/ . Embryol. exp. Morph. Vol. 24, 1, pp. 109-118, 1970
Printed in Great Britain
109
Isolation and partial characterization of
amphibian tyrosine oxidase polysomes
By E. L. TRIPLETT 1 , R. HERZOG 1 AND
L. P. RUSSELL 1
From the Department of Biological Sciences,
University of California, Santa Barbara
SUMMARY
A population of polysomes isolated from frogskinis capable of supporting protein synthesis
in a cell-free system containing an energy generating system,' soluble components', and amino
acids.
These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they
also have antigenic determinants attributable to tyrosine oxidase.
Skin polysomes sedimented in 10-30 % sucrose gradients contain tyrosine oxidase peaks of
enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine
oxidase antigenic acitvity is found in the 250-350S region of the gradient.
Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250-350S particles capable of supporting the incorporation of [14C]amino
acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable
that 25O-35OS tyrosine oxidase antibody precipitates contain only polysomes for this protein.
INTRODUCTION
Switching mechanisms that operate in controlling the tyrosine pathway
during cellular differentiation are completely obscure at present. Yet, such
controls must exist since many cell types (notably those derived from neural
ectoderm) are easily distinguished on the basis of patterns of tyrosine metabolism.
For example, some neurons convert tyrosine to norepinephrine; adrenal medullary cells produce epinephrine; pigment cells produce melanin; and fibrocytes
probably make acetoacetate and fumarate. The switching devices could be of
various types, but it is probable that they consist of systems that operate in
selectively controlling either the catalytic efficiency or rates of synthesis and/or
degradation of enzymes at branch points in the pathway. With this in mind, we
have begun to characterize the machinery involved in the synthesis of amphibian
tyrosine oxidase. This enzyme (at least in Rana pipiens) is capable of oxidizing
tyrosine and 3,4-dihydroxyphenylalanine (DOPA) in the three and four
positions of the ring and 3,4-dihydroxyphenyl-ethyl amine in the beta position of
the side chain (Miller, Newcombe & Triplett, 1969). Thus, it is an enzyme that
operates in two branches of the pathway.
1
Authors' address: Department of Biological Sciences, University of California, Santa
Barbara, California 93106, U.S.A.
110
E. L. TRIPLETT, R. HERZOG AND L. P. RUSSELL
The polysomes that participate in the synthesis of tyrosine oxidase were
isolated from adult frog skins by means of sedimentation in sucrose gradients
and precipitation with antisera directed toward tyrosine oxidase. Some of the
properties of these polysomes are reported here.
MATERIALS AND METHODS
Preparation of bulk polysomes
Polysomes were isolated from frog skins at a temperature never exceeding 4 °C.
In a typical experiment, 100 frogs were decapitated and skinned to yield about
400 g of tissue. Bentonite, 4 0 mg/ml, was blended into the tissue, and it was
passed through a motor-driven meat grinder six times. Twenty ml of a solution
(MSBRBH) containing 0-25 M-KCI, 001 M-MgCl2, 0 0 1 M Tris (pH 7-4),
500 units/ml heparin and 50 mg/ml rat liver proteins was then added to the
preparation along with 4 0 mg/ml of bentonite. Sucrose, 0-25 M, was used in
preliminary experiments, but it was left out in the experiments reported here
because it renders skin cells resistant to breakage. Deoxycholate was also not
used in later experiments since it could not be shown to facilitate recovery of
polysomes or to affect priming capacity of polysomes.
Rat liver proteins were prepared by homogenizing rat livers in three volumes
of a solution containing salts in the above concentrations, centrifuging at 34000 g
for 5min, precipitating the supernatant solution by addition of crystalline
ammonium sulfate to a concentration of 3-3 M, centrifuging at 34000 g for
5 min and dissolving the pellet in a minimal volume of the extraction medium.
The solution was dialysed against large volumes of extraction medium cleared
by centrifuging (105 kg, 90 min) and stored in liquid nitrogen until needed.
This preparation was adjusted to 50 mg/ml protein and used both as a source of
a peptide inhibitor of ribonuclease and the 'soluble components' required for
cell-free protein synthesis. Rat liver was used in preference to frog liver, because,
while the frog preparation is equally effective as an additive to the cell-free
protein synthesizing system, it does not contain the ribonuclease inhibitor found
in the rat system. The omission of rat liver proteins or bentonite during the
first extraction of skin yielded a preparation with very high concentrations of
monosomes but almost no polysomes as judged by sucrose gradient sedimentation studies.
Lowering the potassium concentration to 0-0125 M, a concentration commonly used for the preparation of liver polysomes, yields a skin preparation
containing no polysomes but the usual amount of monosomes. We postulate
that, as in the case of myosin polysomes (Heywood, Dowben & Rich, 1967), low
potassium allows the aggregation and precipitation of skin polysomes (perhaps
in concert with collagen).
Homogenization was carried out in an omnixer (5 min at 80 V) since gentler
procedures could not be found for breaking the tough connective tissue. The
Tyrosine oxidase polysomes
111
shearing forces generated probably degraded some polysomes, but enough
remained intact to perform the experiments. The homogenate was centrifuged
(13200g, 90 min), and the supernatant solution was recentrifuged (105000g,
90 min). The pellet, a clear amber-colored gel with a black central area, was gently
suspended in 30 ml of MSBRBH by means of a glass-teflon, Dounce-type homogenizer. The preparation was centrifuged (34000 g, 5 min) and the supernatant
solution was recentrifuged (105000 g, 90 min). The pellet was gently resuspended
in about 10 ml MSBRBH with the Dounce homogenizer and the suspension
of polysomes was stored in liquid nitrogen until needed (never more than
48 h). Our preliminary experiments had established that the newly frozen preparation is actually a better primer for protein synthesis than freshly prepared, unfrozen polysomes. The frozen polysomes lose about one-third of their priming
capability in 7 days.
Assay for polysome dependent protein synthesis
The cell-free system used to assay the priming capability of polysome preparations was that of Wust & Novelli (1964) with minor modifications. The
assay mixture was constituted in an ice bath and contained these components:
magnesium acetate 002 M, potassium chloride 005M, /?-mercaptoethanol
0 0 0 0 1 3 M, Tris (pH 7 0) 01 M, adenosine triphosphate 0 0 0 7 4 M, guanosine
triphosphate 00038 M, creatine phosphate 00022 M, creatine phosphokinase
005 % (w/v), algal protein hydrolysate-14C (New England Nuclear) 1 /tCi, rat
liver preparation 30 mg/ml protein, polysomes 2-5 mg nucleic acid. At time
zero the temperature of the mixture was raised to 30 °C. Aliquots of 0-1 ml
were taken at predetermined times, and plated on paper discs. The dried discs
were washed by the Mans-Novelli technique (1961) with cold and hot 5 % trichloroacetic acid, ethanol and ether, and counted by scintillation spectroscopy.
The slope of a radioactivity (disintegrations per min/mg polysomal nucleic
acid) time curve extrapolated to time zero was a measure of the rate of
protein synthesis. All assays were done in duplicate. Controls in each set of
experiments established that protein synthesis was dependent on the presence of
rat liver preparation, the energy-generating system and polysomes. Background
was obtained by substituting MSBRBH for polysomes, and figures reported
below have background subtracted from them.
Protein (Lowry, Rosenbrough, Farr & Randall, 1951) and nucleic acid
(Munro & Fleck, 1966) were determined according to the references given or by
optical density (O.D.) at 280 and 260 nm.
Sucrose gradients
Linear 10—30 % sucrose gradients containing 0-25M-KC1, 001 M-MgCl2,
0-01 M Tris (pH 7-4), 50 units/ml heparin and 5 mg/ml rat liver protein were used
for sedimentation studies on polysomes. Centrifugation was routinely per-
112
E. L. TRIPLETT, R. HERZOG AND L. P. RUSSELL
formed at 25000 rev/min for 120 min in a spinco SW25 rotor at a temperature of
approximately 4 °C. Samples were removed from the bottoms of the tubes and
monitored with a flow cell at 260 nm. Sedimentation values were calculated
from the tables of McEwen (1967).
Preparation of tyrosine oxidase antibody
Tyrosine oxidase was purified by the method of Miller, Newcombe & Triplett
(1970) and injected into rabbits according to the schedule of Campbell, Garvey,
Cremer & Sussdorf (1963). Blood from immunized animals was obtained by
cardiac puncture, and serum was separated from cell and clot by centrifugation
at 10000g for 30 min. Immunoglobulins were precipitated by adding ammonium
sulfate to a final concentration of 1-47 Mand centrifuging at 34000 gvfor 5 min.
The pellet was dissolved in about one-fourth the original serum volume of
002 M phosphate (pH 7-2) and dialysed overnight against this same buffer.
Preparations were stored at 4 °C after adding 1:10000 (w/v) merthiolate.
Control bloods were treated similarly. Antibody titers were determined according
to Campbell et al. (1963).
Enzyme assay
Tyrosine oxidase was assayed spectrophotometrically measuring the appearance of 2-carboxy-2,3 dihydroxyindole-5,6 quinone (Dopachrome) at 475 nm in
a reaction mixture containing 20 mM 3,4-dihydroxyphenylalanine (DOPA),
0-2 ml; 0-2 M phosphate, pH 7-2, 1-0 ml; water, 0-5 ml; enzyme solution, 0-3 ml.
Before substrate was added, the reaction mixture was preincubated for 20 min
at 37 °C in 002 mg/ml trypsin in order to activate the enzyme fully (Miller,
Newcombe & Triplett, 1970). The assay was also performed at 37 °C. The slope
of the absorbance time curve at time zero was a measure of enzyme activity.
RESULTS
Experiments were first performed to determine whether or notantityrosine oxidase serum globulins would react immunologically with skin polysome preparations. The template capability of skin polysomes in the presence of antibody
was also tested. Control experiments consisted of repeating the experiment with
globulins from an unimmunized rabbit substituted for the antibody preparation.
In a typical experiment, 5-5 ml of polysomes containing 40 mg/ml protein and
3-6 mg/ml RNA were mixed with 41 ml of serum containing 67 mg/ml protein.
The mixture was gently agitated for 2 h at 4 °C and then centrifuged at 4400 g
for 10 min. The pellets of the experimental (called experimental polysome Ab
precipitate) and the control (called control polysome Ab precipitate) systems
were resuspended in MSBRBH by gentle Dounce homogenization. The supernatant solutions (called experimental and control Ab supernates) were centrifuged at 105000£ for 90 min, and the pellets were dissolved in MSBRBH.
Each of these types of polysome was then used as a primer for a cell-free system.
Tyrosine oxidase polysomes
113
The results are summarized in Table 1. In the control system, almost all RNA
remains in the supernatant solution and all protein priming capacity is in this
fraction. A specific activity is not given for the precipitate fraction because the
radioactivity of this system was not statistically different from background.
Table 1. Priming capacity of skin polysomes treated and untreated
with anti-tyrosine oxidase antibody
DPM/
Specific
activity
RNA (Minus (DPM/min/mg
Protein
(mg/ml) (mg/ml) bgd.)
RNA)
min
Untreated polysomes
Experimental polysome Ab precipitate
Experimental polysome Ab supernate
Control polysome Ab precipitate
Control polysome Ab supernate
40
11
38
5-5
37
3-6
0-45
2-7
01
30
770
345
535
30
700
214
768
198
—
350
006 -
Tube position (ml.)
Fig. 1. Skin polysomes centrifuged in 10-30% sucrose gradients (SW25 rotor,
25000 rev/min., 120 min). Upper line is O.D.26onm- Triangles are RNA-bound
tyrosine oxidase enzyme activity and circles are antigen activity for the same
preparation.
RNA and protein priming capability in the experimental system is present in
both fractions. The specific activity of the experimental supernatant fraction, as
expected, is slightly less than that of the untreated polysomes, but the experimental precipitate fraction has a much higher specific activity than would be
expected. Complexing with antibody protein seems to enhance the activity of
8
E MB 2 4
114
E. L. TRIPLETT, R. HERZOG AND L. P. RUSSELL
these polysomes. The precipitate obtained with anti-tyrosinase globulins is a
result of the presence of this specific antibody activity since very little RNA was
precipitated in the control system.
Experiments were then performed to determine the sedimentation behaviour
of skin polysomes in sucrose gradients. 0-2 ml aliquots of polysomes (containing
3-6 mg/ml RNA) were sedimented, and the profile shown in Fig. 1 was recorded.
Larger amounts of polysomes (1 0 ml/tube) were sedimented similarly and enzyme assays for tyrosine oxidase were performed on aliquots of the eluant.
Tyrosine oxidase activity, also plotted in Fig. 1, has peaks at the bottom and
top of the tube and also at about 250S. Quantitative precipitation studies
(Campbell et al. 1963) using anti-tyrosine oxidase globulins were performed on
similar preparations, and the plot of tyrosine oxidase antigen v. tube position is
also present in Fig. 1. It was noted that profiles for tyrosine oxidase enzyme and
antigen activity were similar in that both have peaks in the 250-350S region.
The enzyme activity seen at the top and bottom of the tube was absent in the
antigen assay.
Another set of experiments was done to determine the relative priming
capabilities of different regions of the sucrose gradient profile and to determine
how much of the priming capability in each fraction could be attributed to
polysomes precipitable with anti-tyrosine oxidase globulins. 10 ml aliquots of
skin polysomes were centrifuged and four fractions were collected as indicated
in Fig. 1. Each fraction was centrifuged at 105000 g for 90 min and the pellets
were each resuspended in 10 ml MSBRBH. Each fraction was mixed with
2-0 ml of anti-tyrosine oxidase globulins (48 mg/ml protein) and gently agitated for 2 h at 4 °C. The preparations were spun at 32000 g for 5 min, and the
pellets were suspended in 10 ml of MSBRBH and called 'Ab treated precipitate'. The supernatant solutions were centrifuged at 105000g for 90 min,
and the pellets, after resuspension in 10 ml MSBRBH, were labelled 'Ab
treated supernate'. Each preparation was then tested for its ability to support
protein synthesis in a cell-free system. Untreated polysomes were similarly
assayed. The results reported in Table 2 demonstrated that larger polysomes have
a higher specific activity, and that there are peaks of activity for anti-tyrosine
oxidase precipitable polysomes near the bottom of the tube and in the region
containing 250S particles. The sum of precipitate and supernatant activity is
greater in each fraction than that of an untreated preparation, and, as in experiments reported above, the discrepancy appears to be attributable to the 'Ab
treated precipitate' systems.
A final experiment was performed to estimate how much of the labelled
amino acid incorporated into each of the cell-free systems described above
could be attributed to tyrosine oxidase synthesis. The method was to liberate all
nascent protein in a reaction mixture after a period of incubation, treat this
preparation with anti-tyrosine oxidase globulins, and to measure the amount of
radioactivity precipitated and the amount remaining in solution. The radio-
Tyrosine oxidase poly somes
115
activity in the precipitate should be a measure of tyrosine oxidase synthesis and
the hot-TCA-insoluble material remaining in solution should be a measure of
protein synthesis other than tyrosine oxidase.
Table 2. Priming capacity of skin polysomes fractionated by
centrifugation in sucrose gradients
Specific activity (DPM/min/mg RNA)
Fraction
Untreated
Ab-treated
precipitate
1
2
3
4
84
35
32
25
231
143
208
140
Ab-treated
supernate
70
29
16-6
77-5
Table 3. Tyrosine oxidase precipitable and non-precipitable radioactivity in
cell-free systems utilizing skin polysomes resolved by centrifugation on sucrose
gradients. Reaction mixtures were treated with RNase prior to addition of antibody
Fraction
Ppt.
(DPM/disc)
1
2
3
4
8514
3475
5471
5638
Super.
(DPM/disc)
6111
8245
—
—
Cell-free systems constituted as above (0-6 ml aliquots) were treated with
ribonuclease (80 u./ml for 20 min at 37 °C) after the kinetics studies were completed. Carrier tyrosine oxidase (10 mg) and anti-tyrosine oxidase antibody
(2 ml at 48 mg/ml) were added to each system and they were incubated at
20 °C for 2 h. The preparations were then centrifuged (34000 #, 5 min), and the
pellets were dissolved in 10N-NaOH and counted in the liquid scintillator.
The results in Table 3 indicate that all the protein synthesized by antibodyprecipitated polysomes 35OS or lighter was tyrosine oxidase. No tyrosine
oxidase was apparently made by polysomes in this region that are not precipitable by antibody. In contrast, the heavier fractions contained polysomes, both
precipitable and non-precipitable, that synthesized tyrosine oxidase. We
propose that the non-precipitable polysomes of this type are aggregates
containing both tyrosine oxidase polysomes and others that mask antigenic
determinants required for specific immunological precipitation.
The reaction mixture for a cell-free system containing untreated polysomes
from sucrose gradient fraction 3 was treated as before with pancreatic ribonuclease to liberate all nascent protein, and this preparation was used as
antigen in gel diffusion studies (Ouchterlony, 1949). Purified tyrosine oxidase
8-2
116
E. L. TRIPLETT, R. HERZOG AND L. P. RUSSELL
'peak I ' (Miller et al. 1970) was placed in a well adjacent to the one containing
the ribonuclease-treated cell-free system. Precipitation arcs were formed in both
systems, and they exhibited reactions of identity. Precipitin arcs failed to form in
preparations that were not treated with ribonuclease.
DISCUSSION
We propose that a class of polyribosomes which we have isolated by sucrosegradient centrifugation followed by precipitation with anti-tyrosine antibodies
consists almost entirely of the translational machinery for tyrosine oxidase.
Several lines of evidence support this contention.
In identifying these polysomes we made use of an observation made some
years ago by Seiji, Shimao, Birbeck & Fitzpatrick (1963) that tyrosine oxidase
enzyme activity could be found (in addition to other places) in isolated microsomal fractions of pigment cells. Since differentiated pigment cells do not have
active tyrosine oxidase in melanin granules, we postulated that the tyrosine
oxidase must be present in inactive polysomes that were fully loaded with
nascent tyrosine oxidase. Some of the nascent peptide must have been sufficiently
finished to assume the secondary folding necessary to confer substrate binding
specificity after activation by trypsin. Furthermore, the nascent peptide must
have bound copper before being released from the polysome since this metal is
needed for the catalysis. If the nascent protein were as complete as this, it could
also be expected to have acquired the antigenic specificity characteristic of tyrosine oxidase. Accordingly, it should be possible to identify tyrosine oxidase polysomes by three powerful criteria—sedimentation velocity, enzymic capabilities
and antigenic properties. The particles described above met all three criteria.
The sedimentation studies indicated that an area in the 250-350S region of
the profile is especially rich in tyrosine oxidase enzymic and antigenic activities.
This region contains, among others, the class of polysomes with 7-9 ribosomes.
This is the size range that would code for a peptide of about 30000 molecular
weight, and amphibian tyrosine oxidase (L. O. Miller, R. Newcombe and
E. L. Triplett, unpublished) is a tetramer of about 120000 molecular weight
composed of four identical subunits. These polysomes, therefore, meet the
size requirement for the tyrosine oxidase system.
Enzyme activity was also observed at the bottom and at the top of the gradient.
We propose that the former are aggregated polysomes, and the latter are degraded polysomes (monosomes) with nascent protein attached.
It was calculated that about 2-3 % of the nucleic acid in the stock skin polysomal preparations was precipitable with tyrosine oxidase antibody. Of the
RNA sedimented in sucrose gradients about 20% is found in the 250-350 S
region, and 39 % of this region is precipitable with anti-tyrosine oxidase
antibody (about 7 % of the total RNA recovered from the gradient). The difference between percentage RNA in antibody precipitates of stock polysomes
Tyrosine oxidase polysomes
111
and sucrose gradient preparations probably resulted from the fact that some
material packed tightly at the bottom of the gradients and was not recovered.
While it is not possible to estimate at this time how much skin polysomal
material participates in tyrosine oxidase synthesis in vivo, 2-3 % is certainly not an
unreasonable figure.
Skin polysomes retain the ability to participate in the incorporation of radioactive amino acids into hot-TCA-insoluble material after precipitation with
antibody. When different regions of the sucrose gradient are examined for the
types of protein they make, it can be demonstrated that all capacity in the 250350S region to prime for the synthesis of tyrosine oxidase is precipitable with
specific antibody. Unfortunately, we were unable to count the supernate of this
system after all labelled tyrosine oxidase was precipitated. Therefore, although
the antibody precipitated 250-350S polysomes contain all the tyrosine oxidase
polysomes for this region, other types of polysomes may also be present as
minor contaminants.
We have no explanation at present for the fact that binding with antibody
enhances the activity of polysomes. One possibility, however, is that antibody
behaves in a manner that is analogous to the lx factor' isolated from reticulocytes (Beard & Armentrout, 1967) and liver cells (Hoagland & Askonas, 1963).
This factor appears to be a protein of about 20S, and stimulates translation of
both synthetic and natural message. These workers postulate that this agent
might operate in nature as a device for the control of protein synthesis at the
translational level. Whether antibody mimics a natural control device of this
type cannot be stated at present, but the system offers an attractive tool for the
study of such phenomena.
Another possible explanation is that antibody aids in the release of nascent
protein just as the presence of /^-chains of hemoglobin appears to facilitate the
release of a-chains by polymerization on the a-chain polysome (Colombo &
Baglioni, 1966). Similar observations have been made with respect to L-chain
binding of H-chain polysomes (Shapiro, Scharff, Maizel & Uhr, 1966) and to the
assembly of /?-galactoside subunits (Zipser, 1963; Kiho & Rich, 1964).
RESUME
Isolement et caracterisation partielle des polysomes de la
tyrosme oxydase des amphibiens
Une population de polysomes isoles a partir de peau de grenouille est capable de promouvoir
une synthese dans un systeme acellulaire comprenant une source d'energie, des 'composes
solubles' et des acides amines.
Par action modere de la trypsine, on separe des polysomes une proteine qui catalyse
1'oxydation de la tyrosine en DOPA. Ces polysomes ont aussi des proprietes antigeniques
attribuables a la tyrosine oxydase.
Les polysomes de la peau qui sedimentent dans des gradients de saccharose (10-30%)
presentent des pics d'activite enzymatique tyrosine oxydase au fond et au sommet du tube et
dans les regions 250S. Un pic d'activite antigenique de tyrosine oxydase apparait dans la
region 250-350 S du gradient.
118
E. L. TRIPLETT, R. H E R Z O G AND L. P. RUSSELL
Les polysomes separes dans le gradient conservent la capacite de promouvoir la synthese
proteique dans un systeme acellulaire. Toutes les particules 25O-35OS capables de promouvoir
l'incorporation d'acides amines 14C dans la tyrosine oxydase sont precipitables par les anticorps de la tyrosine oxydase. II est probable que les fractions 250-350S contiennent seulement des polysomes pour la proteine tyrosine oxydase.
Supported by a contract (N 00014-67-0120-0003) between the Office of Naval Research and
the Regents of the University of California.
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