Abstract Strong evidence for the compartmentation ofarginine in oth
unfertilized and fertilized seaurhin egge wasotined yopaine
arginine content of an extract obtained from permeabilizeeggs with the
arginine content of an extract produced by extracting the same eggswith
105e heat. A significant portion of the total arginine of the egg was found in
the hestentract. This suggests that some arginine is sequestered within
1yhanging compartments
Introduction: The unfertilized sea urchin egg is a very sleepy cell
metabolically, but fertilization triggers an activation of the egg which is
chafacterized by a sharp increase in oxidative phosphorylation, protein
synthesis and other metabolic processes. Another effect of activation is a rise
in the level of arginine phosphate (Chambers and Mende 1953) Arginine
phosphate often occurs in invertebrates and is analogous to creatine
phosphate in vertebrates. In the sea urchin egg, arginine phosphate serves
as a buffer of high energy phosphate and is closely linked to the energ
status of the cell. When arginine phosphate is depleted, several cellular
metabolic activities such as protein synthesis (Winklerand Linde.
unpublishedandea(Taiasa68)areth
AlP lewelsafe not depleted
Arginine phosphate ie synthesized from arginine by ATP via the
ollowng reaction
Afg+ATP «------Arg-P4ADP4Ht
Arg A- Arginine
2-P AP - Arginine phosphste
ATP -Adenocine triphocphste
This teaction is catalyzed by the enzyme arginine kinase. Since this is the
only reaction by which arginine is phosphorwlated, the equation for the
equilibrium constant kaqof thie reaction,
Arg-PIADPIIH
— —-- -------- -
14
ArgheiP
signifies that the rise in the ratio of arginine phosphate to arginine (AP/A)as
is seen at fertilization should signify a corresponding rise in the ratio of ATF
to ADP. By rearranging the above equation, this concept becomes more clear.
Arg-Pl
keg lATP
---------—--------
Argl
ADPIH
fe
At
nged equation also demonstrates, the rise in IATPI/IADP ratio
brought about by the rise in the AP/A ratio at fertilization is dependant
upon the values for keq and for H' remaining constant. The value for keg
would rmain constant if arginine kinase were actiwe both before and after
ferti
ation An experiment performed by Winkler and Linde
1
111.
ipublicheddemonstrates that hen ATPyhe
11.
nfertil
lition u
ininephophateleed
e
ili
ine kinae
ative befoe ferunsation
ADP - Adenosine diphosphate
Permeabilzanon studies show that anumber of other enzymesare, in fact, sctivated
uponfernlization (Swezeyand Epel 1567) Arginine kinese wesnotoneoftheenavmes
udied
The second condition for a rise in the lAPI/A ratio to signal a similar
increase in IATPIIADPI ratio is that the Htion concentration must remain
constant. However, at fertilization the internal pHofthegin
While in theory this incresse in pHshould cause arise in AP/A rauo on its
Stn, Winkler and Linder found though that the rise in pH at fertil
ns
notenough to account for therise in arginine phosphate atfer
ization.
1
lhey also found that a rise in arginine phoplate from artificiel ati
ation in
sodiumifeseawater inthepreene of A23187ionpoe
eteof activatingh
eegg whilekeeping phonstantpodu
in arginine phosphate nearly equal to that obserwed in fertilized controis
Assuming that arginine kinase is active both before and after
fertilization and assuming that the increase in pH at fertilization does not
+1
getyaee
nthesis of argininephosphate in vivo theieine
APIA ratio that occurs at fertilization should signal a similar rise in the
ratio of ATP to ALP. However, such an increase in the IATPI/IADP! ratio is
not generally observed. There is littie consensus as to what actually happens
to the ATP/ADP ratio. For instance, Epel (1969) has observed that it remains
constant while Winkler and Linder minhr
fertilization
The hypothesis that this study auggests as an explanation for the
pafent lack of coffelation betwwen these two fatios is that arginine is
sequestefed wthin slowyehanging compartentintheeariie


Pattme
nihzedthenprevious measurements of cellular at
mine
hich a
he all argininetbethin theytopa
the actual quantity of arginine available to the cell for arginine phosphate
synthesis, and consequently, misrepresent the cytoplasmic lAPlAl ratio.
Materials and Methods
Materials NAHe pyruateLrginie an
purchased from Sigma.
Handling of gametes Gametes were collected by injecting Stronsyhentrus
purpuratus with 1 mi of 0.5 mi KCl intracoelomically Eggs were colleted
into 150 C sea water, filtered through a 90 micron mesh, diluted with filtered
seawater to à 23 suspension, and kept suspended by constant stirring with;
motorized paddie. Dry sperm was collected with a Pasteur pipette and
stored at5oc
Fertilization procedure Fertilizedegre ie
0000 dilution of sperm, then were allowed to stand 8 minutes and kept
suspended by aeration with a Pasteur pipette.
eation Procedure Both fertilized and unfertilized eggs were then
Permebina
washed twice in Catartificial seawater, washed once in permeabilization
medium prepared according to Swezey and Epel (1983) then concentrated to
à wolume of 1 ml in the same permeabilization medium. The cells were then
permeabilized according to the method described by Swezey and Epel
1933) Three 1000-voit pulses wereused to permeabilize theegs
Permeabilized cells were allowed to stand for three minutes and then
centri
ed Thesupernatant wscollected andhe
edfor 10 minuteat
1050 Thiefraction wastermed the permeabilized fraction which would be
indicative of cytoplasmie arginine One  of permabiization mediuma
Nicotinimide Adenine Dinucleotide (reducedform
added to the remaining pellet These were miedeated for 10 minutes at
105° and centrifuged This supernatant as colleted and termedthe pelet
fraction. This fraction would beindicatiwe of the amount of arginine in
clowy-exchanging compartment
Determination of Arginine Levels Arginineleeleineahfat
estimate by themethod desribedinGaedandGriehaber175
Readings were obtinedom a PerkinEe
pectrophotometer set at 40nm excitation and 460 nm emittance. The
feaction was performed in acuwette wth a light path of lem contsining
200ulof 25mMpyruvate 200ulof 5 Mtriethanolamine buffer wth 25mM
EDTA, 400ul of 015 mM NADH, 5 unit of octopine dehydrogenase (in 5Oul),
50ulof extractand 14miof distilled water. The final volume in the cuwette
was 2.5 ml. After thereaction reached equilibrium, 10 ulof 2 mM arginine
was added as an internal standard.
Results: The change in fluorescence of NADH brought about by cell extract
wascompared tothe change of fluorescence caused by an internal standard
of 002 umolesofarginine. Since the change in fluorescence is diree
proportionalto the arginine present,
ity of rginine
han
Molar quat
t
------------------------------------- 4 g -
t
+++.
Changein Huoeseneoandd
Using a value of 4K 100 packed celle per mithe molar quantity of arginine
per egg was determinedThetotalarginine per ce
cell ior the unfertilized egge and 0.47 picomoles per cell for the fertilized
—
e Arginine content for each individual fraction is outlined in the table
below and in the attached graph.
Unfertilized
Fertilized
Permeabilized
26+- 0005 pmel
17 + 006 pmcell
Pellet
30+-005 pmell
24- 915pmel
Discussion: These results give strong evidence that arginine is significantiy
compartmentalized in the sea urchin egg. The values of total arginine per
cell of 50 and 47 picomoles of arginine per cell compare favorably with
results of Gross and Fry as repoted by Giudice (1973) of .32 picomoles of
inine per cell. Some research tends to suggest that compartments of
ginine and other amino acids could exist in the sea urchin egg. Lee and
++4--
Epel (1983) found acidie granules in fertilized Strongyspurpuratus
and lytechinus pietus eggs which were stained by acridine orange. These

ar
anulecould conceivably accumulate basic amino acids such as alennne
lan and llan (in press) discovered that when methionine is present in an
egenus medium to Lpitus egge, it is incorporated into proteins at a
higher rate than intracellularly injected methionine. This result suggests
hatannno add channeling a process which could require dlowly-enchanging
patentationan
heuhine

Although thedata in this paper dosuggest that arginine is
———++-
Cnparunentahzed in the sea urchin egg there is another possible
planation fof the observed result. Because arginine is a very stronely
positively charged ami
in thecenl The data do not distinguish between electrical binding and
— —
copar
thentation sofurther experimentation is necessary. One possible
enperiment to distinguih beteen
e topossibilities istouse detergent
to discolve all the membranes of the cell inuding the propsediie
Jini
compaftments Ithe afgnnne werecompartmentaliethenaltheelu
arginine would be feleased as a reeult of this detergent procedure. If the
arginine ereelectrically bounthen arginin
and could be removed by boiline
Further experimentation would alo berequired to determine whethe
tomeoftheother aspects of the reeults are true effects of compartmentation
of arginine or mere artifacts. No other strong inferences can be drawn from
these fesulte because only two trials were run for each type of extract,
Although the agreement between all of the sets of trials was very good, more
trals need to berun to determine whether or not the variation between the
measured arginine lewels is due to actual changes in compartmentalized and
cytoplasmiarginine ortoexperimentalerorForinstane thelevelsof
compartmentalized arginine seemto rise after fertilization. This would
assume an uptake of free arginine from the cytoplasm to the compartmente
or proteolysis of proteins contained within the compartments. The increase
howeeris ufficiently amall sothatit cannot be considered significant over
such a emall sample size
Amore significant feature atleatinmanude
iwhatappea

be adrop in free arginine lewels aiter fertilization. Afterfetia
lewelof cytoplasmicarginine per cellis 0 picomoles loter than before
eftilization ifthis value provesto besignnneant,then it could reeult from
++
atint
theincease in arginine phosphatehate
piconoe pere
gasmeasured by Chambers and Mende (1953
—
As with most data, the data reported in this paper bring up far more
questions than they answer, requiring more experiments to be performed.
I nothing else, the data did grant some strong evidence for a hypothesis that
was completely overlooked by mi
needed to determine whether or not arginine is sequesteredinto slowdy-
hanging compartmentsin the seaurhineggeperimentthatud
obtain this evidence would be relatively simple to carry out. The
significance of the actual finding of arginine compartrents would extend
beyond the study of egg activation. It could also lead to studies into the
channeling of amino acids, into the energy needs of the developing embrye
and perhaps even to differentiated cells.
Acknowledgements: I would like to thank my advisor, David Epel for his
keen suggestions and strong encouragement. I would also like to thank Rob
yor his invaluabelpithprocedures andeneaardy
Finally, lwould like to recognize Nat Timmins, who had to put up with me at
home, Steve Verbinski and Saween Singh, who had to put up with me in lab.
and Leonard D'Amico, who had the singular honor of having to put up with
me in both places. To everyone, l'm sorry about my horrible singing voice
and the frequency wth hich use it
0.2
004
Unfertilized

Fertilized
Fermeablized
Pellet
-
WorksCited:
rationeoftheinaniephohat
Chambers, EL and T.Mende.
+++
Mrrete oontenenised ufchin eggsiolowing
and arginine phosphat
lication Emp Cell Res 5. 500-519 (1953
ration folowing fer
pel D Loes ATP regulatete
funationofseaufh
Enp Cell Res.
5312-319 (1959
Gaede, G and M. Grieshaber. Afapid and epedific enzymatio method fort-
estimation of Larginine Anal i
Giudici GDevelopmental BilegyoftheSea Urhin EmbryAademi
Prese. New Tork and Londen, 1973. p 300
11-
lian,and Ian: Preferentielchannehng of
methionine into protein by sea urchin embryes (Inpe
Lee Ho and DEpel Changes in intracellular adidie compartmentsin
win
ti-
ater aedvation Devel. Bio. 98. 445-454 (1983

meactiwit eealed inepated
and D.Epel E
Permeabil
edseiurchinesge Proo Nat Acad S USA 35,812-11
1955
Winkler, M. and C Linder: Arginine phosphate ratios in the sea urchine
unpublished atthistime)
Tanigasawa T. Studies on echinoderm phosphagens. Enp Cell Res. 53.
a
6 (1963)