/ . Embryol. exp. Morph. Vol. 19, 1, pp. 1-8, February 1968
With 1 plate
Printed in Great Britain
The tyrosinases of Fundulus heteroclitus at
different stages of embryonic development
By LESLIE M. SPITZ 1 & JEAN B.BURNETT 1
From the Department of Dermatology, Harvard Medical School
The study described here was undertaken in an effort to determine whether
multiple forms of tyrosinase (EC 1.10.3.1) are present in Fundulus heteroclitus
during embryonic development. If they are present, do they appear simultaneously in the course of development and do they continue to be produced and
maintained without change during the part of the life cycle when tyrosinase
should normally be active? Multiple forms of tyrosinase have been found in
Neurospora crassa (Sussman, 1961; Fox & Burnett, 1962; Fling, Horowitz &
Heinemann, 1963), mushroom (Smith & Kruger, 1962; Bouchilloux, McMahill
& Mason, 1963; Jolley & Mason, 1965), potato (Patil & Zucker, 1965), Drosophila melanogaster (Lewis & Lewis, 1963; Mitchell & Weber, 1965), the eggs of
Rana pipiens (Turney, 1964), goldfish (Kim & Tchen, 1962; Kim, Tchen &
Chavin, 1962), hamster melanoma (Pomerantz, 1963,1966) and mouse melanoma
(Shimao, 1962; Burnett, Seiler & Brown, 1967). By using a variety of physical
and chemical techniques to isolate the tyrosinases present in various organisms
and organs, the investigators cited above have been able to establish numerous
specific characteristics of the enzymes. Their published data suggest that in both
plants and animals there may be variation in the chemical and physical characteristics of individual, enzymically active tyrosinase molecules. Experiments
have not yet revealed, however, the metabolic or functional significance of the
various forms of an enzyme within a single system. Since the presence of multiple
forms of tyrosinase within a single system seems to be universal, it was anticipated from the outset that multiple forms of tyrosinase would be found in the
embryos of Fundulus heteroclitus.
The formation of melanin is catalysed in both plants and animals by the
enzyme tyrosinase. In the presence of this enzyme, tyrosine is oxidized to
3,4-dihydroxyphenylalanine (DOPA) and DOPA, in turn, is oxidized to DOPAquinone. The conversion of DOPA-quinone to melanin proceeds in several
steps (Lerner & Fitzpatrick, 1950).
The presence of multiple forms of tyrosinase within a single organism or organ
could be the result of chance mutations. Such mutations might cause subtle
1
Authors' address: The Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114, U.S.A.
I
JEEM 19
2
L. M. SPITZ & J. B. BURNETT
changes that would not significantly affect the metabolic function of the enzyme.
If, however, two or more forms of tyrosinase were characteristically to become
active at different stages of development and thereafter remain independent,
one might infer that there are functional differences between the various forms
of the enzyme.
MATERIALS AND METHODS
Fundulus heteroclitus was chosen for study because manifestations of tyrosinase
activity, namely the appearance and accumulation of the pigment melanin, are
easily discernible during its early embryonic development and continue to be
Table 1. Characterization of Fundulus heteroclitus at various
stages of embryonic development at room temperature (22 °C)
Stage of
development*
Age of
embryo (h)
22
56
23
25
27
29f
31f
33f
66
84
112
144
168
216
34
39
228
384
Major identifying characteristics
Main divisions of the brain are differentiated
and optic lobes are prominent
Integumentary melanophores are first visible
Circulation begins
Dendrites of melanophores are clearly visible
Eyes begin to be pigmented
Heart chambers are differentiated
Ectoderm of the yolk sac is detached
anteriorly at the lower level of the forebrain
Fish has hatched
Yolk sac is completely absorbed
* See Armstrong & Child (1965).
f Stages at which eyes were removed from duplicate samples for comparison.
evident throughout larval life. The specimens used in this study were obtained
from the Marine Biological Laboratory, Woods Hole, Massachusetts, during
the spawning season (June through August). Ova and milt were expressed from
females and males and mixed in sea-water. When milt had been in contact with
the eggs for 30 min, the water was changed and the eggs were allowed to stand
in milt-free sea-water at room temperature (22 °C) for 1-2 h. At the end of this
period, the eggs were rolled on paper towelling to remove their outer coat,
which hampers observation of development. The developing zygotes were
maintained in sea-water at room temperature. At the various stages of development (Armstrong & Child, 1965) listed in Table 1, samples of approximately
300 were removed for analysis. During the later stages of embryonic development (stages 29-31), pigment is recognizable in two major locales: in the retinal
pigment epithelium and in melanophores scattered throughout the body tissues.
At stages 29, 31 and 33 two samples of 300 zygotes were removed so that the
tyrosinase activity from intact embryos could be compared with that from
embryos from which the eyes had been removed. Apart from this, no attempt
Tyrosinases of Fundulus
3
Table 2. Isolation and fractionation of tyrosinase from Fundulus heteroclitus
Unless otherwise specified, all procedures were carried out at 5 °C and all dialysis
was performed against 1 1. of 0-05 M tris-glycine buffer, pH 8-2.
300 EMBRYOS
(In 2 ml of 0 0 5 M tris-glycine buffer, pH 8-2,containing 0-25M sucrose)
I
I
Dialyse for 12 h
I
Homoeenize for 10 min
Centrifuge for 20 min at 35000 g
SUPERNATANT (S,)
PELLET (PJ
I
Add 1/10 vol. saturated (NH4)2SO, and
1 vol. of ice-cold acetone equal to the
vol. of the supernatant plus the (NH,)2SO4
I
I
Let stand for 1 h a t - 20"C
Centrifuge for 20 min at 35000g
I
SUPERNATANT (S.,)
I
Dialyse for 2 h
I
Evaporate under No to 0-7 ml
I
Add 0-2 g sucrose
PELLET (P.,)
I
Suspend in 1 ml of
tris-glycine buffer
I
Dialyse for 2 h
Suspend P l in P2 dialysate
I
I
Fraction I
Soluble tyrosinase
(T\T*-)
Homogenize for 5 min
I
Add 5 ml ice-cold acetone
Let stand for 30 min at - 2 0 X
I
Centrifuge for 20 min at 35000£
PELLET (P3)
SUPERNATANT (S,)
I
I
Evaporate under N« to 0-7 ml
I
Suspend in 1 ml tris-glycine
buffer containing 6 M urea
I
Homogenize for 5 min
I
Add 0-2 g sucrose
I
Fraction II
Centrifuge for 10 min at 35000 g
SUPERNATANT (S.,)
I
Evaporate under NL, to 0-7 ml
I
Add 0-2 g sucrose
I
Fraction III
Solubilized tvrosinase
(T\T2J3)
PELLET (P,)
4
L. M. SPITZ & J. B. BURNETT
was made to determine the localization of tyrosinase activity in specific parts
of the body. Each sample was frozen immediately in a minimum volume of seawater and stored at — 20 °C until analysed.
Solutions containing active tyrosinase were prepared from the various
300-egg samples by a modification of the procedure of Brown & Ward (1957)
(Table 2). It was found impracticable to study fresh homogenates of the various
samples because extraneous material interfered with proper band development
within the gel. When prepared by the technique outlined in Table 2 fraction I
contains soluble enzyme from the initial homogenate; fraction II contains little
or no enzyme; and fraction III may contain soluble enzyme that was trapped in
the particulate material during sedimentation, or enzyme that had been bound to
particulate material and released (solubilized) by the action of urea, or enzyme
from both these sources. Duplicate aliquots of each fraction were subjected to
acrylamide-gel electrophoresis (Davis, 1964; Ornstein, 1964) at room temperature for 30-60 min at 5 mA/tube. Bromphenol blue, added to the buffer in the
cathode reservoir, served as a tracking dye. After electrophoresis the gels were
neutralized in 0-1 M phosphate buffer, pH 6-8, for 30 min and then immersed in
0-15% L - D O P A in 0-1 M phosphate buffer, pH 6-8. Since the manifestation of
tyrosinase activity in the tubes does not appear for more than 24 h when tyrosinase is the substrate, L - D O P A was routinely used as the substrate in these
experiments. The brown bands of pigment (melanin) that form where tyrosinase
reacts with L-DOPA serve to indicate the location of the enzyme(s) in the tubes.
The Rx value for the enzyme, which is defined as the ratio between the distance
traveled by the enzyme into the gel and the distance traveled by the tracking dye,
constitutes a means of characterizing the enzyme, while the relative density of
the pigment bands produced constitutes a somewhat quantitative measure of
the amount of enzyme present.
RESULTS
The presence of tyrosinase activity was first seen as two very faint brown bands
in fraction I (the soluble fraction) at stage 23. The tyrosinases responsible for
the formation of these bands have Rx values of 0-75 (the more anodic) and
0-64; they are here designated T 1 and T 2 respectively. With minor variations,
this pattern of band formation continued through stage 27; thereafter the bands
became much less distinct.
From stage 29 through stage 33, three bands of tyrosinase activity were
consistently found in fraction III. The Rx values of the tyrosinase in two of these
bands were identical with those of the T 1 and T 2 from fraction I; that of the
least anodic band was 0-31 (Plate 1). Preparations from eyeless embryos were
compared with preparations from whole embryos at stages 29, 31 and 33. The
enzymic pattern of eyeless embryos did not differ consistently from that of
whole embryos.
The amount of tyrosinase activity (T1 and T2) in fraction I decreased steadily
PLATE 1
J. Embryol. exp. Morph., Vol. 19, Part 1
Dye front
A
B
Patterns of the activity of soluble (A) and solubilized (B) tyrosinase derived from Fundulus
heteroclitus at stage 29 of embryonic development as shown by acrylamide-gel electrophoresis.
As oriented here, the enzyme migrated from the bottom (cathode) toward the top (anode)
of the gel.
A. Fraction I. Rapidly migrating, bromphenol blue tracking dye forms the upper band. The
position of this band (front) in the tube provides a basis for calculation of the Rx values of
the enzymes. The bands of soluble T1 and T2 are clearly visible below the front.
B. Fraction III. The tracking dye (front) is in the same position as in Fraction I. The bands of
solubilized T1 and T2 are in the same position as those of soluble T] and T2. A third band, that
of solubilized T3, is present below them.
L. M. SPITZ & J. B. BURNETT
facing p. 4
Tyrosinases of Fundulus
5
from stage 29 through stage 39 (the last stage studied), as shown by the rate of
development of color and the density of pigment bands within the gel. Concurrently, the activity of tyrosinase (T1, T2, T3) in fraction III seemed to remain
constant as the age of the embryo increased. Throughout the period of embryonic
development under investigation, no change could be observed in the relative
activity of the two forms of tyrosinase in fraction I or of the three forms in
fraction III.
DISCUSSION
The number of fertilized eggs available for these experiments was sufficient
for only one series of enzyme extractions at each stage of development. Results
are consistent, however, although the 300-egg samples provided only enough
enzyme to produce faint bands of pigment.
Work by other investigators (Kim & Tchen, 1962; Kim et ah 1962; Lewis &
Lewis, 1963; Seiji, Shimao, Birbeck & Fitzpatrick, 1963; Chian & Wilgram,
1967) has shown that activators and inhibitors play a role in the functioning of
tyrosinase in other organisms, and it is possible that the synthesis and activation
of tyrosinase during the embryonic development of Fundulus heteroclitus may
depend on equally complex mechanisms. Whatever the mechanism by which
active tyrosinase may be formed within this fish, T 1 and T 2 become active at the
same time in extracts prepared from F. heteroclitus during the early stages of
pigment production. This suggests that in vivo there may exist simultaneously
two enzymes, corresponding to T 1 and T 2 in our preparation, that have identical
schedules of synthesis and activation.
In stage 29 and thereafter, three forms of active tyrosinase could be simultaneously extracted from fraction III. It is possible, but not probable, that the
T 1 and T 2 of this fraction had been trapped during pellet formation in the first
two centrifugations (see Table 2) and later released when the pellets were gently
resuspended. From stage 29 to stage 39 the density of the bands of T 1 and T 2 of
fraction III remained constant whereas that of the isologous bands of fraction I
gradually decreased. It would appear, therefore, that most of the T 1 and T 2 of
fraction III was released from particulate material when urea was added.
Since T3 was not detected in fraction I, which contained the easily extractable,
soluble cell components, it is unlikely that T3 is formed de now within the
melanophore. The presence of T3 in fraction III is probably due to the action of
urea on the bonding of this form of the enzyme to particulate material.
Although the interrelationships of these various tyrosinases have not yet been
determined in detail, the data reported here suggest that, as melanin is formed in
living systems, soluble enzymes may become associated with particulate material
(e.g., premelanosomes, melanosomes). This concept is well supported by the
work of Seiji et al. (1963). These investigators demonstrated that in the melanocytes of B-16 mouse melanoma tyrosinase is synthesized by 'small granules'
(presumably particles of ribonucleoprotein) and stored in distinctive cytoplasmic
6
L. M. SPITZ & J. B. BURNETT
organelles (melanosomes). The enzyme found only in solubilized form may be
derived from either of the two soluble enzymes or from a combination of the
two. This concept is supported by recent experimental evidence: Burnett et al.
(1967) have found that the solubilized tyrosinase of mouse melanoma is a
unique, complex enzyme which contains at least four distinct components. In
the study described here some of the particle-bound tyrosinase released in
soluble form by urea seems to be electrophoretically indistinguishable from
tyrosinases that were not particle-bound.The persistence of active forms of
tyrosinase in fraction III is consistent with the work of Kim et al. (1962), who
found that in the mature goldfish {Carassius auratus L.) the hyperpigmentationassociated stress correlated more closely with an increase in the tyrosinase
activity of a particulate fraction than with an increase in the activity of a
soluble fraction.
SUMMARY
1. The study described here was undertaken in an effort to determine:
(a) whether multiple forms of tyrosinase are present in the embryos of Fundulus
heteroclitus at different stages of development; (b) the stages of development at
which active tyrosinases first appear in the embryos, and (c) the persistence of
the tyrosinases found.
2. The material studied was prepared from developing embryos by differential
centrifugation and analysed by acrylamide-gel electrophoresis.
3. Two forms of active soluble tyrosinase (T1, T2) were first detected at
stage 23 when pigment cells first became visible in the embryo; they persisted
with apparently diminishing activity throughout stage 39, the last stage of
development studied.
4. Three forms of active tyrosinase (T1, T2, T3) could be released from particulate material. They were first detected at stage 29 soon after the dendrites of
the melanophores became clearly visible. These solubilized tyrosinases continued to be active and appeared to remain unchanged throughout the course of
these experiments.
5. Solubilized T 1 and T 2 appear to be electrophoretically identical with
soluble T 1 and T2. It is possible that T3 may be derived from T 1 or T 2 or from
the combination of T 1 and T2. It is unlikely that T 3 is synthesized de novo within
the melanophore.
ZUSAMMENFASSUNG
1. Der Grund fur die hier beschriebene Untersuchung war, festzustellen:
(a) ob die vielfachen Formen der Tyrosinasen in den verschiedenen Stadien des
Embryos vom Fundulus heteroclitus vorhanden sind; (b) zu welcher Zeit der
Entwicklung des Embryos die aktiven Tyrosinasen auftretten, und (c) die
Bestandigkeit des Auftrettens der gefundenen Tyrosinasen.
2. Das studierte Material war durch ultrazentrifugations Techniken von den
Tyrosinases of Fundulus
7
verschiedenen Stadien befindenden Embryos gewonnen und analysiert mit
Acrylamid Gel Elektrophorese.
3. Die zwei Formen der aktiven, loslichen Tyrosinase (T1, T2) wurden im 23.
Stadium zum ersten Mai gefunden, wenn die pigmentierte Zelle erstmals sichtbar
wurde im Embryo. Die Formen waren anwesend vom 23. bis zum 39. Stadium,
das letzte untersuchte. Die Aktivitat nahm langsam wahrend diesen Stadien ab.
4. Drei Formen von aktiver Tyrosinase (T1, T2, T3) wurden vom unloslichen
Niederschlag freigesetzt. Diese drei Formen wurden zuerst im 29. Stadium
gefunden, wenn die Dendrite des Melanophors sichtbar waren. Diese losbar
gemachten Tyrosinasen waren aktiv und unverandert vom 29. bis zum 39.
Stadium.
5. Die losbar gemachten T 1 and T 2 sind elektrophoretisch identisch mit den
loslichen T 1 und T2. Es ist moglich, dass das T 3 vom T 1 oder T 2 oder von der
Kombination T 1 und T 2 entsteht. Es ist unwahrscheinlich, dass das T 3 in
Melanophor de novo entsteht.
This investigation was supported by United States Public Health Service Research Grants:
Grant no. CA-08292 from the National Cancer Institute and Grant no. 5 T 5-GM-1651 from
the National Institute of General Medical Sciences.
The authors wish to thank Dr George Szabo, Assistant Professor of Anatomy in the
Department of Dermatology, Harvard Medical School, for his interest in this study and for
his hospitality in allowing them to use his laboratory at Woods Hole for procedures related
to the fertilization of the Fundulus heteroclitus eggs.
Mr Hans Seiler of the Department of Dermatology of the Harvard Medical School has
kindly translated the summary of this paper into German.
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{Manuscript received 31 May 1967, revised 11 September 1967)