/ . Embryol. exp. Morph. Vol. 37, pp. 159-172, 1977
Printed in Great Britain
\ 59
Characterization of an organ-specific differentiator
substance in the planarian Dugesia etrusca
ByVERNON E. STEELE 12 AND
CHRISTOPHER S. LANGE 1
SUMMARY
A substance which inhibits brain formation in decapitated regenerating planarians (Dugesia
etrusca) was characterized and partially purified. The substance's inhibitory activity was
followed during each purification procedure by adding freshly decapitated animals of a
standard size to each fraction, and later measuring the resultant regenerated brain volume.
The inhibitory activity remained in the supernatant after a 10000 g centrifugation of a cellfree homogenate. Most of the activity sedimented when the 10000 g supernatant was
centrifuged at 32000 g. The degree of inhibitory activity increased with increased numbers of
animals in the initial homogenate. The substance has an apparent molecular weight between
2x 105 and 4x 105 daltons. Digestion by pronase destroyed the activity, but treatment with
RNase, DNase I, or lipase had no significant effect. The inhibiting substance has an isoelectric
point (pi) of between 4-75 and 5-38 and migrates to the anode when electrophorezed in
pH 6-8 buffer.
INTRODUCTION
In a wide variety of animals, many investigators have shown that the differentiated cell population controls the mitotic and tissue determinative processes of
the undifferentiated cell population. It is believed that the differentiated cells
of the planarian control the course of stem cell-mediated tissue renewal by the
secretion of promotor and/or inhibitor substances. One such substance, which
has been shown to be present in crude extracts of planarians (Lender, 1955,1956,
1960), controls the differentiation of stem cells into brain tissue. Using Dugesia
lugubris and Polycelis nigra, Lender demonstrated that homogenates of heads
had a much greater capacity to inhibit brain tissue formation (in animals
whose brains had been removed) than homogenates of tails. In addition, he
showed that:
(1) If the crude extract was centrifuged at 10000 rpm for 20 min, the inhibitory activity was found in the supernatant.
(2) If this supernatant was then centrifuged at 20000 rpm for 20 min, the
inhibitory activity was found in the pellet.
1
Authors'1 address: Departments of Experimental Radiology and Radiation Biology and
Biophysics, The University of Rochester School of Medicine and Dentistry, Rochester,
New York 14642, U.S.A. (Reprint requests to Christopher S. Lange.)
2
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
160
V. E. STEELE AND C. S. LANGE
(3) The head homogenate was not inactivated by treatment with 70 % alcohol,
lyophilization, or heating to 60 °C for 2 min.
(4) However, higher temperatures reduced the activity, and boiling for 30
min destroyed it.
(5) The substance does not exhibit zoological specificity, since ground brains
of Dugesia lugubris, Dugesia gonocephala, or even a minced 9-day embryonic
chick brain (Lender & Deutsch, 1961) inhibited the differentiation of the brain
in Polycelis nigra.
The brain has been shown to be the first organ to regenerate in an anterior
blastema (Wolff & Lender, 1950). If, however, an intact brain were implanted
into the anterior region of a decapitated animal, either the implanted brain
became the brain of the regenerated head, or the animal regenerated two heads,
one containing the implanted brain and one head containing no brain tissue
(Wolff, Lender & Ziller-Sengel, 1964). Regeneration of the brain not only determines the anterior-posterior polarity, but has been hypothesized to be the primary
organizer which, once formed, begins a series of inductions and differentiations
to replace all other missing tissues (Wolff, 1962).
This study attempts to characterize further the diffusible brain inhibitory
substance in Dugesia etrusca.
MATERIALS AND METHODS
1. Water
Filtered water is tap water which has been filtered through both a Calgon
no. 9 and a Culligan 'Flavr-Gard' water filter. Planarian saline is similar to
McConnell's (1967) with the exception that it contains 2-5 ml from each of the
two stock salt solutions per liter in distilled water.
2. Stock and experimental animals
Stock cultures of the planarian, Dugesia etrusca, were maintained in filtered
tap water at 19 ± 1 °C and fed canine liver biweekly.
All experimental animals were starved for between 1 and 2 weeks prior to
use, to insure minimal effects of metabolic variations and of nutritional wastes.
The standard size animal used for the assay procedure was 5-0 ±0-5 mm in
length. These animals were of such size that they would not and did not fission
during the time course of the experiment. Fully grown fissioning animals were
8-10 mm in length and were chosen for homogenization.
3. Assay pro cedure
The 'assay' planarians were decapitated immediately behind the auricles
(see Fig. 1). The remaining tails were placed in shallow Petri dishes or vials to
which were added the various fractions to be tested (2-5 ml/animal). The animals
were then allowed to regenerate for 9 days (except where noted) before sacrificing
with 2 % HNO 3 . They were then fixed, embedded, serially sectioned (the first
Characterization of differentiator substance in Dugesia
161
d.l.
ep.
Fig. 1. Nervous System of Dugesia etrusca. (A) Dorsal view of head region: a.c,
anterior commissure; e, pigmented eye spot; b., 'brain' (defined by medial fusion
of right and left cerebral ganglia); d.l., decapitation level; m.n.c, main nerve cords;
p.c, posterior commissure. (B) Cross-sectional view through eyes and brain: b, brain;
ep., epithelial layer; o.n., optic nerve. NOTE: For the sake of clarity, all nerve fibers
radiating from the cerebral ganglia (or main nerve cords) and any extracerebral
ganglia have been omitted.
40 transverse sections from the anterior end are used), and stained for nervous
tissue using the method of Betchaku (1960). Regenerated brain volumes were
measured using a projecting microscope (Bioscope, Inc.) and a compensating
polar planimeter (Keuffel & Esser Co.).
4. Centrifugation studies
A crude homogenate was prepared by homogenizing intact planarians in
a Potter-Elvehjem type tissue grinder in sterile distilled water at 0-4 °C. This
homogenate was then centrifuged at 10000 g for 20 min. The pellet was resuspended in sterile filtered water. A portion of the supernatant was then centrifuged
162
V. E. STEELE AND C. S. LANGE
at 32000 g for 20 min. Aliquots from each fraction in addition to similar aliquots of sterile filtered water for controls, were placed separately in vials. One
decapitated animal (5 mm original length) was then added to each vial. The
animals were then assayed as described in section 3.
5. Extract concentration vs. inhibitory activity
Varying proportions of the 32000 g pellet (described in section 4) were diluted
with sterile filtered water. The protein concentration of each fraction was then
determined using the method of Waddell (1956). Bacterial growth in this and succeeding experiments was inhibited by the addition of lO/^g/ml Kanamycin (Bristol
Laboratories) to each vial (Lange, 1968). Four animals were assayed per fraction.
6. Continuousflowelectrophoresis
A 10000 g supernatant extract was first filtered through a 8-0 jum pore
Millipore filter to remove remaining cells, then ultrafiltered using a Dow hollow
fiber beaker b/HFU-1 (retains molecules > 30000 daltons). This ^ 30000 M.W.
retenate extract was then electrophorezed using a Karler-Misco electrophoresis
apparatus. The background electrolyte buffer was 0-1 M phosphate buffer
(pH 6-8). Perpendicular to the direction of the buffer flow was a 2 mA-280 V
direct current field. Fraction tubes and electrode reservoirs were changed daily
during the 72 h period of operation and stored at 2 °C. The fractions and
reservoirs were combined into five groups: + reservoir, fractions 1-9 (positive
side), fractions 10-13 (ca. neutral), fractions 14-22 (negative side), -reservoir.
The fractions were concentrated to 30 ml (at the same time dialyzed against
sterilized planarian saline to remove the phosphate buffer) again using the Dow
hollow fiber b/HFU-1. These five groups plus two control groups were then
assayed for inhibitory activity.
7. Molecular weight determination
Since previous gel filtration studies (Steele & Lange, unpublished) have shown
that the active substance was eluted at the void volume using Sephadex G50 and
G75, the molecular weight must be over 80000 daltons. Therefore a 1-6 x 100
column, filled with BioGel A-l-5 m gel filtration media, was equilibrated at room
temperature with sterilized planarian saline. The void volume, Vo, was measured
using Blue Dextran 2000. The elution volumes, Ve, of four molecular weight
markers (Calbiochem) were measured (Fig. 4). The partition coefficients, K&v,
between the liquid phase and the gel phase were determined using the formula:
where Vt is the total volume of gel bed. An extract containing only those
molecules ^ 30000 M.W. was prepared as described in section 6 and applied to
the column. The fractions collected were combined into seven groups and assayed
for inhibitory activity.
Characterization of differentiator substance in Dugesia
163
8. Enzyme digestion studies
All enzymes used were tested for activity using planarian extracts, and the
amounts used were in great excess for each experiment.
8.1. Digestion with pronase and RNase. Pronase, B grade (Calbiochem no.
537088) was added to a resuspended 32000 g pellet containing a known amount
of protein. After 12 h at room temperature the digestion mixture was extracted
with cold ethanol (Green & Hughes, 1955) and dialyzed. The original extract, the
digested extract, and a sterilized planarian saline control were then assayed for
inhibitory activity.
Ribonuclease A (Sigma Chem. Co. no. R4875) was added to a resuspended
32000 g pellet and incubated for 12 h at room temperature. The original extract,
the RNase-treated extract, RNase alone, and sterilized planarian saline control
were then assayed for inhibitory activity.
8.2. Digestion with DNase and lipase. DNase I (Worthington Biochem. Corp.
no. 2007) was added to a ^ 30000 M.W. extract (section 6) in 10~3 M MgCl2
and incubated 24 h at 37 °C, along with extract alone, DNase alone, and
sterilized planarian saline alone controls. After the incubation, the digested
extract and the controls were ultrafiltered and dialyzed to remove the MgCl2.
These extracts were then tested for inhibitory activity.
A similar procedure was performed for Lipase (Sigma Chem. Co. no. L2253).
9. Isoelectric focusing
A ^ 30000 M.W. extract (section 6) was dialyzed against a solution of 1%
glycine for 24 h, then added to an ampholine solution of pH 3-5-10 (LKB, Inc.).
This mixture was then added to a linear sucrose gradient in a 440 ml electrofocusing column (LKB 8102). The final ampholine concentration was 1 %. After
electrofocusing had been achieved, the column was fractionated and the volume,
pH, and absorbance (280 nm) of each fraction were measured. The 40 fractions
were then combined into 12 groups according to pH, concentrated, and placed
separately on a BioGel P-60 column (0-7 x 60 cm) and eluted with pH 7-2
phosphate buffer (1 M-NaCl). The extract proteins in each group were eluted at
the exclusion volume while the ampholytes were eluted separately at various
later times. After concentration and dialysis against sterilized planarian saline,
the extract proteins were tested for inhibitory activity.
RESULTS
1. Centrifugation studies
All animals placed in the resuspended 10000 g pellet (10 kgP) died after 3-4
days. However, an averge reduction in regenerated brain volume of about 42%
was observed for animals placed in the 10000 g supernatant (10 kgS) (see Table 1).
A value somewhat higher, although not significantly different (P > 0-05), was
observed for the resuspended 32000 g pellet (32 kgP). The slight difference in
164
V. E. STEELE AND C. S. LANGE
Extract concentration (//g/ml,protein)
Fig. 2. Regenerated brain volume vs. extract concentration. Protein concentration
was measured spectrophotometrically (Waddell, 1956). The dashed line represents
the linear regression line using the mean regenerated brain volume at each extract
concentration.
Table 1. Extract centrifugation fractions with resultant
regenerated brain volumes
Fraction
lOkgP
lOkgS
32kgP
32kgS
Control
No.
No. Regenerated brain
animals animals
volume ± S.E.
treated measured
(x 105 /*m3)
4
4
4
4
4
0
4
4
4
4
—
1-90 ±013
2051008
2-76 ±008
3-27 ±0-16
activities is possibly due to diffusion of the inhibitory substance into the
supernatant (32 kgS), since this fraction is slightly lower than the control value.
2. Effects of extract concentration on regenerated brain volume
As the extract concentration increased from 0 to approximately 12-5 /*g/ml
protein, the volume of the regenerated brain tissue decreased significantly by
40% (Fig. 2). At the highest protein concentration used, an 82-6 % decrease in
Characterization of differentiator substance in Dugesia
165
O
rh
-1 6 -
rh
©
x
-5-i
rh
4
2 -
b/HFU-l
extract
+
Res.
Fractions
Fractions
Fractions
10-13
14-22
(mod. + ) (weak+ or — )(mod.—)
1-9
Control
Res.
Fig. 3. Regenerated brain volume resulting from combined fractions of an electrophoretic separation of the extract. b/HFU-l Extract, the > 30000 M.W. extract;
retained by b/HFU-l Dow Hollow Fiber Ultrafiltrator; +Res, fraction collected
from the anodal reservoir; Fractions 1-9, fractions containing substances which
migrated with moderate speed toward the anode; Fractions 10-13, fractions which
showed little or no migration in the electric field; Fractions 14-22, fractions
containing substances which migrated with moderate speed towards the cathode;
— Res, fraction collected from the cathodal reservoir; Control, contained no fractions, sterilized planarian saline only.
brain volume was observed due to the addition of the extract. A regression line
fitted to these data yields the equation
Y = (2-85 ±0-185) x 10 5 -(4-32 ±0-71) x 103X,
where Y is the regenerated brain volume in /*m3 and X is the extract protein
concentration in /*g/ml (coefficient of determination = 0-902). This equation
predicts that an extract concentration (prepared as described) of approximately
66 /Ag/ml protein is needed to completely inhibit all brain regeneration. This
concentration would correspond to approximately 12 animals homogenized per
milliliter extract produced. This approaches well within an order of magnitude,
the in vivo concentration. However, we should not assume a homogeneous
in vivo concentration exists.
3. Continuous paper electrophoretic separation of the inhibitory substance
Electrophoretic separation of the ^ 30000 M.W. extract into five general
classes of electrophoretic mobilities resulted in no significant loss of brain
inhibitory activity. Addition of these five groups of substances to regenerating
assay animals resulted in the mean regenerated brain volumes shown in Fig. 3.
The substances which migrated to the anodal reservoir ( + Res.) inhibited brain
formation nearly as efficiently as the unseparated extract (b/HFU-l Extract).
The remaining four groups were not significantly different from control.
166
V. E. STEELE AND C. S. LANGE
« 4
+1
X3
•Si
105
106
Molecular weight (daltons)
107
Fig. 4. Apparent molecular weight measurement of the inhibitory substance(s).
Top: R.B.V. (Regenerated Brain Volume) is plotted against the log molecular weight
(daltons) scale of the bottom graph. Each of the eight values shown represents the
mean (±one standard error) regenerated brain volume. The control value was
determined from animals which regenerated in sterile planarian saline only.
Bottom: KAV (partition coefficient, see Methods, §7) is plotted against the logarithm
of molecular weight in this column calibration curve. The molecular weights of the
five standards are those stated by Calbiochem and/or Parmacia, Inc. (BSA = Bovine
Serum Albumin). Three measurements of the partition coefficient were performed
for each standard, with the exception of apoferritin where six measurements were
made. The standard deviation associated with each point was no larger than the
diameter of the illustrated point.
4. Molecular weight determination
Substances which demonstrated significant inhibitory activity were found in
elution volumes similar to those of globular proteins whose molecular weight
ranged from 2 x 105 to 4 x 105 (as determined by the calibration curve, Fig. 4).
The regenerated brain volumes of five fractions which should contain higher or
lower molecular weight substances did not differ significantly from the control
volume. The histogram (Fig. 4, top) also shows that two fractions exhibited
Characterization of differentiator substance in Dugesia
167
Table 2. Extract enzyme digestions and resultant regenerated
brain volumes
oo oo oo
A. Pronase digestion*
Control
Untreated extract
Pronased extract
B. RNase I digestion
Control
Untreated extract
RNased extract
RNase onlv
C. DNase 1 digestion
Control!
Untreated extract!
DNased extract
DNase only
D. Lipase digestion
Control!
Untreated extract!
Lipased extract
Lipase only
8
4
7
2-27 ±0061
1-84 ±0032
215 ±0041
8
8
8
8
8
5
8
8
3-318 ±0-143
1-970 ±0-072
2098 ± 0-224
3-650 ±0-237
OO OO 00 OO
Fraction
Regenerated brain
No.
No.
volume + S.E.
animals animals
(xlO5/*m3)
treated measured
8
5
8
8
3-481 ±0-111
2025 ± 0064
2-251 ±0-077
3-223 ±0-144
8
8
8
8
8
5
3
8
3-481 ±0-111
2025 ± 0064
2038 ± 0064
3-551 ±0136
* A 7-day regeneration period was allowed for the pronase experiment. RNase, DNase,
and lipase digestion fractions were allowed 9 days.
t The DNase digestion and lipase digestion experiments were performed simultaneously
with one control group of animals and one untreated extract group of animals.
significant inhibitory activity. Therefore the apparent molecular weight is
estimated at slightly greater than 300000 daltons. It should be noted that this
apparent molecular weight may be an aggregate of two or more active molecules,
however, such aggregation, had it occurred, had no observable inactivating
effect.
5. Enzyme digestions
5.1. Pronase and RNase. As seen in Table 2, the addition of pronase to the
extract significantly destroyed the inhibitory activity (P < 0-01). The regenerated
brain volumes resulting from decapitated animals exposed to the pronasetreated extract did not differ significantly from decapitated animals exposed to
sterilized planarian saline. Although the 'extract' brain volume is similar to that
of the RNase study the 'control' volume is noticeably smaller. This is due to the
7-day regeneration period used instead of a 9-day period. Comparison of the
control volumes demonstrates a 46-2 % increase in mean brain size resulting from
the additional 2-day regeneration period. However, the 'extract only' controls
168
V. E. STEELE AND C. S. LANGE
4r-
f*i-5-
l-5-i
X
X
rol
+1 3 --
c
o
U
i
200
i
300
Elution volume (ml)
i
1
1
400
Fig. 5. Extract electrofocusing and effects of fractions on regenerated brain volume.
Top: results of assaying fractions for brain inhibitory activity. The fractions at
either extreme actually contained those substances which electrofocused at < 413
and ^ 10-37 respectively. Control group contained sterile planarian saline only.
Bottom: The pH and absorbance of fractions as eluted from the isoelectric focusing
column containing ampholytes, pH range 3-5-100. • , pH; O, Absorbance at
280 nm (A28o); pi, Isoelectric points of absorbance peaks.
did not significantly increase in size during this period. This evidence argues
strongly against any form of cytotoxic delay resulting from exposure to the
extract.
Addition of RNase to the regeneration media did not significantly alter the
resultant regenerated brain volume when compared to control values. Incubation of the extract with RNase for 12 h did not produce significantly different
results when compared to the untreated extract. However, the regenerated brain
volumes resulting from the extract and RNase-treated extract are significantly
Characterization of differentiator substance in Dugesia
169
different from both the RNase only or control values (P < 0-01) for each of the
four cases.
5.2. DNase and lipase digestion. The extract incubated with DNase did not
show a significantly different amount of inhibitory activity from that of the
untreated extract (Table 2). Addition of only the DNase to the decapitated
animals did not produce an effect significantly different from control.
Also, the inhibitory activity of the lipase-treated extract was not significantly
different from that of the untreated extract. Lipase alone had no inhibitory
effect nor did it differ from the mean control R.B.V.
6. Jsoelectric focusing of the extract
A nearly linear pH gradient (r2 = 0-984) was established during the 48-h
electrofocusing period for the range of the ampholine mixture used (i.e. pH
3-5-10). Absorbance measurement (280 nm) of each eluted fraction revealed five
regions which could be identified as containing substances at their isoelectric
points. These absorbance peaks (pi's) were at pH 4-3, 4-9, 5-6, 6-3, and 7-8.
Before fractionation, visual observation of the electrofocusing column's contents
revealed eight bands or zones. Of the 11 groups tested, the group containing
substances whose isoelectric points ranged from pH 4-75 to 5-38 was the only
group exhibiting brain inhibitory activity resulting in significantly lower R.B.V.'s
(Fig. 5). Most of the substances in this group would correspond to those which
exhibited a pi of 4-90 as determined by the absorbance peak. The remaining ten
groups were not significantly different from control.
DISCUSSION
Lender (1955, 1956, 1960) has previously shown that a diffusible brain
inhibitory substance exists in several planarian species. This was demonstrated
by the excision of the head ganglia only, the addition of head or tail extracts
every other day, and the measurement of regenerated brain length after 9 days.
The experiments we present in this paper show that one can excise the entire
head and still observe a significant amount of brain inhibitory activity in
homogenates or extracts. Our use of decapitation demonstrates that existing head
tissues are not necessary for the inhibitory action of the substance. This method
also has the advantage of being certain that : (a) no former brain tissue or head
ganglia exist prior to the extract treatment, (b) repeatable tissue volumes are
excised, and (c) no effects from anesthesia are present. In Lender's experiments
the head homogenates were replenished on the 3rd, 5th, and 7th day which, in
some cases, completely inhibited brain regeneration. Although the differences in
brain size, we present in our data, are not as large in some cases as Lender's, we
have shown that a single exposure to our extracts containing the brain inhibitor
immediately after decapitation did produce significant differences. It is also
highly probable that once the wound surface has healed (see Morita & Best,
170
V. E. STEELE AND C. S. LANGE
1974), the passage of any such inhibiting substances into the regenerating
blastema is severely restricted. Since the entire planarian body contains the
brain inhibitory substance, the whole animal was homogenized. While Lender
measured brain length, we felt that the measurement of brain volume would
reflect more accurately any differences in inhibitory activities present in our
various extracts. Preliminary tests using our assay system in Dugesia etrusca
showed that when differences in mean brain length were not significant, differences in mean brain volume were highly significant (P < 0-01). All animals in
our tests regenerated brains, although a number of animals, especially in the
extract-treated groups, died before the 9th day of regeneration and were
discarded. Eyespots, or pigmented cupules characteristic of this species, were
regenerated in all instances. We did not attempt to further characterize or possibly
separate the eye inducer substance(s) from the brain inhibitor substance in this
study. Excision of the brain without including the eyes would be technically very
difficult in D. etrusca.
Since the inhibitory substance remains in the 10000 g supernatant but will
pellet at 32000 g, it is possible to assume that in homogenates, as prepared, it is
not bound to cell membrane fragments or large organelles, but could be
associated with something approximately the size of a ribosome. Since some of
the activity still remains in the 32000 g supernatant, this association may be one
in equilibrium with other bound or unbound states. As extract concentration
increased, the amount of protein present increased linearly, and the resultant
regenerated brain volume decreased. Perhaps each potential brain tissue neoblast
binds or removes from the intercellular fluid pool one or more inhibitory
substance molecule(s). The data from these experiments cannot resolve whether
the action of the inhibitory substance is a persistent or a temporary effect.
Perhaps the fact that Lender (1956) found some animals with no brain tissue
when he replenished the extract every other day, and we, with our single treatment,
did not, could support the idea of a temporary mode of inhibition.
The initial gel filtration studies indicated that (1) the inhibitory substance
could be partially purified by gel filtration, and (2) the elution at the void volume
of Sephadex G75 meant that the inhibitory substance, free or in its bound (but
still active) state, was a comparatively large molecule (^ 80000 daltons) (Steele
& Lange, unpublished results).
In the electrophoretically separated extract the inhibitory substance migrated
to the anodal reservoir and quantitatively effected a diminished regenerated brain
volume comparable to that of the unseparated extract (b/HFU-1). We can
therefore state with a high degree of certainty that the inhibitory substance is
a negatively charged molecule. It should be noted that substances which migrated
to the cathodal reservoir were promotive with respect to brain regeneration at
the 90 % confidence level. It follows that this promoting substance(s) would also
be ^ 30000 daltons in size.
An apparently opposite situation exists in Hydra littoralis. Lenique & Lund-
Characterization of differentiator substance in Dugesia
171
blad (1966a, b), using agar electrophoresis of homogenized stems, reported that
electronegative proteins were growth promoters and electropositive proteins
were growth inhibitors. Rose (1966) reported similar results in Tubularia.
Evidence in higher animals, such as the salamander, the adult frog, and the
rodent (Becker, 1961; Becker & Spadaro, 1972), suggest that some form of
electrical control over growth and differentiation does exist.
The apparent molecular weight of the inhibitory substance(s), as determined
by the calibrated agarose gel column, confirmed the earlier results using
Sephadex gels. The apparently high molecular weight (2-4 x 105) observed is that
of the substance in its active undenatured state. It is possible that while in
sterilized planarian saline, ionic conditions may be such as to facilitate aggregation.
If aggregates were present, the aggregation did not decrease the inhibitory
powers of the substance. It should be noted that slightly larger R.B.V.'s (higher
at the 90 % confidence level) were observed for substances in the molecular weight
range of 4-8 x 105 daltons. These promoting substances are possibly identical to
the electropositive promoting substances observed in the extract electrophoretic
separation.
It is suggested by the enzyme digestion studies that the brain inhibitory
substance is, at least in part, protein or polypeptide in nature. At least the
site(s) necessary for its action is labile to digestion by pronase. Since this
substance's activity is not destroyed by DNase or RNase, it is probably not
a nucleoprotein (or any associated nucleic acids are not necessary for its activity
as assayed). The inability of pancreatic lipase to alter its activity reduces the
probability that it is some form of lipoprotein complex. The possibility still
remains that the a and a' ester linkages of a triglyceride portion are obscured by
a protein complex and are not susceptible to pancreatic lipase attack. Also
remaining are the possibilities that theinhibitor substance may be a glycoprotein,
metalloprotein, or a chromoprotein. Since many of the active extracts used in
these experiments were colorless, the possibility of having a pigment prosthetic
group (chromoprotein) has a low probability. Since many growth regulating
hormones, such as thyroglobulin or FSH in higher animals, are glycoprotein in
nature, this class of conjugated proteins remains a strong possibility. Experiments
are in progress to explore this likelihood.
The isoelectric point of the inhibitory substance reaffirms the results of the
electrophoretic separation. The low pi value indicated suggests that a large
number of negative charges on the molecule must be balanced by a high
hydrogen ion density before the net charge is zero. Experiments in progress, with
a narrower pH range of ampholytes, should give a more accurate determination
of the isoelectric point.
The characterization, as presented, does not attempt to make any resolution
between the eye inducer substance and brain inhibitor substance, found to
exhibit many common properties by Lender (1955,1956, 1960). A highly concentrated extract, purified by centrifugation, ultrafiltration, and electrophoresis,
172
V. E. STEELE AND C. S. LANGE
should completely inhibit brain formation. If eye formation is then delayed
(compared to control) or absent, a good probability exists that both activities
are not possessed by the same substance.
V. E. Steele acknowledges the support of the Rochester A.E.C. Laboratory Graduate
Participant Program and C. S. Lange the support of an NIH Research Career Development
Award. This paper is based on work performed under contract with the U.S. Energy Research and Development Administration in the Department of Experimental Radiology
(Contract No. AT(30-l)-4284) and University of Rochester Biomedical and Environmental
Research Project and has been assigned Report No. UR-3490-766.
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BECKER,
(Received 4 July 1975, revised 6 August 1976)