels were run at constant voltages of 325 V for 5 hours at
4°C. Tris-citrate gels were run at the same voltage and temperature but for 20
hours. All electrophoreses were performed on a horizontal gel system using
Heathkit IP-17 constant-voltage power supplies. Controls for non-specific staining
were not done.
Nomenclature
Each locus will be dealt with individually to facilitate discussion. The
nomenclature used to designate each allozyme, a variant of the same locus,
corresponds to its relative electrophoretic mobility in the specified buffer system.
Loci are assigned capital letters: the slowest in mobility is given by A, followed by B
for the next slowest, and so on. Allelic variants at each locus are assigned lowercase
superscripts: the fastest is given by a, followed by b, c, etc. For example, isozyme
GPI-B has the alleles Baä, Ba°, and B°°. To describe the phenotype of a particular
isozyme all capital letters were used (e.g. IDH for isocitrate dehydrogenase or PGM
for phosphoglucomutase).
RESULTS
Phosphoglucomutase:
In this enzyme system no genetic variation was found within or between the
species examined. Electrophoretic phenotypes encoded by two loci are shown Fig. 1
and 2. Consistent with previous studies (e.g. Place and Powers, 1977), the major
isozyme, PGM-A, was present in both muscle and liver tissue. These PGM-A
electromorphs were identical in liver and muscle tissue of S. paucispinis and S.
goodei. The other enzyme phenotype, PGM-B, was detected in very low quantities
in liver and only after extensive incubation. It was surprising to detect minor
amounts of PGM-B activity in muscle tissue which had not been observed previously
in fish. This activity detected could have been a proteolytic by-product that still had
catalytic activity but a different net charge. PGM-B had the same electrophoretic
mobility between tissues and between species.
Glucosephosphate isomerase:
Two loci were expressed for glucosephosphate isomerase, GPI-A, and GPI-B
(Fig. 3 and 4). GPI-A had the same electrophoretic mobility in both species but was
specific for muscle tissue. Variation exists between the individual’s muscle and liver
tissues. Interestingly, this differential expression of loci occurs for both species.
The GPI-B locus was expressed in both the liver and muscle tissues of the
two species. Allelic variation was observed at this locus in S. paucispinis in both
tissues. Lanes 1 and 4 (Fig. 5) show the heterozygote. One out of the twenty fish
tested had this phenotype, and therefore possessed all the allozymes of the GPI-B
isozyme: Baa Bab BbP. Lanes 2 and 5 show the predominant genotype for GPI-B,
the homozygote GPI-Baa. Nineteen of twenty fish expressed this allozyme. Lanes 3
and 6 are predictions of the third phenotype, not seen in the tested sample size, but
which would exist in natural populations.
The three electrophoretic bands present in the heterozygote suggest that the
enzyme GPI is a dimer. The band in the middle is heavier in color because its
phenotype (B2b) can be formed two ways, Ba0 or B0a. Assuming the same Keq for
all the dimerization reactions.
Esterase:
Three enzyme phenotypes were observed that presumably code for three
loci: EST-A, EST-B, EST-C (Fig. 6 and 7). No genetic variation was found within
or between the species for this enzyme. The liver contained all three electromorphs
with identical electromorphic mobility. This is not likely to indicate heterozygousity
because every fish tested was shown to have these three bands, and the probability
of every individual of the sample size (n=20) being a heterozygote is very low.
Resolution of the esterase stain in muscle was difficult and no definitive results were
reported.
Isocitrate dehydrogenase:
Two electrophoretic phenotypes were observed in this enzyme system which are
presumably encoded by two loci, IDH-A, and IDH-B (Fig. 8 and 9) The expression
of the IDH-A locus, like GPI-A, was tissue dependent. IDH-A was present
exclusively in the liver tissue of both species, whereas IDH-B was specific for muscle
tissue. No interspecific differences were evident in either electromorph.
Sorbitol Dehydrogenase:
In this enzyme system only one isozyme was detected in both liver and
muscle, SDH-A, and both species had the same phenotype (Fig. 10 and 11).
Malate dehydrogenase:
This was the only enzyme of six tested that showed genetic variation between
species. Electrophoretic phenotypes encoded for by four loci were observed, MDH-
A, MDH-B, MDH-C, MDH-D (Fig. 12 and 13). Resolution in liver was difficult for
both species. The MDH-D isozyme was clearly visible in S. goodei in both liver and
muscle tissue (Fig. 12), but it was completely absent in S. paucispinis, and thus it was
a distinquishing isozyme between the two species.
Isozymes MDH-A, MDH-B, and MDH-C do not exhibit the variation nor the
clarity in resolution that MDH-D provides, thus speculation is required. Presumed
differences are detected within individuals of S. goodei. MDH-A is expressed in
muscle but not in liver. The same is true for MDH-C. More analyses could be
made from Fig. 12 but much speculation is needed, and therefore will not be
discussed further. Table 1 is a summary of the analyses made in this experiment.
DISCUSSION
The fact that five of six enzymes systems were shown to be identical between
S. paucispinis and S. goodei is very unusual (Dennis Powers, personal
communication). One would expect that two species of fish, whose external
morphology is very different, would not show such biochemical similarity. It must
be pointed out that this similarity refers only to the net charge of the enzymes, and
not to the entire amino acid sequence. Two proteins which show identical
electrophoretic movement may contain different amino acid sequences.
Alternatively, substitution of a single amino acid could alter the mobility of the
protein greatly. Yet the similarity that does exist between the two species, for these
enzymes, is striking.
PGM, EST, and SDH were the three enzyme systems that showed no
variation of any kind in the two species. The other enzymes showed minor
variation, with the exception of MDH. This could give us implications about their
speciation. Perhaps they evolved into separate species relatively recently (within the
last million years as versus 5 million years). Although their morphology has
diverged, their biochemistry may not have "caught up" with their external changes.
The enzymes tested may be highly selected for and therefore conserved over the
millions of years. On the other hand, the morphological differences that exist must
have some biochemical basis therefore there are some biochemical differences.
More enzymes would have to be examined to be more definitive.
The IDH isozymes varied within individual tissues. The IDH-A locus was
expressed only in the liver tissue (both species) and was absent in the muscle
sample. IDH-B was present exclusively in muscle tissues of the two species. This
variation tells us that a different locus is expressed in each of the two tissues. It is
important to realize that both species exhibited the identical variation between the
tissues. IDH-A had the same electrophoretic mobility in both species. IDH-B also
showed matching electrophoretic movement between the species.
GPI was the only system to show variation within a species. A heterozygote
was detected in S. paucispinis in both liver and muscle tissue. The other fish tested
were homozygotes of the fast allele (GPI-B44). The rare allele, b, was seen to have
a frequency of 2.5%. From this, the chance of the rare homozygote appearing can
be predicted. The frequency of allele GPI-B°° appearing is 0.0625%. Besides this
one case, no other variation was seen for glucosephosphate isomerase.
MDH showed the only variation between species of the six enzyme systems
tested (Fig. 12 and 13). S. goodei possessed the isozyme MDH-D and S. paucispinis
did not. The fact that this enzyme system was different between the two can be a
powerful diagnostic tool. Given a tissue sample of either of these two species one
could deduce the species the tissue sample came from just by staining for MDH.
This may be especially helpful when trying to determine the correct species of
juvenile fish. When fish are young their morphology has not developed to the point
of correctly distinguishing them from each other. Thus finding an electrophoretic
difference in MDH between S. paucispinis and S. goodei becomes a powerful
resource of information, provided that there is no change in the expression of loci
during development.
Malate dehydrogenase is different from the other five enzymes in that there
is a mitochondrial and cytoplasmic form. Further experiments would have to be
done to determine the difference in electromobility of the two distinct forms for
these two species.
After examining the electrophoretic variability of six enzymes in S.
paucispinis and S. goodei, and finding five of the six identical between species one
may conclude that there is great selection pressure for these enzymes. MDH,
contrarily, showed variation between species and may prove to be a useful
diagnostic tool.
ACKNOWI
EMENTS
I am greatful for the help, support, and, encouragement of: Dr. Dennis
Powers, Professor Charles Baxter, the employees of Randy’s Fishing Trips, Anja
Bachmann and Evon Asforis. Thank you all.
BIBLIOGRAPHY
Eschmeyer, W., and Herald, E. (1983). A Field Guide to Pacific Coast Fishes,
Houghton Mifflin Company, Boston.
Martin, E.A. (1983). Dictionary of Life Sciences, Market House Books Ltd, Hong
Kong.
Place, A. R., and Powers, D. A. (1978). Genetic bases for protein polymorphism in
Fundulus heteroclitus (L.).I. Lactate dehydrogenase (Ldh-B), malate
dehydrogenase (Mdh-A), glucosephosphate isomerase (Gpi-B), and
phosphoglucomutase (Pgm-A). Biochem. Genet. 16:577.
Selander, R. K., Hunt, W. G., and Yang, S. Y. (1969). Protein polymorphism and
genetic heterozygosity in two European subspecies of the house mouse.
Evolution 23:379.
Shaw, C. R., and Prasad, R. (1970). Starch gel electrophoresis of enzymes: A
compilation of recipes. Biochem. Genet. 4:297.
C
TAELE 1
SUMMORY OF ELECTROFHORETIC ANALYSIS
SFOUCISFINIS
B
3EBASTES GOODEI
VARIATION
LIVER MUSCLE
LIVER MUSCLE
GM-A
++++
++++++
+ +++
+ + + +
NE
GM-E ++
+-
0
+ + 4
GFI-4 —
+++ +

F- ++++

+ + +
+- ++ ++
+ ++
EST-+
+-++ +
MO



0
-


S7- +
N
MO
N
IDH-A++++
HO
++ ++++
IDH-E
++++ ++
+- + + +
NO
HDH—A —
++
+ +
NO
+ + +
++
MDH-E +++
++++
MO
HDH-C++++
+++
+++
YES (ES)
—
++++++
++ +
MDH-D
E
+— ++

++++

+ -++
0
MS: MITHIM EFECIE
BE: EETMEEN EFECIE
++ HELOTIVE EAHD INTE
VOFIATION
NO
NO

O
O
HO
NO
NO
NO
NO
NO
VES (ES)
YES (ES)
0
FIGURE LEGENDS
Fig. 1: Histochemical staining for PGM of S.goodei (C), and S. paucispinis (B),
muscle (M), and liver (L). The PGM-B band can be seen faintly above the liver
samples of both species. The muscle PGM-B electromorph is present but it cannot
be seen in this photograph. Actual gel size is 4X larger than photograph.
2: Composite drawing for all gels stained for PGM. No electrophoretic
variation can be detected for this locus.
Fig. 3: Starch gel stained for GPI. Genetic variation is seen at the GPI-A isozyme,
GPI-A activity is detected in muscle tissue (both species), and is absent in the liver
samples. This differential loci expression between tissues is genetic variation within
an individual.
Fig. 4: Summary drawing of gels stained for GPI. Genetic variation within each
individual (for both species) is detected at the isozyme GPI-A. Muscle tissue
expresses loci GPI-A and GPI-B. Liver tissue shows only the GPI-A phenotype.
Fig. 5: Drawing showing the polymorphism found at the GPI-B electromorph in
both liver and muscle. Columns 1 and 4 show the heterozygote phenotype, columns
2 and 5 the abundant homozygote (95% of the fish I tested), and columns 3 and 6
the unseen homozygote. The allozymes of GPI-B are GPI-Bda, GPI-Ba
and GPI¬
BDb
Fig. 6: Histochemical staining for EST. No electrophoretic variation can be
detected for this enzyme.
Fig. 7: Summary of EST stains. No electromorphs are shown in the muscle columns
because results could not be obtained for this tissue.
Fig. 8: Gel stained for IDH activity. Muscle and liver tissue each express different
loci. IDH-A activity is detected in the liver and IDH-B is expressed solely in the
muscle. Both species show this trend.
Fig. 9: Composite drawing of gels stained for the IDH-A and IDH-B phenotypes.
Fig.10: Sorbitol dehydrogenase activity detected in the muscle and liver of S.
paucispini and S. goodei. No detectable electrophoretic variation exists between
tissues, individuals, or species.
Fig.11: Drawing of SDH stain showing absence of genetic variation.
Fig.12: Gel, stained for MDH activity, shows genetic variation between species.
The MDH-D locus is only expressed in S. goodei, and is completely absent in S.
paucispini.
Fig.13: Drawing of MDH loci. Phenotype MDH-D shows genetic variation
between the species S. paucispini and S. goodei.
O
(
ORGN
. .
Cn  6n
6 C  6
PEM  2
-B
PGM-A
(0
ORGN
—
—


muscle
liver
Sebastes paucispini
— —


muscle
liver
Sebastes goodei
PGM-B
PGM-A
ORGN 8. 6. 0.
cer. Lste
C Cr.
GPB
GP-A
Fig
0
ORGN
—

—
muscle
liver
Sebastes paucispini
—

—.
muscle
liver
Sebastes goodei
GPI-B
GPI-A
Fig
ORGN
(
Sebastes paucispini
— —
— —
—
—
—
—
—
—
— —
—
— — —
3
6
2
muscle
iver
GPtgod.
gb
GPIB
GP-A
Fig l
ORGN
Sebastes
goodei
jver
Sebastes
paucispini
ZEST-C
—EST-B
EST-A
Fig
(
ORGN
O
—
muscle
liver
Sebastes paucispini
—

muscle
liver
Sebastes goodei
EST-C
EST-B
EST-A
E.
(0
ORGN
/DB
D+A
k   8. Cm. Cn.
oe
Fig
0
ORN
—

muscle
liver
Sebastes paucispini
—

muscle
liver
Sebastes goodei
IDH-B
JOH-A
10
O
ORGN
SDH-A
—

/Sebastes
Sebästes
paucispiri
goodei
liver
musck msck
Fc
0
ORGN
—
—
muscle
liver
Sebastes paucispini
—
—
muscle
liver
Sebastes goodei
SDH-A
Fig 7
ORGN
(-.
C Cn.
C 6. 8,
—
NOD
NDC
ADHB
MHA
Fig 3
0
ORGN
—


—
—
muscle
liver
Sebastes paucispini


—
—
—
muscle
liver
Sebastes goodei
MDH-D
MDH-C
MDH-B
MOH-A