ABSTRAC
The noxious yellow slime secreted by the marine snail
Calliostoma canaliculatum contains mostly small
constituents (less than 12Kd) that are heat stable and
partially hydrophobic. There are at least two bioactive
fractions in the slime. One has a molecular weight of
approximately 700 - 800 daltons, and the other contains the
characteristic yellow color.
INTRODUCTION
The marine snail Calliostoma canaliculatum secretes a
vellow slime as a defense against a sea star predator,
Pycnapodia helianthoides. In response to the noxious slime
P. helianthoides retracts the arm exposed to the slime and
nay move away. Behavioral observations on this interaction
(McDevitt, personal communication) indicate that the slime
release is the final method of defense used by the C.
canaliculatum, after the snail has attempted to flee and
work itself free from the sea star. This indicates that
the slime may be an energy expensive compound to make. The
snail does not need dietary precursors to produce the
slime, which seems to be synthesized di novo and stored for
later secretion(McDevitt, personal communication).
The purpose of this study was to characterize this slime
biochemically and fractionate it, isolating those fractions
with bioactivity. P. helianthoides, was used to test
bioactivity, because it is the natural recipient of the
sline.
3 -
MATERIALS & METHODS
Bioassays
Bioassays were conducted on the sea star Pycnopodia
helianthiodes. The sea star was placed in a fingerbowl
with 1-2 inches of sea water, and 50 ul of the test
substance was applied to the tip of a small arm (1.5-3
inches). There was a minimum recovery period of 3 minutes
between trials. The reaction was classified on a scale of
O - 3, with O equal to no response, 1 equal to weak
response (closing of arm around tube feet and slight
retraction of arm), 2 equal to a moderate response
(retraction and curling up of arm), and 3 equal to a strong
response (strong curling or retraction of arm - often
coupled with running away from stimulus).
Collection
C. Canaliculatum were collected on the kelp off Cabrillo
Point at Hopkins Marine Station (Pacific Grove,
California). The snails were induced to secrete the yellow
slime in air by placing a tube foot of the P. helianthodes
— 4
on the tentacles of the snails so that they retracted with
the tube foot when prodded with the pipet. This caused the
secretion of sea water, and in most cases the yellow
slime. This crude yellow slime/sea water mixture was
collected and refrigerated in flasks.
Ultraviolet Spectra/isible spectre
Ultraviolet and visible spectra were taken on a Hewlett
Packard 8452A array diode spectrophotometer connected to an
IBM PC. The spectra were then loaded into the Lotus
Worksheet package for analysis and graphing.
Solubilities
The crude yellow slime was centrifuged at 7500g for 10
minutes. The pellet was mixed with a pipet in the test
solvent. Distilled water, distilled water with O.iM HCI,
and distilled water with O.1M NaoH, ethanol, acetone,
ether, and benzene were the test solvents. The ethanol
soluable constituents were tested for bioactivity by freeze
drying the ethanol away and resuspending the dry solids in
filtered sea water (to the original volume of the slime).
Heat Stability
Epindorph tubes containing the crude yellow slime were put
in a boiling water bath (98 C). At 1, 2, 5, and 10 minutes
tubes were remove and put on ice. Bioassays of serial
dilutions(1, 1/5, 1/25, 1/125) were taken of the tubes
boiled for 5 and 10 minutes and of the original slime
control.
Washing by Centrifugation
Each wash consisted of centrifuging 2.0 ml of the crude
vellow slime at 150,000g for 1 hour (4 C) in a Beckmann
Ultracentrifuge, pipeting off the supernatant, and
resuspending the pellet in 2.0 ml of filtered sea water.
Two series of four washes were done, one started with the
crude slime sonicated for 10 brief (approxinately 1 sec
each) pulses, and one started with unsonicated crude slime
as a control. The four supernatants and the final pellet
(resuspended in 2.0 ml of filtered sea water) were tested
for bioactivity and analyzed were analyzed by the
UV/visible light spectroscopy.
Coomassie Blue Stainfor Protein
-6
A 2.5ul sample was applied to a Whatman No. i filter paper,
dried, stained with Coomassie Blue R-250 dissolved in 252
isopropanol, 10acetic acid and 65 distilled water for 15
minutes. The stained paper was washed in distilled water
for 10 minutes, and then dried. (Esen, 1978)
Dialysis
The crude slime was centrifuged at 7,500g for 10 minutes
and 1.0 ml of the supernatant (the rest of the sup. Was
kept as a control) was loaded into a dialysis bag (with an
approximate cutoff of 12Kd), which was stirred in 100Oml of
filtered sea water. After 6 hours the bath was poured out
and replaced. This was left overnight and the retentate in
the bag and the control were compared on the UV/visible
spectra, in bioactivity, and in protein concentration.
Gel Column Chromatography
A 40ml Sephadex G-50 column was used and 30 drop fractions
(about 1.7m1 each) were collected. For one experiment
filtered sea water was used as the eluent. The crude
vellow slime was centrifuged at 7500g for 10 minutes and
40Oul of the supernatant were loaded onto the column.
Fractions were then analyzed by UV/visible spectroscopy,
tested for bioactivity, and protein concentration. In a
second experiment distilled water was used as the eluent,
and 2.Oml of the supernatant was loaded onto the column.
Spectrophotometer readings were taken of the fractions at
234nm. The regions of peak absorbance were pooled (
fraction numbers 20-27, 28-40, 46-54) and lyophylized down
to solids and then resuspended in 2.0 ml of filtered sea
water. These concentrated fractions were analyzed by
UV/visible spectroscopy, and tested for bioactivity. The
column was calibrated using dinitrophenol (M.W. 185),
aspartane (M.W. 294), bromophenylblue (M.W. 670), vitamin
Bi2(M.W. 1250), aprotinin(M.W. 6,500), cytochrome C(M.W.
12.400), carbonic anhydrase (M.W. 29,000), and blue
dextran(M.W. 2,000,000) (see Figure #15). A 40Oul sample
of each standard was loaded on the column, and the region
of peak UV/visible spectroscopic absorbance was the elution
place. (see Cooper,1977 for explanation of techniques)
Infrared Spectra
Two sets of concentrated fractions (20-27) and (28-40) from
above, were lyophylized again, dissolved in methanol and
filtered to remove salts. The methanol was evaporated off
and the residual solid was made into a Nujol mull to take
the infrared spectrum on a Perkin-Elmer Spectrophotomer
with salt plates.
Gel Electrophoresis
Two acrylamide gels were run, a 10-182 gradient gel, and a
123 gel (see Cooper, 1977 for techniques). The gels were
calibrated with both high and low molecular weight
standards (see Figures #11 and #12). On both gels the
experimental lanes consisted of crude supernatant (7,500g
for 10 minutes), and fractions 23 and 47 from the gel
colunn eluted with sea water. The gels were stained with
the silver chloride technique. (Morrissey, 1981)
RESULT!
Solubilities
The yellow pellet was very soluble in ethanol, soluble in
distilled water, slightly soluble in acetone, and insoluble
in ether and benzene. Distilled water with Naoh
precipitated the pellet, and HCl brought it back into
solution. The portion which was dissolved in ethanol
produced a strong response from the sea star when the
residue was transferred into filtered sea water. It had a
spectrum almost identical to the supernatant from the
original centrifuged crude slime (see Figure l and control
on Figure #6). The ph of the slime (as it was collected
crudely in its sea water mixture) was 7.4.
Heat Stability
The tubes which were boiled for 5 and 10 minutes showed no
significant decrease in bioactivity as compared to the
untreated control over the range of dilutions tested. (see
Figure #2)
Washing b Centrifugation
- 10
There was bioactivity in all the supernatants, except one,
as well as in the final pellets (Figure #3). There was no
significant difference in the sonicated versus unsonicated
slimes. The biological activity did not decrease
significantly with progressive washes, although the
absorbances in the UV/visible spectra were lower with
progressive washes (Figures #4 and #5).
Dialysis
The retentate showed no bioactivity, while the undialyzed
control produced a strong response from the sea star. The
UV/visible spectrum of the retentate showed that it had
lost much of its absorbance. (Figure #6)
Gel Column Chromatography
The Sephadex G-50 column, eluted with filtered sea water,
separated the slime into two major components with peaks
centered at fractions 23 and 47 (as determined by
UV/visible spectra - see Figure #10). The bioactivity of
these fractions were higher than that of other fractions
(Figure #9). Fraction 47 stained stronger with coomassie
blue than did fraction 23.
- 11
Elution with distilled water separated the slime into 3
components centered at fractions 23, 31, and 50. When
concentrated, fractions 20-27 produced moderate to strong
reactions, fractions 28-40 produced strong reactions, and
fractions 46-54 produced moderate to strong reactions. The
original supernatant produced strong reactions.
Gel Electrophoresis
In both gels run, the lanes loaded with fractions 23 and 47
showed no protein bands, and the lanes with the yellow
sline supernatant showed 2 major protein bands having
molecular weights of approximately 50,000d, and 18,000d
(Figures #11 and #12)
UV and Visible Spectra
Peak absorbances of crude slime were a double peak at
30Onm, and a shoulder at 380-400nm (see control, Figure
46). Peak absorbances for fraction 23 were at 234nm, 260nm,
318nm (Figure #7), and peak absorbances for fraction 47
were at 234nm, 308nm, and 36Onm (Figure #8). The retentate
from the dialysis had a sharp peak at 220-230nm and very
small shoulders around 30Onm and 400nm (Figure #6).
- 12 -
Infrared Spectra
Fractions (20-27) (Figure #14) and (28-40) (Figure #15) seem
to have nearly identical infrared spectral profiles. They
both show a broad absorbance centered at 3350 cm-1, a
narrower absorbance at 1660 cm-1, and a small peak at 1150
cm-1. Other peaks at 2900 cm-1, 1460 cm-1, 1380 cm-1, 1020
cm-1, and 720 cm-1 are all artifacts due to Nujol.
DISCUSSION
Much of the data indicate that the slime is amphiphilic.
The initial collection showed that the slime was somewhat
inniscible with the sea water secreted with it. In the
solubility tests, the fact that the slime dissolved more
easily in ethanol (including an active component) than in
distilled water indicates that the slime is slightly
hydrophobic. But it is not entirely hydrophobic, or it
would have been more soluble in acetone, ether, and
benzene.
Washing the pellet (the progressive washes experiment)
indicated that the bioactive component was in both the
pellet and in solution. Sonication made no difference with
this data, so the slime was not slowly being released from
inside a nembrane-bound compartment. More likely the slime
was saturating the water, and the excess was left in the
pellet. This again supports the possibility that the
compound is partially hydrophobic.
Also, a component of the slime(fract 47) seemed to stick
to the gel chromatography column, because its elution
volune exceeded the column volume. This indicates that
this portion has some hydrophobic characteristics.
Also relevant to the amphiphilic character of the sline,
are observations made on detergent-like properties of it.
The slime seemed to destroy sea urchin egg membranes. No
quantitative analysis of this was performed.
Behavioral observations show that this hydrophobicity may
make sense. The snail has been observed to wrap itself in
its own slime as it secretes it (McDevitt, personal
communication). The slime could better serve its purpose
of deterring the P. helianthoides, because every time the
sea star touched the shell it would sense the slime. The
slime also would be harder to wash away.
The slime is fairly pH neutral (7.4 in sea water). The
fact that it is more soluable in acid than in base
indicates that it might contain a basic functional group
(amino?).
The bioactive molecules are not large proteins. They are
dialyzable ( therefore « 12Kd M.W.), heat stable, and do
not appear on an acrylamide gel (possibly running through,
or not staining silver chloride), and are soluble in
precipitate in
proteins
usually
ethanol (large
ethanol) (Cooper, 1977).
All of the bioactive components are smaller than 12,000
daltons as evidenced by the dialysis experiments. Fraction
23 has a molecular weight of 700 - 800d, calculated from
its elution volume on the gel column. (763.53d calculated
- 15.
from linear regression of the calibration) (see Figure
+15). Fraction 47 is beyond the linear separating portion
of the column, so no molecular weight can be assigned to
this fraction.
The gel electrophoresis data shows that both fractions 23
and 47(whose columns show no bands) contain no proteins or
peptides with molecular weights of greater than 2,500
daltons (the lowest molecular weight standard band on the
gel).
Whether the active components are peptides or other small
molecules was not determined. There was conflicting data.
There appear to be many small peptides in the slime; the
crude supernatant of the slime strongly stained with
coomassie blue while after dialysis the retentate stained
weakly if at all. The active fractions 23 and 47 stained
vith coomassie blue more strongly than do the other eluted
fractions. So the fractions either contain peptide or
other compounds which stain coomassie blue.
None of the spectra indicate that the bioactive fractions
are peptides. In the UV/visible spectra, both active
fractions have a peak near 230nm but are lacking peaks at
280nm (Figures #7 and #8). Proteins and peptides
characteristically absorb at both peaks (Pavia, 1979). The
peaks at 260nm, 308nm and 318nm are a mystery. The
16
shoulder at 360-400 could be the yellow color absorbance.
The infrared spectra of both fractions do have a carbonyl
peak at 1660 cm-1, but are lacking both the characteristic
carboxylic acid hydroxyl absorbance and the characteristic
amino group absorbance. The peak at 3350 cm-1 shows an
alcohol-like hydroxyl absorbance (Pavia, 1979).
There are data indicating that the slime components block
both sodium and potassium channels at 1/100 dilution of the
crude slime, and that the fractions from the second gel
chromatography column (concentrated fractions 20-27, and
46-54) have separate, different effects. Fractions 20-27
seem to block the sodium current, and fractions 46-54 seem
to block a potassium current of Lolligo opalesceas
chemoreceptor cells (Dr. William Gilly, personal contact).
The slime also seems to block the potassium current in
neurons from the Doriopsila albopunctata (Dr. Stuart
Thompson, personal communication). It is possible that the
compound shares structural characteristics with
tetrodotoxin, or saxotoxin.
Summary of Characteristics
The characteristics of the yellow slime shown in this
paper are:
17
1. There are at least two active components in the slime
as separated by the Sephadex G-50 column.
2. The slime is amphiphilic, showing both hydrophobic and
hydorphilic traits.
3. The active components of the slime are a relatively
small particles. All are less than 12,000d. One bioactive
component has a molecular weight of 700-800d.
4. There is no conclusive data to show whether the active
components are peptides or other other small molecules.
Further Experiments
Further purification of the slime should be attempted.
Fractions from the Sephadex G-50 column could be run on an
ion exchange column to separate the slime according to
charge if it has any. The bioassay should be made more
quantitative. Possibilities include developing
neurological, or sea urchin egg lysis assay. It also might
be possible improve the P. helianthoides assay by attaching
force transducer to an arm of the sea star to measure the
force of arm retraction. Infrared specs should be taken of
more highly purified fractions. The HPLC could be run to
analyze amino acids of acid hydrolyzates, and to analyze
the similarities between the slime and TTX and PSP.
18
References:
Cooper, Terrance G., The Tools of Biochemistry, (John
Wiley and Sons, 1977).
Esen, A. 1978. A simple method for quantitative,
semi-quantitative, and qualitative assay of protein. Anal.
Biochem. 89: 264-273.
McDevitt, Christine, The dynamics of the escape response
of gastropod Calliostoma canaliculatum to seastar
Pycnapodia helianthoides., Spring Class 1988, Hopkins
Marine Station.
Morrissey, J.H. 1981, Silver stain for proteins in
polyacrylanide gels: a modified procedure with enhanced
2: 307-310.
uniform sensitivity. Anal. Biochem. 1!
Pavia, Donald L., Lampman, Gary M., Kriz, George S. Jr.,
Introduction to Spectroscopy: A Guide for Students of
Biochemistry, (Saunders College Publishing, 1979).
EIGURE LEGENDS
Eigure l
Ultraviolet and visible spectrum (190 - 820nm), of slime
components soluble in ethanol.
Figure 12
Heat Stability of Slime.
Strength of reaction of P. helianthoides vs. dilution
strength of slime. A - control slime, B - boiled for 5
minutes, C - boiled for 10 minutes.
Eigure 13
Bioactivity of Successive Elutions from Slime Solids.
Strength of P. helianthoides reaction of four successive
washes and final pellets. A - centrifuged at 12,500g for 5
minutes, B - Initially sonicated, centrifuged at 150,000g
for 1 hour, C - centrifuged at 150,000g for 1 hour.
20
Eigure 4
UV/visible Spectra of Successive Elutions from
Sonicated Slime Solids.
Ultraviolet and visible spectrum (190 - 820nm), of
supernatants of sonicated slime. A - sup. from wash fl, B
- sup. from wash #2, C - sup. from wash #3, D - sup.
from wash #4.
Eigure 25
UV/visible Spectra of Successive Elutions from
Unsonicated Slime Solids.
Ultraviolet and visible spectrum (190 - 820nm), of
supernatants of control (unsonicated) slime. A - sup.
from wash #1, B - sup. from wash #2, C - sup. from wash
#3, D - sup. from wash #4.
Eigure
UV/visible spectra of Dialyzed Slime and Control Slime.
Ultraviolet and visible spectrum (190 - 820nm), of A -
- 21 -
control slime, and B - the retentate slime after dialysis.
Eigure t7
Ultraviolet and visible spectrum (190 - 820nm), of fraction
23, from Sephadex G-50 column chromatography, eluted with
filtered sea water.
Eigure 8
Ultraviolet and visible spectrum (190 - 820nm), of fraction
t47, from Sephadex G-50 column chromatography (eluted with
filtered sea water).
Eigure 9
Bioactivity of Chromatographic Fractionation.
Strength of P. helianthoides reaction vs. fraction number
from Sephadex G-50 column (eluted with filtered sea
water). (Notice Y axis range is none to moderate).
Eigure 19
Absorbance at 234nm vs. fraction number from Sephadex G-50
column eluted with filtered sea water.
Eigure ll
Analysis of Slime by SDS-polyacrylamide Gel
Electrophoresis: 10 - 182 acrylamide gradient.
Calibration of gradient gel (10 - 182) with standards i,
myocin n.w. 205 Kd. 2, B-galactosidase m.w. 116 Kd. 3,
Phosphoglutarate m.w. 97 Kd. 4, BSA m.w. 66 Kd. 5,
Ovalbumin m.w 45 Kd. 6, Carbonic anhydrase m.w. 29 Kd. 7,
Soybean Trypsin Inh. m.w. 20 Kd. Log of molecular weight
ys. migration distance divided by total migration of gel.
Line is a linear regression of the 7 standard points on the
graph.
Eigure #12
Analyais of Slime by SDS-polyacrylamide Gel
Electrophoresis: 122 acrylamide.
Calibration of 122 gel with standards 1, myocin m.w. 205
Kd. 2. B-galactosidase m.w. 116 Kd. 3, Phosphoglutarate
m.w. 97 Kd. 4, BSA m.w. 66 Kd. 5, Ovalbumin m.w 45 Kd. 6,
Carbonic anhydrase m.w. 29 Kd. 7, Soybean Trypsin Inh.
23 -
m.w. 20 Kd. Log of molecular weight vs. migration
distance divided by total migration of gel. Line is a
linear regression of the 5 middle points on graph.
Eigure #13
Analysis of Slime by Sephadex G-50 Gel Chromatography
Calibration of Sephadex G-50 gel column with standards
1,dinitrophenol (M.W. 185) 2, aspartame (M.W. 294) 3,
bromophenylblue(M.W. 670) 4, vitamin Bi2(M.W. 1250) 5,
aprotinin(M.W. 6,500) 6, cytochrome C(M.W. 12,400) 7,
carbonic anhydrase(M.W. 29,000) 8, blue dextran(M.W.
2.000,000). Log of molecular weight versus K - (elution
volune - void volume)/(total volume - void volume).
Eigure 1
Infrared spectrum of concentrated fractions (20 - 27) from
Sephadex G-50 column in taken in a Nujol mull with salt
plates.
Eigure #15
Infrared spectrum of consentrated fractions (28 - 40) from
24
plates
colu
take!
Nujol
salt


4



.
ACKNOWLEDGEMENTS
I want to thank my advisors, Dr. Robert Swezey and Dr.
Charles Baxter. At the start of this project I was a
newcomer to both biochemical techniques and the marine life
at Hopkins Marine Station. Rob taught me the biochemical
technigues and was always around the lab and willing to help
me. And Chuck was an invaluable source for information on
the species I studied as well as other species of the
Monterey Bay. I also want to thank Dennis Larochelle, a
graduate student, who helped me with the laboratory
technigues, and answered the infinite questions which I
asked. Lastly, I have to thank Tom Otis (one of the
Teaching Assistants for the course) who helped collect
hundreds of snails, and my "partner in slime" (as she coined
the term), Christy McDevitt, who was fun to work with, even
when she was seasick.
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