The synthesis and activity of tyrosinase during development of the frog Rana pipiens

DEVELOPMENTAL

BIOLOGY

40, 270-282 (1974)

The Synthesis

and Activity

Development

of Tyrosinase

during

of the Frog Rana pipiens

STEPHEN C. BENSON AND EDWARD L. TRIPLET Department

of Biological

Sciences, University

of California,

Santa Barbara,

California

93106

Accepted June 20, 1974 We have established by radioimmunoprecipitation that tyrosine-DOPA oxidase @DO, tyrosinase) [EC 1.14.18.11 is first synthesized by frog embryos at the early neurula stage soon after embryonic induction of the neural plate by the underlying chordamesoderm. The DOPA moiety of the enzyme, at the time of its first appearance, is almost inactive enzymatically and can be activated by mild proteolysis (with trypsin). A very large increase in the amount of active DOPA oxidizing enzyme (without trypsinization) is observed at hatching (stage 21), and this is accompanied by melanin deposition in pigment cells. The tyrosine moiety of the enzyme is also partially inactive at the time of first synthesis, but the ratio of active to inactive enzyme remains approximately constant throughout early development. DOPA decarboxylase enzymatic activity is first detected at neurula stage, and this activity is accompanied by the first appearance of catechol amines. INTRODUCTION

Some neural crest and neural plate derivatives use tyrosine in unique ways. Melanophores convert tyrosine to melanin with a single enzyme, tyrosine-DOPA oxidase (TDO), which oxidizes tyrosine to DOPA and DOPA to its quinone. Conversion of DOPA quinone to melanin is autocatalytic. Some neurons and adrenal medullary cells use TDO for the conversion of tyrosine to DOPA, and DOPA decarboxylase catalyzes the production of DOPAmine which may, in turn, be catalytically transformed into other catecholamines, such as norepinephrine and epinephrine. TDO thus operates at an important branch point in a pathway that may lead either to melanin or catecholamines depending upon cell type. We report here some observations on the turnover and catalytic efficiency of this enzyme. A review (Table 1) of the expression of the neural crest differentiated state in Rana pipiens as concerns tyrosine-DOPA metabolism reveals that catecholamine synthesis occurs at stage 15, neurula (Caston, 1962), and pigment synthesis at stage 21, late hatching (Smith-Gill et al., 1972).

However, it has been shown that determination of the neural crest occurs much earlier between stage 11 (mid-gastrula) and stage 12 (late gastrula) (Raven, 1935; Lehman and Youngs, 1952; Bagnara, 1973). There is, therefore, a temporal gap of about 80 hr between neural crest determination and the onset of overt pigment cell differentiation. The question then arises as to what types of events occur at the mid-gastrula stage that cause neural crest cells to embark upon a path of differentiation that is expressed days later. The question in this context can be restated as: What is the nature of the response of presumptive neural crest cells to inducing action as concerns tyrosine metabolism? With regard to tyrosine metabolism, neural crest cells could respond to the inducing action of the chordamesoderm in at least one of three ways. The first is that the tyrosinase gene(s) is selectively programmed at mid-late gastrula for transcription and translation at neurula (stage 15) and hatching (stage 21) when overt cell differentiation occurs. A second possibility is that the tyrosinase gene(s) is transcribed in response to the inducer’s influence at the conclusion of gastrulation and the messen270

Copyright All rights

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

BENSON AND TRIPLETT

Tyrosinase

TABLE RELATIONSHIP

BETWEEN EMBRYONIC DEVELOPMENT

Morphology

Hours after fertilization at 22”

9 10 11

Blastula Early gastrula Mid-gastrula

16 22 29

12 13 14

Late gastrula Neural plate Neural fold

36 46 52

15 16 17 18 19 20 21 22 23 25

Rotation Neural tube Tail bud Muscle response Heart beat Hatching Mouth open Tail fin circulation Opercular fold Operculum closed

271

Frog Deuelopment

1

AND NEURAL CREST DETERMINATION AND DIFFERENTIATION

REGARD TO TYROSINE

Shumway stage

during

Appearance of special products of tyrosine metabolism in Rana pipiens embryos

Reference

Spemann (1938)

Embryonic induction Neural crest determination

Lehman and Youngs (1952) Bagnara and Hadley (1973) Caston (1962) Caston (1962) Caston (1962)

62 67 77 89 108 120 140 162 182 240

ger RNA is translated at a later stage of development. In this case the mRNA would need to be stabilized, perhaps as an inactive polyribosome complex, an “informosome” (Spirin, 1966) or an “informofer complex” (Lukanidin et al., 1972). A third possibility is that both transcription and translation of the tyrosinase gene product occurs as the immediate response to the inductive events and that the enzyme is catalytically inactive until the stage at which cell differentiation occurs. Evidence will be presented in this paper and its companion (Benson and Triplett, 1974) that the latter hypothesis is correct. In this paper we demonstrate that presumptive neural crest cells respond to the induction by underlying chordamesoderm with the synthesis of TDO. The enzyme which appears at the early neurula stage is characterized by an enzymatically partially active tyrosine moiety and an almost completely inactive DOPA moiety. Dramatically increased DOPA oxidase activity is observed at the hatching stage.

WITH

METABOLISM

Stevens (1954) Smith-Gill et al. (1972)

Preliminary studies indicate that DOPA decarboxylase appears in concert with catecholamines during development. METHODS

Enzyme Extraction

AND

MATERIALS

and Purification

Adult frog skin TDO. TDO was purified from adult Rana pipiens skins by the method of Miller et al. (1970) as modified by Mikkelsen and Triplett (unpublished). All procedures were done at 4°C in a darkened room or under a red photographic safe light in order to avoid photoactivation of the enzyme (Mikkelsen and Triplett, unpublished). Briefly, the homogenate was centrifuged and crude enzyme was precipitated from the supernatant solution with ammonium sulfate at values between 40 and 70% saturation. The solubilized, dialyzed pellet was passed through a DEAE-cellulose column and active fractions, after concentration by ultrafiltration, were passed successively through CM-cellulose, columns and Bio-Gel P-300 columns. The preparations were homoge-

272

DEVELOPMENTALBIOLOGY

neous as judged by equilibrium sedimentation and electrophoresis on standard acrylamide gels (Miller et al., 1970), SDS-acrylamide gels, and urea-acrylamide gels at low and high pH. Preparation of crude embryonic TDO. For the study of the developmental changes in TDO and for some of the immunochemical studies, subsequent TDO was partially purified from Rana pipiens embryos. Five thousand embryos at different stages of development were dejellied as follows. Embryos were cut into clumps of 30-50 and placed in finger bowls containing 0.3% (w/v) sodium borohydride in distilled water. The reducing action of the sodium borohydride dissolved the jelly in 20-30 min, allowing the embryos to settle to the bottom of the finger bowl where they are removed with a transfer pipette to a bowl containing Millipore-filtered stream water. Embryos were washed in Millipore-filtered stream water and then in 5 volumes of 20 mM sodium phosphate buffer, pH 7.2. They were homogenized in 3 volumes of 20 mM sodium phosphate buffer, pH 7.2, centrifuged (35,000 g, 10 min), and the supernatant solution was concentrated to Ko the original volume by ultrafiltration or precipitated with ammonium sulfate. The protein precipitating between 40 and 70% ammonium sulfate saturation was resuspended in %othe original volume in 20 mM sodium phosphate pH 7.2. Spectrophotometric and polarographic assays for TDO activity were done with the 40-70s ammonium sulfate preparation, and radiometric assays were done with the ultrafiltered concentrated preparation. of embryonic and adult Extraction DOPA decarboxylase (DDC). Adult frog adrenal glands were removed and chilled to l”C, and embryos were dejellied and washed as described. Adrenals or embryos were homogenized at 0-4°C in 3 volumes of 10 mM sodium phosphate buffer, pH 7.0, containing 0.32 M sucrose and 1.0 mM

VOLUME 40.1974

@-mercaptoethanol. The homogenate was centrifuged (35,000 g, 10 min), the lipids were removed by aspiration, and the supernatant solution was concentrated by ultrafiltration to go the original volume. Any precipitate which formed was removed by centrifugation (35,000 g, 10 min). This soluble enzyme extract was labile upon freezing but could be kept for 24 hr at 4°C without loss in activity. Enzyme Assays Spectrophotometric assays for DOPA oxidase activity were performed according to the method of Pomerantz (1963) as modified by Miller et al. (1970). A polarographic assay was also developed to measure the oxidation of tyrosine and DOPA. The following reaction mixture was incubated at 37°C prior to the addition of substrate: 1.8 ml of 0.2 M sodium phosphate buffer pH 7.2, 0.2 ml enzyme preparation, 0.1 ml double-distilled water or trypsin at 1 mg/ml, 0.6 ml of substrate (20 mM DOPA or 3.6 mM tyrosine) at 37°C was added to initiate the reaction. The loss of molecular oxygen from the reaction was measured polarographically with a membrane type oxygen electrode augmented with a microrange extender. The slope of the amperage change as a function of time extrapolated to time zero was used as a measure of substrate oxidation. DOPA decarboxylase activity was measured by the method of Lloyd and Hornykiewicz (1970) with the exception that assays were conducted in air, as replacement of air by pure nitrogen did not influence the degree of decarboxylation. Protein determinations. All protein determinations were done according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Immunological

Procedures

Electrophoretically pure skin TDO was used for all immunological procedures unless otherwise indicated. Rabbits were in

BENSON AND TRIPLETI.

Tyrosinase

jetted subscapularly with 0.3 to 0.5 mg of TDO at daily intervals for 5 days, and after 1 week the injection series was repeated. Rabbits were bled as early as one week and as late as a month after the last injection. Immunoglobulins were partially purified by precipitation in ammonium sulfate at values between 0 and 33% saturation. Preparations were stored at 4°C after dialysis against 0.14 M sodium chloride, 10 mM sodium phosphate (pH 7.2), 1: 10,000 (w/v) Merthiolate. Control globulins were prepared similarly from the serum of unimmunized rabbits. Ouchterlony double-diffusion determinations were performed as outlined by Kabat and Mayer (1956). Tyrosine-DOPA oxidase could be detected in agar gels, polyacrylamide gels, and in anti-TDO precipitating lines by the DOPA oxidation reaction. Acrylamide gels or agar plates were covered with a solution of 5 mM L-DOPA and incubated at 37” for 6 hr. TDO was revealed by black lines at the site of melanin deposition. Alternatively, gels were stained with Coomassie brilliant blue to reveal protein. Purified adult skin TDO was used for immunotitration equivalence point determination (Kabat and Mayer, 1956). Nonspecific precipitation with control serum was reduced to negligible amounts by centrifuging the antibody and enzyme preparations at 34,000 g for 10 min before performing the incubation. The tubes were incubated at room temperature for 60 min and then 10 hr at 4°C. Precipitates were removed by centrifugation (17,000 g, 15 min), washed extensively in PBS, and assayed for protein; the supernatant solutions were assayed for DOPA oxidase activity. Isotopic Labeling and Immune of Radioactive TDO Precipitation One hundred embryos were dejellied and washed as described. The embryos were then rinsed four times in sterile 10% Stein-

during Frog Development

273

berg’s (1957) solution and incubated 3 hr in sterile one-third strength Steinberg’s solution containing 250 pg of streptomycin sulfate per milliliter (Nutritional Biothem) and 1000 IU/ml penicillin G (nutritional Bio-them). Neutralized tritiated algal protein hydrolyzate (0.9 mCi/ml, Schwarz-Mann) was injected into the embryos with a microsyringe apparatus calibrated to deliver 0.1 ~1 of solution. Embryos from stage 9 (blastula) through stage 14+ (early neural tube) were injected in the animal hemisphere. Subsequent developmental stages were injected in the midtrunk region. When the effect of protein synthesis inhibition was studied, puromytin (Sigma) was added to the algal protein hydrolyzate to a final concentration of 500 pg/ml. After injection the embryos were transferred to sterile 10% Steinberg’s solution containing 50 pg/ml streptomycin sulfate and 100 IU penicillin G, and allowed to incorporate label at 21°C for the desired labeling period. Leakage of injected isotope was examined at two developmental stages, Stage 12 (late gastrula) and stage 20 (hatching). During the first hour after injection, leakage was 10% of the total injected cpm, decreasing to approximately 5% of the total per each additional hour of incubation. Most of the loss of injected isotope after the first hour is due to diffusion since wound closure is complete within 1 hour in Steinberg’s. The injection procedure apparently did not reduce the viability of the embryos since both injected and control embryos developed normally and at the same rate. After the labeling period, embryos were washed extensively in sterile 10% Steinberg solution and homogenized with several strokes of a Potter-Elvehjem homogenizer in 2.0 ml of phosphate-buffered saline (PBS) (0.14 M NaCl, 10 mM sodium phosphate buffer, pH 7.2). The homogenate was centrifuged (34,OOOg, 10 min), the lipids were removed by aspiration, and the supernatant solution was recovered.

274

DEVELOPMENTALBIOLOGY

For the precipitation of labeled TDO from the PBS extract a modification of the procedure described by Segal and Kim (1963) was used. To a 500-~1 aliquot of the PBS extract was added purified carrier adult TDO and a volume of antibody in slight excess of the equivalence point. To a duplicate tube was added adult carrier TDO and control serum. Incubation mixtures were adjusted to 0.1% Triton X-100 to discourage nonspecific precipitation, and the tubes were incubated at 25°C for 1 hr and 4 hr at 0-4°C. The precipitate was collected by centrifugation (12,000 g, 10 min), and supernatant solution was saved. The immune precipitates were washed extensively with cold PBS. All precipitates were hydrolyzed in 1 N NaOH for 14 hr at 37°C. Radioactivity in an aliquot of neutralized hydrolyzate was determined by scintillation spectroscopy. In order to estimate total PBS protein synthesis and the amount of free unincorporated label, an aliquot of the 34,000 g PBS supernatant was adjusted to 10% TCA, and the precipitate which formed at 4°C after 4 hr was collected by centrifugation as above. Radioactivity in an aliquot of the TCA supernatant was a measure of the total unincorporated label. The TCA precipitates were washed 3 times with cold 10% TCA, hydrolyzed, neutralized, and counted as above to give a value for total PBS extractable radioactive protein. Acrylamide

Gel Electrophoresis

Acrylamide gel electrophoresis of purified adult TDO was performed according to Miller et al. (1970). Sodium dodecyl sulfate (SDS) electrophoresis of antigen-antibody precipitates was performed according to Palmiter et al. (1971). Preparation

of [“Cltyrosine

DOPA oxidase

Purified adult TDO was made radioactive by acetylation with [1-“Clacetic anhydride (5 mCi/mmole, New England Nuclear) by the method of Fraenkel-Conrat et

VOLUME 40,1974

al. (1949). The reaction was carried out for 40 min and the enzyme was then dialyzed against 20 mM sodium phosphate, pH 8.0, and passed through a Sephadex G-25 column equilibrated with the same buffer. The radioactivity applied to the column was resolved into two peaks, one coinciding with the absorbance at 280 nm. This pooled peak had a specific activity of 31,300 dpmlmg enzyme protein. Autoradiography The procedure was a modification of that used by Ecker and Smith (1968). Embryos were dejellied, washed, and injected with 0.1 ~1 of neutralized SH-algal protein hydrolyzate (500 pCi/ml). Embryos were fixed at 1, 10, 30, 90 min after injection in 2% glutaraldehyde, 50 mM sodium phosphate pH 7.3, 5 mM MgCl, for 45 min at 4°C and then washed for 1 hr at 4°C in the above buffer minus glutaraldehyde. Embryos were then dehydrated, cleared, rinsed in toluene, and infiltrated with Paraplast. Sections were bleached of melanin in 10% H,O, in 63% ethanol for 20 hr (Moore, 1963). Slides were dipped in Kodak NTB-3 track emulsion at 40°C for 5 set, dried, and stored in a light-tight box at 23°C for 6 weeks. Developed slides were stained with methyl green. Materials All chemicals were of reagent grade. Rana pipiens were purchased as adults and eggs were obtained by pituitary injection into gravid female frogs (Rugh, 1948). RESULTS

Tyrosine DOPA Oxidase Activity Development

during

Experiments were first performed to determine TDO activity as a function of developmental stage. Assays were done both spectrophotometrically, using DOPA and polarographically, as a substrate, using either tyrosine or DOPA as substrate.

BENSON AND TRIPLETT

Tyrosinuse

during Frog Development

275

FIG. 1. The developmental change in DOPA oxidase activity from total embryos. The developmental change in TDO specific activity as measured by the DOPA-oxidase spectrophotometric assay was performed on the 40-70’S ammonium sulfate fraction. Each point represents the mean and standard deviation of the results from four different experiments. Assays were performed with (-) and without (-----) the addition of trypsin as described in Materials and Methods.

FIG. 2. The developmental change in TDO specific activiy as measured polarographically. The tyrosine (A) and DOPA (A) oxidation activity in the 40-70s ammonium sulfate fraction was measured polarographically as described in Materials and Methods. Fractions were assayed with (-) and without (-----) the addition of trypsin. Each point represents the average of two different experiments.

Since adult frog skin TDO can exist in an inactive state and can be activated by mild trypsinization (McGuire, 1970; Miller et al., 1970; Mikkelsen and Triplett, unpublished), all embryo TDO assays were done in duplicate; one preparation was trypsinized and the other was not. Results of the spectrophotometric assays are summarized in Fig. 1. Low and decreasing DOPA oxidase specific activity, both in the trypsinized and nontrypsinized assay reactions, was observed during the initial 29 hr of development from the unfertilized egg to the mid-gastrula stage. By mid-gastrula no DOPA oxidase activity could be measured with either assay. DOPA oxidase activity in trypsinized preparations reappears at the neurula stage of development following the completion of neural induction. This activity continues to rise dramatically to the tail-bud stage. On the other hand, DOPA oxidase activity of the untrypsinited preparations is very low at neurula and remains low until hatching, at which time it undergoes a significant increase. Identical results were obtained using the polarographic assay (Fig. 2). The same general trend was obtained

using tyrosine as substrate in the polarographic assay system (Fig. 2). There is decreasing activity until mid-gastrula at which time no tyrosine oxidase activity could be measured. Activity in both trypsinized and untrypsinized preparations reappears at the early neural plate stage. The difference in specific activity between trypsinized and nontrypsinized preparations from neurula to hatching in the tyrosine oxidation is not as marked as in the DOPA oxidase system but is nevertheless significant. In order to be assured that observed differences in DOPA and tyrosine oxidation activities during development were representative of the enzyme concentration, enzyme activity as a function of total protein concentration was determined spectrophotometrically using DOPA as a substrate (data not shown) and polarographically using both tyrosine and DOPA as substrates (Fig. 3). There is a linear relationship in each case between enzyme activity and total protein. These data negate the possibility that differences in enzyme protein concentration affected the specific activity results.

276

DEVELOPMENTAL

BIOLOGY

40, 1974

VOLUME

TABLE

2

ASSAYS COMBINING COMPLETE AND HEAT-STABLE EXTRACTS FROM STAGES WITH HIGH AND Low DOPA OXIDASE ACTIVITIES~ Assay system”

FIG. 3. Tyrosine and DOPA oxidation as a function of enzyme concentration. The TDO was purified through the 40-70’S ammonium sulfate step from the following embryonic stages: stage 17, tail bud, 77 hr (0); stage 25, operculum closed, 240 hr (0). Tyrosine (-) and DOPA (-----) oxidation was monitored polarographically after trypsin treatment.

The possibility was also examined that the increase in activity following embryonic induction was due to the elimination of inhibitors present in preinduction developmental stages. To test this possibility, low activity extracts from an early developmental stage, early gastrula, Shumway stage 10 (24 hr), were incubated prior to assay under various conditions (Table 2) with the higher specific activity extract of a later, postinductive stage, mouth open, stage 21 (140 hr). The incubation of complete untreated early-stage extract or the soluble component of boiled early-stage extracts with stage 21 extracts reveals an effect of mere dilution, not of inhibition, on the postinduction DOPA oxidase activity. DOPA Decarboxylase Development

Activity

A B C D E F G H

Stage 10 early gastrula (24 hr)

Stage 21 mouth open (140 hr)

Heat

COIlplete extract (ml)

Heatstable extract (ml)

COIIplete extract (ml)

stable extract (ml)

0 0 0.5 0.25 0.25 0 0.25 0

0 0 0 0.25 0 0.25 0 0.25

0.50 0.25 0 0 0.25 0.25 0 0

0 0.25 0 0 0 0 0.25 0.25

DOPA oxidase activity’

12.2 6.2 2.1 1.2 6.4 6.0 1.1 0.1

“The 34,000 g supematant solutions from stages with high (stage 21, 140 hr) and low (stage 11, 29 hr) DOPA oxidase activities were incubated together for 1 hr at 22’ and assayed spectrophotometrically for DOPA oxidase activity. The heat-stable extract was prepared by heating the complete homogenate for 30 min at 95” followed by centrifugation at 34,000 g for 15 min to remove coagulated protein. bThe protein concentration in each extract was identical. The standard DOPA oxidase assay was used with the exception that the volume of enzyme assayed was 0.5 or 0.25 ml. Extracts were not trypsinized prior to assay. c Each value for activity ( AOD 475 nm/min x 10Z).

A linear relationship was observed between DDC activity and protein concentration (data not shown) reducing the possibility that protein concentration effects such as enzyme aggregation or dissociation contributed to the observed specific activity changes in DDC specific activity.

during

Experiments were also performed to determine DOPA decarboxylase (DDC) activity as a function of developmental stage. DDC activity is completely absent in the embryos prior to the late neural fold stage of development (stage 15). Stages subsequent to the neural fold show a steady increase in the specific activity of DDC throughout the remaining developmental stages (Table 3).

Preparation

of TDO Antibody

Adult frog skin TDO, purified to homogeneity, was used exclusively as an antigen to produce an antibody in rabbits. Prior to using these antibodies to precipitate embryonic TDO it was necessary to demonstrate antigenic cross reactivity between embryonic and adult TDO since there was no assurance that embryonic and adult TDO shared the same antigenic determinants. It was also necessary to deter-

BENSON AND TRIPLETT TABLE

3

DEVELOPMENTAL.CHANGES IN DOPA ACTIVITF Developmental stage

HOWS of developmerit

8+ Late cleavage

14

12 Late gastrula

36

15 Late neural fold 21+ Tail fin circulation 25 Operculum complete

60 162 242

Tyrosinnse

Protein ‘3’

7.67 6.44 8.53 7.12 10.50 9.81 8.90 9.65 12.50 13.30

DECARBOXYLASE Picomoles DOPA decarbox+ ated in 30 min’

Specific activityC

0.43 0.69 0.53 0.41 8.02 7.23 9.68 18.45

0.06 0.06 0.06 0.76 0.73 1.09 1.91

49.31

3.96

43.51

3.27

0.11

“Embryos were homogenized and assayed for DOPA decarboxylase (DDC) as described in Materials and Methods. Duplicate assays were performed at each stage of development. b Based on the specific activity of the “CO,-DOPA a loss of 100 dpm was equivalent to 1.875 picomoles of DOPA decarboxylated. c Specific activity is defined as picomoles of DOPA decarboxylated in 30 min/mg/ml protein.

the equivalence point ratio of antigen and antibody to facilitate complete precipitation of the embryonic and added adult carrier TDO. The specificity of the antibody toward partially purified adult skin and embryonic TDO from several developmental stages was examined in the Ouchterlony double diffusion analysis (data not shown). A single precipitin band was obtained when the antibody against adult TDO in the center well was allowed to react with partially purified adult and partially purified embryonic TDO from various stages of development and all exhibited reactions of identity. The precipitin band was stained with DOPA to reveal DOPA oxidase activity or for protein with Coomassie brilliant blue. In both cases a single band was detected. These results indicate that the TDO from adult and embryonic sources is mine

during Frog Development

277

immunologically very similar if not identical. Accordingly, antibody directed against adult TDO was used with confidence to isolate embryonic TDO by immunoprecipitation in radioactive labeling experiments. Adult TDO was used as a carrier to ensure maximum immunoprecipitation. The equivalence point (that is, the ratio at which enzyme can no longer be detected in the supernatant solution) in our system is I.2 ~1 antibody and 1.0 ~1 (130 pg) TDO. Nonimmune control serum globulins precipitated less than 5% of the enzyme activity. Radioactive Labeling of Phosphate-Buffered Saline (PBS) Extractable Embryonic Proteins To establish the time of maximum incorporation into PBS extractable proteins, embryos at four widely separated stages of development were injected with L[sH]amino acids and allowed to incorporate these amino acids into protein. At times up to 6 hr post injection, embryos were homogenized and the PBS extractable proteins were examined for trichloroacetic (TCA) precipitable radioactivity. The results (Fig. 4) indicate that by 300 min blastula embryos have approached a steady state whereas an additional 60 min of incubation was required before neurula, tail-bud, and tail-fin circulation stages approached a steady state. Therefore, in subsequent isotope immunoprecipitation studies investigating the stage-specific synthesis of TDO, an incorporation time of 6 hr was chosen to maximize incorporation and yet not traverse the temporal gap from one developmental stage to another. The possibility that amino acids are incorporated to any significant extent into macromolecules other than protein is eliminated by the data in Table 4. Embryos from the late neural fold (56 hr) and tail-fin circulation (160 hr) stages of development were injected as above with 0.1 ~1 of

278

DEVELOPMENTALBIOLOGY VOLUME40,1974 .

*

.

3

.

0

c

*

FIG. 4. Kinetics of incorporation of [3H]amino acids into PBS-extractable proteins. One hundred embryos were injected with 0.1 ~1 of [3H]amino acids (0.9 mCi/ml) and allowed to incubate at 22°C in one-tenth strength Steinberg’s medium. At the indicated times, 10 embryos were sacrificed; a PBS extract was prepared, and the total TCA-precipitable radioactivity was determined as described under Methods. Blastula (0), stage 9, 17 hr; neurula (A), stage 15, 61 hr; tailbud (O), stage 17, 72 hr; and tail fin circulation (A), stage 22, 145 hr. Counts are expressed as dpm per 10 embryos. TABLE

4

EFFECTOF PUROMYCINON THE INCORPORATION OF [SH ]AMINO ACIDS INTO “PBS''-EXTRCTABLE PROTEIN@ Developmental stage

14+ Late neural fold

22 Tail fin circulation

Expt. NOS.

PWOmycin

1

+ -

2

+ -

1

+ -

2

+

dpm per 5 embryos

4142 27628 2193 24372 4896 36916 6261 38419

Percent inhibition

85 91 a7 83

n Embryos were injected at the indicated stages of development with 0.1 ~1 of a solution containing 0.09 PCi of [3H]amino acids and 0.05 /.rg of puromycin sulfate. Control embryos received only the radioactive amino acids. The embryos were incubated for 6 hr at 22”C, at which time a PBS extract was prepared and the TCA-insoluble radioactivity was determined as described in Materials and Methods. Duplicate assays were performed at each stage.

3H-amino acids containing 500 pg/ml of puromycin. Puromycin at 0.05 pg per embryo inhibited incorporation into TCAprecipitable radioactivity by an average of

88% in late neural fold and 85% in tail-fin circulation stages after 6 hr of incubation. The residual incorporation is presumably due to the fact that puromycin was injected with the isotope and required time to equilibrate within the embryo. Implicit in the microinjection technique is the assumption that the injected isotope equilibrates relatively quickly with the endogenous amino acid pool and that the incorporation measured is synthesis representative of the free amino acid pool in the embryos. Autoradiographs (not shown) of stage 11, mid-gastrula sectioned embryos showed that there were virtually no grains produced over sections made from embryos fixed immediately after injection. By 90 min the total number of grains had increased and the difference between the site of injection and the other regions of the embryo was small. There was a higher concentration of grains in the dorsal half of the embryo which is consistent with Deuchar’s (1956) observation that there is a decreasing dorsal-ventral concentration of free amino acid in amphibian embryos. Specificity of Isotopic Immune Precipitation Reaction The following experiment was performed to establish with certainty that only TDO was precipitated from embryonic PBS extracts by anti-TDO antibodies. Adult [l’C]TDO was added to PBS extracts of 3H-labeled embryos from three widely separated stages of development. The precipitate which formed upon the addition of an equivalence point amount of anti-TDO antibody was recovered by centrifugation, washed in PBS and dissolved in a Trisglycerol-SDS solution. The preparations were then electrophoresed on SDS acrylamide gels, sliced, and counted for radioactivity. Figure 5 shows the distribution of radioactivity of gels from different stages of development. About 96% of the tritium radioactivity migrated with the purified [“‘CITDO. These experiments demon-

BENSON AND TRIPLETT

Tyrosinase

during Frog Development

279

skate that embryonic and adult TDO contain similar or identical antigenic determinants and that the addition of carrier adult TDO to achieve complete precipitation of embryonic TDO at various developmental stages can be used with confidence. TDO Synthesis

FIG. 5. Electrophoresis of 3H-labeled embryonic TDO precipitated immunologically from embryonic PBS extracts with authentic “C-labeled adult skin TDO. Embryos at (A) neural plate, stage 13+, 46 hr, (B) tail bud, stage 17, 74 hr, and (C) hatching, stage 20, 120 hr were injected with [3H]amino acids and allowed to incubate 6 hr at 22°C as described in Materials and Methods. The embryos were homogenized and a PBS extract was prepared. An aliquot of the PBS supernatant was incubated with anti-TDO and 0.1% (final concentration) of Triton X-100. Approximately 10 pg of authentic “C-TDO prepared as described in Materials and Methods was added and the entire mixture was incubated at 22O for 4 hr. The precipitate was collected, washed, dissolved, and electrophoresed on SDS acrylamide gels as described

during Development

TDO synthesis as a function of developmental stage was determined by immunoprecipitation of isotopically labeled TDO utilizing purified adult TDO as a carrier for the anti-TDO antibody precipitation reaction. Embryos were dejellied, microinjetted with a [3H]amino acid mixture and cultured for 6 hr at 22”. After this a PBS extract was prepared, and an aliquot was added to purified adult carrier TDO and TDO antibody in slight excess of the equivalence point concentration. Nonimmune serum at an identical protein concentration was used in parallel control experiments. The resulting immune precipitates and supernatant solutions were processed as outlined in Methods and Methods, and the results are illustrated in Table 5. The amount of label incorporated into the soluble PBS extracts and measured as TCAinsoluble radioactivity showed, in several cases, considerable variation between closely related stages, and thus any individual variation in the labeling of PBS extracts may be reflected in the labeling of TDO. Therefore the last column in Table 5 expresses the results in terms of the relative incorporation into TDO. The counts precipitated by control serum were negligible. Furthermore, after the introduction of additional unlabeled carrier TDO to the labeled PBS supernatanb solutions, devoid of all labeled embryonic TDO, the counts precipitated were 510% of that obtained in the first precipitation of radioactive TDO. All counts exunder Materials and Methods. Gels were sliced and counted with a 9H counting efficiency of 35% and a *VI efficiency of 67%. Tritium radioactivity was corrected for “C spillover.

280

DEVELOPMENTAL

TABLE RELATIVE

stage of development

9 Blastula 10 Early gastrula 11 Mid-gastrula 13+ Neural plate 14+ Early neural tube 17 Tail bud

5

ACCUMULATION OF TDO DEVELOPMENTS HOUS of deV&p merit

16 22 29 51 60 77

19 Heart beat

108

21 Mouth open 25 Operculum closed

140 240

BIOLOGY

DURING

“COr-

Total TCA insoluble radioactivity c3H dpm per 100 embryos)

I ,ected” b i mmunov&;i-

Percent total incorporation as TDO

TDO ( $H dpm per 100 e mbryos)

417212 356467 561515

5 64 72 4

647380 594497 593604 564471 655413 648340 703684 677400 688071 694282 435320 528912

1254 1045 1285 1345 2428 1961 3932 3652 3736 3859 1992 2574

266193

i

0.00 0.00 0.02 0.00 0.194 0.175 0.217 0.238 0.369 0.301 0.558 0.539 0.543 0.516 0.457 0.486

n One hundred embryos were injected with [3H]amino acids (0.09 &i/embryo) at the indicated hours after fertilization, incubated for 6 hr and a PBS extract prepared as indicated in Materials and Methods. A sample of the PBS-soluble supernatant solution was precipitated with TCA as a measure of the total PBS-soluble protein accumulated during the labeling period, and another sample was assayed by immunoprecipitation techniques for TDO synthesis. Duplicate experiments were done simultaneously. b“Corrected TDO dpm” refers to radioactivity incorporated into TDO after subtracting any nonspecific immunoprecipitated radioactivity. Nonspecific precipitation is the sum of counts precipitated by the control antibody plus the counts in the second immunoprecipitation of the labeled PBS extract from which radioactive TDO had been removed by the first precipitation.

pressed in TDO are thus corrected for these two controls of nonspecific precipitation. The first detectable accumulation of radioactivity in TDO occurs at the early neural-fold stage of development (51 hr after fertilization) at which time TDO accounts for 0.184% of the total labeled

VOLUME

40. 1974

protein. TDO labeling continues to account for an increasing percentage of the total labeled protein reaching a maximum of 0.548% at stage 19 just prior to hatching. Thus for the 2-3-fold increase in the enzymatic specific activity of TDO during this period (Figs. 1 and 2) there is an equivalent 3-fold increase in the incorporation of radioactive amino acids into the enzyme. After hatching, however, the relative labeling of TDO decreases until, at stage 25, only 0.471% of the total labeled protein is TDO. The rate of increase in the enzymatic specific activity of TDO during development likewise becomes less during this period but then rises to a maximum by stage 25. The results in Table 5 indicate that the appearance and rise in TDO specific activity seen in the post inductive stages following gastrulation (Figs. 1 and 2) correlates closely with the onset of new enzyme synthesis. These data also establish that the low level of TDO activity observed prior to the completion of embryonic induction is not due to new enzyme synthesis following fertilization but supports the proposition that this “early” enzyme activity is the result of synthetic activity during oogenesis . DISCUSSION

The close temporal correlation between the emergence of TDO and DDC activity and neural crest determination after primary induction suggest the importance of these enzymes in neural crest determination. On the basis of the experiments described here we propose the following model to account for the differentiation of neural crest cells with respect to tyrosine metabolism. The model predicts that an important event in neural crest differentiation following primary induction is the transcriptionally dependent appearance and immediate translation of TDO messenger RNA. The

BENSON AND TRIPLETT

Tyrosinase

during Frog Development

281

The fact that the substrate preference of determination of the neural crest by the TDO changes developmentally with reinducing chordamesoderm is then partly spect to its two substrates, tyrosine and characterized by the appearance of TDO DOPA, suggests that the catalytic effiwith very low enzymatic activity. Tyrosine oxidizing activity of TDO be- ciency of the enzyme for these substrates comes significant at the late neural fold can be independently modulated by environmental stimuli, and the nature of this (stage 14+, 55 hr) stage of development. This is accompanied by the appearance environmental control is presently under of the first enzymatically detectable DOPA investigation. We know, for example, that decarboxylase activity and the production inactive purified TDO can be activated in of catecholamines at stage 15, 64 hr (Cas- several ways with resulting differences in ton, 1962). Thus, overt differentiation of substrate preference (Mikkelsen and Trithe sympathetic nervous system is maniplett, unpublished). The DOPA, but not fest at this time. the tyrosine-binding sites, are activated by A large increase in the amount of DOPAultraviolet irradiation at 334 nm or 290 nm. oxidase activity (without trypsinization) of Trypsin treatment of TDO activates both TDO is observed at the hatching stage binding sites. The lipids normally associ(stage 21, 120 hr), and this results in the ated with TDO and an intermediate in the appearance of melanin in determined pigmelanin pathway modify these effects. ment cells. Melanophores are thus overtly Various agencies have been postulated differentiated at this time. as devices for the activation of TDO in An explanation for the fact that the diverse biological systems. They include synthesis of TDO declines slightly while differential substrate permeability of preenzyme concentration (activity) continues melanosome membranes (Van Woert et al., 1971; Whittaker, 1973) and natural inhibito increase is not yet available although some possibilities may be considered. A tors of TDO activity (Chian and Wilgram, and Okhawara, 1966; most obvious possibility is that after a 1966; Halprin 1969; Menon and Haberman, maximum rate of synthesis has been McGuire, 1970). Our mixing experiments with prereached the degradation of the enzyme embryos (Table 2) decreases markedly. Only limited data are and postdifferentiation available concerning the in uiuo stability of and the fact that TDO purified to complete homogeneity may be inactive, speak embryonic TDO. Unpublished observations indicate an increased stability of the against these possibilities with respect to enzyme after neurulation. Crude uncenmelanophore differentiation in Rana pipiens . trifuged homogenates of stage 22, swimWe do not yet have a clear picture of the ming tadpoles stored at 22” retained of 50-60s of their TDO activity after 48 hr. means by which the large activation DOPA oxidase activity in Rana pipiens is Crude extracts of stages prior to neurulaaccomplished at the hatching stage of detion (stage 8, mid-cleavage; stage 10, early velopment, but some progress has been gastrula) retained only 20% of their original activity by 48 hr when stored at the same made on this problem (Slaughter and Tritemperature. A very rough estimation of plett, unpublished). We know, for examTDO half-life gives a value of 30 hr for ple, that young Rana pipiens embryos stages prior to neurulation and 54 hr after contain a very potent trypsin inhibitor neurulation. Whether or not in vitro stabilassociated primarily with yolk platelets. ity is a reflection of in vivo, stability The inhibitor is no longer detectable in the remains to be determined. pigmented retina at the hatching stage

282

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MCGUIRE, J. S. (1970). Activation of epidermal tyrosinase. Biochem. Biophys. Res. Commun. 40, 1084-1089. MENON, I. A., and HABERMAN, H. F. (1970). Activation of tyrosinase in microsomes and melanosomes from B-16 and Hardy-Passey melanosomes. Arch. Biothem. Biophys. 137, 231-242. MILLER, L. O., NEWCOMBE, R., and TRIPLETT, E. L. (1970). Isolation and partial characterization of amphibian tyrosine oxidase. Comp. Biochem. Physiol. 32, 555-567. MOORE, B. C. (1963). Histones and differentiation. Proc. Nat. Acad. Sci. U.S. 50, 1018-1026. PALMITER, R. D., OKA, T., and SCHIMKE, R. (1971). Modulation of ovalbumin synthesis by estradiol178 and actinomycin D as studied in explants of chick oviduct in culture. J. Biol. Chem. 246, 724-737. POMERANTZ, S. H. (1963). Separation, purification, and properties of two tyrosinases from hamster melanoma. J. Biol. Chem. 238, 2351-2357. RAVEN, C. P. (1935). Zur Entwicklung der Ganglienleiste. IV. Untersuchungen iiber Zeitpunkt und Verlauf der “materiellen Determination” des presumptiven Kopfganglien leistenmaterials der Urodelen. Wilhelm Roux Arch. Entwicklungsmech. Organismen 132, 510-575. RUGH, R. (1948). “Experimental Embryology.” Burgess Publishing Co., Minneapolis, MN. SEGAL, H. L., and KIM, Y. S. (1963). Glucocorticoid stimulation of the biosynthesis of glutamic-alanine transaminase. Z’roc. Nat. Acad. Sci. U.S. 50, 912-917. SMITH-GILL, S., RICHARDS, C., and NACE, G. (1972). Genetic and metabolic basis of two “albino” phenotypes in the leopard frog, Rana pipiens. J. Exp. 2001. 180, 157-168. SPIRIN, A. (1966). On “masked” forms of messenger RNA in early embryogenesis and in other differentiating systems. Curr. Top. Deuelop. Biol. 1, 2-38. VAN WOERT, M. H., KORB, F., and PRASAD,‘K. N. (1971). Regulation of tyrosinase activity in mouse melanoma and skin by changes in melanosomal membrane permeability. J. Znuert. Dermatol. 56, 343-348. WHI~AKER, J. R. (1973). Tyrosinase in the presumptive pigment cells of ascidian embryos: tyrosine accessibility may initiate melanin synthesis. Deuelop. Biol. 30, 441-454.