AM. ZOOLOGIST, 11:503-511 (1971).
Lactate Dehydrogenase Isozymes, Gytochrome Oxidase Activity,
and Muscle Ions of the Rattail (Coryphaenoides sp.)
GREGORY S. WHITT
Department of Zoology, University of Illinois, Urbana, Illinois 61801
AND C. LADD PROSSER
Department of Physiology and Biophysics, University of Illinois,
Urbana, Illinois 61801
SYNOPSIS. Investigations into the behavior of molecules in organisms occupying unique
environments may provide a better insight into the functions of these same molecules
in organisms from more common habitats. An organism well suited for such analyses is
the rattail (Coryphaenoides sp.), a deep-sea teleost. The photoreceptor cells of the
retina are predominantly rods. Although the lactate dehydrogenase A, and B,
isozymes are present in this fish, the E4 isozyme (found in the retina of many teleosts)
is absent in the rattail retina. The rattails possess a lower cytochrome oxidase activity
than shallow water fish. The sodium concentration is higher, and the potassium
concentration lower in the rattail as compared with surface marine fish. The patterns
of molecular synthesis and concentrations in the rattail may be related to such factors
as light intensity, hydrostatic pressure, and temperature which exists in the deep-sea
environment.
INTRODUCTION
Examination of the visual apparatus of
deep-sea fish reveals that the cellular structure of the retina and the properties of its
visual pigments are specifically suited to a
low light habitat (Walls, 1942; Prosser and
Brown, 1961). The retinas of deep-water
fishes of several families consist entirely of
rods, long and closely packed (Verrier,
1931, Contino, 1939). The visual pigment
present in the deep-sea fish retina is rhodopsin (Wald, et al., 1957; Munz, 1958)
which is so densely packed within the rod
that it absorbs over 90% of the blue-green
light which strikes it (Denton, 1959). This
rhodopsin possesses a golden color (Denton and Warren, 1956).
Many fish possess a retinal specific lactate dehydrogenase isozyme (LDH E4)
which is characterized by its highly anodal
migration upon electrophoresis (Nakano
and Whiteley, 1965; Markert and Faulhaber, 1965; Goldberg, 1966; Morrison and
This research was supported by NSF grants
GB 16425 and GB 4005. Thanks to Mr. T. E. Wheat
for photographing the gels.
503
Wright, 1966; Massaro and Markert,
1968). The E4 isozyme is encoded in a
third LDH locus (E) distinct from the A
and B loci functioning in most tissues
(Whitt, 1969, 1970a; Whitt et al., 1971).
The kinetic, physical, and immunochemical characteristics of the E4 isozyme are
more like those of the B4 isozyme (predominant in aerobic tissues) than those of
the A4 isozyme (predominant in anaerobic
tissues) (Whitt, 1968, 1969, 1970a,&).
Thus, the LDH E locus appears to have
arisen by duplication of the B locus
(Whitt, 1969, 1970a). The observation
that this retinal specific E4 isozyme is absent from the primitive non-teleostean fish
examined (e.g., Agnathans, Chondrichthyes, Dipnoans, Chrondrostei, and Holostei) (Whitt and Horowitz, 1970; Horowitz
and Whitt, unpublished) as well as from a
few families in the primitive orders of
teleosts (e.g., Cypriniformes and Clupeiformes) (Whitt and Maeda, 1970) suggests
that the LDH E locus originated prior to
the adaptive radiation of the teleosts.
The E4 isozyme is present in highest
amounts in the cells of the neural retina,
504
GREGORY S. W H I T T AND C. LADD PROSSER
especially the inner segment of the photoreceptor cells (Whitt and Booth, 1970).
The cellular specificity of this isozyme in
addition to its kinetic properties suggests
that its function is important to the visual
metabolism of some fishes (Whitt and
Booth, 1970; Whitt, 1970a).
The occurrence of the E4 isozyme thus
far has not been correlated with any environmental parameter; therefore, it is important to examine the eyes of fish from as
many different habitats as possible in order to determine the role of the E4
isozyme. The LDH isozyme repertory of
the retina from a deep-sea fish has not yet
been reported. The purpose of the present
investigation is to determine whether the
retina of a typical deep-sea teleost (the
rattail) possesses the retinal specific lactate
dehydrogenase isozyme.
Cytochrome oxidase is an enzyme which
has been shown to change in its activity
with temperature acclimation (see Hazel
and Prosser, 1970 for references). It is a
useful measure of oxidative metabolism,
and as a heme enzyme, it should be useful
for studies of the effects of pressure and of
adaptation to cold.
In a number of marine fishes living in
the subarctic at depths where there is little
annual variation in temperature, the
plasma osmoconcentration and muscle sodium are higher than in surface fish and in
freshwater fish (Prosser et al., 1970).
Since the rattails are living at a temperature which probably deviates very little
from 2°C, it is of interest to measure ion
concentrations in their tissues. Comparison
was with mackerel and mugil taken from
16°C water.
MATERIALS AND METHODS
Source of fish
The rattails (Coryph.aenoid.es sp.) were
caught in the Galapagos Archipelago -.vith
a free vehicle long-line from the ship R/V
Alpha Helix (Phleger and Sou tar, 1971).
The specimens were caught at a depth of
about 7000 feet and were brought to the
surface dead, but in good physical condi
tion. The eyes of fish to be examined histologically were fixed in formalin. The tissues to be homogenized prior to electrophoresis for LDH were stored at —20°C.
Cytochrome oxidase activity determination
Activity of cytochrome oxidase was measured in 10 percent homogenates of fresh
tissue in 0.05 mM phosphate buffer at pH
7.4. The assay method was that of Smith
(1955). Cytochrome-c was reduced by bubbling with hydrogen and was stored frozen
under nitrogen. Homogenates were centrifuged at 100 X g for 15 minutes and the
supernatant was assayed at 23°C for oxidation of the cytochrome-c by means of a
Beckman DU spectrophotometer at 550
nra. Readings were taken at 0, 60, and 120
seconds and after complete oxidation by
K3Fe(CN)0. The velocity constant (K)
was calculated as follows:
K —
log(O.D. ti — O.D..J — log(O.D. tj — O.D. M )X2.3
t 2 — ti
Initial rate (I-R.) was calculated from the
relation:
K X concentration of cytochrome— c
T.R. =
Comparisons were made of muscle from
rattails with muscles from two shallowwater fish, i.e., mackerel and mugil.
Ion analysis
For ion analysis, muscles were removed
from fish soon after they were brought to
the ship. They were weighed, dried, and
then transported to the University of Illinois where sodium and potassium concentrations were measured by flame photometry.
Homogenate preparation for electrophoresis
Frozen tissue (skeletal muscle, rardinr
muscle, and retina) was hand-homogenized
in 2 volumes of 0.1 M Tris-HCI buffer, pll
7.0 at 4°C The homownate was rentri-
505
ENZYMES AND MUSCLE IONS OF THE RATTAIL
TABLE 1. Cytochrome oxidase activity in fish muscle.
Velocity constant
Species
Cytochrome-c concentration 7 X 10-3mM
Rattail
15.4 X 10-°
13.0 X 10"°
Mackerel
White muscle
4.0 X 10"6
4.99 X lO"6
Red muscle
Cytoehrome-c concentration 3.5 X 10"2mM
Rattail
7.3 X 10-6
12.6 X 10"5
2.4 X 10^*
Mugil
4.4 X 10"*
3.7 X 10-*
6.5 X 10"*
fuged twice for 30 minutes at 48,200 X g
in an RC-2B Sorvall centrifuge at 4°C to
obtain a clear supernatant fraction which
was employed for antisera addition and
electrophoresis.
Electrophoresis
procedures
Electrophoresis was performed in a vertical gel apparatus (Buchler Instruments,
Inc.) in a 12% electrostarch gel at 4°C for
16-18 hours with a voltage gradient of 8
V/cm. The EBT and Tris-dtrate buffer
systems employed, as well as the procedures
for the staining of the gel, have been previously described (Whitt, 1970a).
Antibody specificity determinations
The preparation of the anti-A and antiB serum is described by Markert and
Holmes (1969). These antisera formed
against the sea trout, Cynoscion regalis,
LDH homopolymers cross-react well with
homologous LDH subunits from different
species of teleosts (Whitt, 1969; Holmes
and Markert, 1969).
Antisera against the A4 and B4 isozymes
were separately mixed with an appropriate volume of enzyme extract. This antibody-antigen mixture was incubated at
room temperature for 15 minutes. After
incubation, the antigen plus antibody mixture was centrifuged as described for the
homogenate preparation in order to precipitate the LDH complexed to the anti-
Initial rate
DIM cyt-c oxidized/sec/gmw
5.7 X 10-*
3.3 X 10-*
1.48 X 10-3
1.85 X 10"3
1.62
2.8
5.2
2.45
4.12
3.6
X 10-* '
X 10-3
X 10"*
X 10-2 :
X lO"22
X 10-
AV; 3.2 X 10-"
Avg:
Avg:
Av, 3.3 X 10"a
bodies. The resultant supernatant was subjected to starch gel electrophoresis. Those
antigen-antibody complexes which are not
sedimented by the centrifugation are removed during electrophoresis by the molecular sieving action of the gel. The absence of specific isozyme bands after electrophoresis is a dramatic demonstration of
immunochemical homology (Whitt, 1969;
Holmes and Markert, 1969).
RESULTS
Histology of the rattail retina
Sections of retina were stained with
methyl blue. Unfortunately, preservation
was poor, but receptor cells were identified
as rods in agreement with previous observations in other deep-water fish (Verrier,
1931; Contino, 1939).
Cytochrome oxidase activity
skeletal muscle
in fish
Table 1 presents the cytochrome oxidase
activity in the muscle samples from different species of fish. The data were obtained
for two concentrations of reduced cytochrome-c.
Ion concentrations in fish skeletal muscle
The data for sodium and potassium in
fish muscle are given in Table 2.
506
GREGORY S. W H I T T AND C. LADD PROSSER
TABLE 2. Sodium and potassium concentrations in fish muscle.
Potassium
Sodium
Species
% dry wt.
mM/kgdrj.
mM/kg wot
Mugils
20.0
19.7
19.6
17.2
22.7
19.8 ± 1.7
13.1
12.4
196
104
101
177
24.3
20.9
28.6
30.6
22.2
25.2 ± 2.5
40.5
31.3
43.2
58.5
35.5
31.3
70.0
40.4
38.5
43.2 ± 7.7
Avg.
Rattails
9.6
13.2
12.4
9.1
Avg.
17.7
14.4
13.4
12.8 ± 2.1
97.5
143.6 ± 9.1
308
248
455
184
288
347
389
280
344
316 ± 48.2
Tissue specificity of lactaie dehydrogenase synthesis in the rattail
JIM/ •kgdry
890
522
557
806
363
636.6 ±41.3
589
488
512
245
433
535
491
497
615
479.5 ± 68.0
mMAgw.t
110.0
103.5
98.0
138.0
86.2
107.1 ± 11.6
78.2
60.7
48.6
78.4
49.2
48.5
88.3
71.5
69.0
65.8 ± 10.3
anti-A serum precipitated the A4 isozymes
of the skeletal muscle (Fig. 2). The anti-B
serum precipitated the anodal B4 isoThe LDH isozyme patterns of skeletal zymes (Fig. 2). As also indicated by the
tismuscle, cardiac muscle, and retina are dis- sue specificity of the isozyme patterns, the
played in Figure 1. All the tissues possessed B isozyme migrates more anodally than
4
a broad diffuse band of LDH B4 isozyme the
A4 isozyme. The multiplicity of the
activity. This broad region was actually isozymes
in the B4 region is observed in
composed of many closely migrating iso- the isozyme pattern
of the untreated
zymes. The A4 isozyme, which remained at skeletal muscle.
the origin during electrophoresis was only
The polypeptide composition of the
detected in the skeletal muscle.
LDH isozymes from the cardiac muscle
The retina did not possess an unique and retina were also determined by the
LDH E4 isozyme with its typically highly application of antibodies followed by elecanodal migration. Both the heart muscle trophoresis. The isozyme pattern of the
and the retina possessed at least two catho- heart and retina were identical (Fig. 3)
dally migrating isozymes which were desig- and their response to the antibody precipinated X and Y because their subunit com- tation was also identical; therefore, only
position was unknown. The isozyme pat- one of these zymograms (the heart muscle)
terns of the heart and retina were almost was chosen (Fig. 3).
identical. The use of the concentrated
The anti-A serum, at the concentrations
Tris-citrate, pH 6.8 buffer for electrophoresis instead of the pH 8.6 EBT buffer indicated, did not precipitate any of the
did not result in a qualitative alteration in heart (and retina) isozymes. However,
when higher concentrations (2 anti-A : 1
the isozyme patterns.
enzyme extract) (not shown) were apEffect of antisera upon the rattail lactate plied, the B4 isozyme was unaffected, but
the X and Y isozymes were precipitated.
dehydrogenase isozymes
The anti-B serum, at the concentrations
The anti-A and anti-B sera were added indicated, precipitated all the isozymes of
separately to the skeletal muscle en/yme the heart (and retina). The B4 isozymes
extract which was then subjected to elec- and the more cathodal X and Y isozymes
trophoresis in order to determine the poly- were all precipitated. The slight smear of
peptide composition of the LDHs. The LDH activity above the origin after the
ENZYMES AND MUSCLE IONS OF THE RATTAIL
507
The high numbers of rods observed in
the rattail retina is similar to the observation made on retinas from different species
of deep-sea fish (Verrier, 1931; Contino,
1939; Walls, 1942).
tails. The circulation had been stopped for
an indeterminate time (one-half to several hours) before the rattail muscles were
obtained. This might alter the intracellular and extracellular distribution of ions
but it should not alter the total ion content of the muscles. In view of similar
elevated muscle sodium concentrations in
subarctic fishes from lesser depths but constant low temperatures, it is suggested that
the uniformly low temperature may be
critical for ion concentrations in the rattails.
Cytochrome oxidase activity of fish muscle
Lactate dehydrogenase isozymes of the
rattail tissues
The cytochrome oxidase values varied
considerably. Part of this was related to the
different concentrations of the two samples of cytochrome-c which were used.
However, the range for the mackerel and
mugil is similar to values of cytochrome
oxidase in goldfish muscle (Freed, 1965).
The average initial activity for surface fish
was 10 times greater than that for the
rattails. The principal conclusion which
can be drawn from these limited results is
that the activity of the enzyme in muscle of
the rattails is much lower than in muscles
of active shallow-water fish. Possibly the
rattails are relatively sluggish fish and have
oxidative enzymes in correlation with
this. An alternative suggestion is that the
cytochrome oxidase system is reduced in
some way because of the hydrostatic pressure and temperature at which the rattails
live.
The broad band of B4 LDH activity
appears to be composed of multiple bands
with very similar electrophoretic mobilities. These multiple B4 bands might be
caused by the presence of two different
codominant alleles at the B locus (Whitt,
1969), duplicated B loci (Holmes and
Markert, 1969), or sub-banding generated
by epigenetic factors (Markert and Holmes,
1969).
The immunochemical data indicate that
none of the tissues possessed isozymes of
intermediate
electrophoretic
mobility
which were precipitated by both antisera.
Therefore, there appear to be no detectable heteropolymeric isozymes.
The retinal specific E4 isozyme present
in many teleosts is not detected in the retina of the rattail. The absence of this
isozyme has been established by several
criteria. There is no highly anodal LDH
isozyme which is restricted to the retina.
The rattail retina does not possess any
LDH isozymes which are not present in
the heart tissue. In a few species of teleosts
there is E subunit synthesis in the heart.
However, in these fish, only the E ^
heteropolymer is observed for heart tissues,
whereas only the retina possesses the more
anodal E4 isozyme (Whitt et al., 1971).
The cathodally migrating X and Y LDH
isozymes of the heart and retina are composed of LDH polypeptides immunochemically related to the B subunits. However,
unlike the B and E polypeptides, the
application of the anti-B serum is due to
the molecular sieving properties of the gel
which retarded the mobility of the partially active antigen-antibody complex.
DISCUSSION
Histology of the rattail retina
Sodium and potassium concentrations of
fish muscle
The measurements of muscle sodium
and potassium concentrations support previous conclusions concerning differences
between deep (cold) water and surface
(fluctuating temperature) marine fish
(Prosser et al., 1970). The sodium concentration is significantly (72 percent) higher
in the rattails than in the mugils. Potassium concentration is 38 per cent lower and
water content slightly higher in the rat-
508
GREGORY S. W H I T T AND C. LADD PROSSER
ORIGIN
\
\
\
\
FIG. 1. Lactate dehydrogenase isozymes of rattail
tissues. Electrophoresis was carried out in the pH
8.6 EBT buffer system. The LDH isozyme pattern
o£ the heart appears identical to that of the retina.
subunits comprising the X and Y isozymes
are also slightly immunochemically related
to A subunits. Thus, the X and Y isozymes
do not appeal- to contain E polypeptides because of their cathodal migration,
their antigenic properties, and their syn-
ENZYMES AND MUSCLE IONS OF THE RATTAIL
509
-B4(RABBIT)
1
ORIGIN-
(-1
*
\
\
\
5.
^.
%
FIG. 2. Effect of anti-LDH A4 and anti-LDH B t
sera upon the rattail skeletal muscle lactate dehydrogenase isozymes. The isozymes present in the
control serum are the rabbit LDH isozymes, one o£
which (the B4) is present in the antisera
enzyme mixtures in the other slots. The ratios
indicate the amount of antiserum added to the
enzyme extract.
thesis in a non-neural tissue. The X and Y
isozymes might be tetramers composed of
subunits encoded in a duplicated LDH locus. The presence of additional LDH loci
has been reported for other species of fish
(Hochachka, 1966; Bailey and Wilson,
1968; Klose et al, 1968; Massaro and Markert, 1968; Ohno et al, 1968).
The failure to detect the retinal specific
E4 isozyme in the rattail does not necessarily indicate the absence of the LDH E
gene because this locus might be present
but functional only at very low levels. Another possibility which must be considered
is that the E4 isozyme is degraded as soon
as it is synthesized. This latter consider-
ation appears unlikely from the point of
view of cellular efficiency as well as the fact
that the E4 isozyme in other species is
more stable than the B4 and A4 isozymes
(Whitt, 1970a).
The absence of the E4 isozyme in the
rattail does not simply reflect the absence
of this unique isozyme from all the Gadiformes, because many of Gadidae possess
this retinal specific isozyme (Odense et al.,
1969; Horowitz and Whitt, unpublished).
The fact that the retina possesses a large
number of rods and lacks the E 4 isozyme
suggests that there is not an obligatory
relationship of the E4 isozyme with rods. It
is not known whether the absence of the
510
GREGORY S. W H I T T AND C. LADD PROSSER
-B4(RABBIT)
ORIGIN
%
°
%
\
\
k
1
\
V
\
V
%
\
k
T$
\
V
*
?L
\
^
\
*
FIG. 3. Effect of anti-LDH A, and anti-LDH B4 sera
upon the rattail heart muscle lactate dehydrogenase isozymes. These results are identical for the
LDH isozymes of the rattail retina. The isozymes
present in the control serum are the rabbit LDH
iso/ymes, one of which (the B,) is present in the
antisera enzyme mixtures in the other slots. The
ratios indicate the amount of antiserum added to
the enzyme extract.
E4 iso/yme in the rattail is due to the types
of photoreceptor cells in the retina, the
type of visual metabolism, the low light
intensity environment, or to some other
ecological parameter. It should be stressed
that only one species of deep-sea fish was
ENZYMES AND MUSCLE IONS OF THE RATTAIL
investigated; thus, future investigations of
other species will be required to determine
whether the absence of an E4 isozyme is
typical of deep-sea fish.
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