Cell Differentiation, 6(1977) 41--51
41
© Elsevier/North-Holland Sci,vntific Publishers Ltd.
INCORPORATION OF SPECIFIC AMINO ACIDS AS MARKERS OF CELL DIFFERENTIATION
S.P. YADAV and H.K. DAS * Division of Biochemistry, Indian Agricultural Research Institute, New Delhi-l l O012, India
Accepted 10 January, 1977 Incorporation of different amino acids into the proteins of wheat embryo has been found to be not uniform during the various stages of seed germination. It is suggested that incorporation maxima o f appropriate amino acids can be used as markers of switching of specific protein synthesis during embryo development. Such markers have been found suitable for defining the functional activity of preformed messenger RNA.
Cell differentiation is the ultimate result of switching on of the synthesis o f new proteins and also possibly switching off of some old ones. An experimental system would be suital)le for analysis, if these changes can be linked to some perceptible markers. !Jsing enzymes as markers is an accepted approach (Ellingson et al., 1971; Morris et al., 1971" Ihle et al. 1972). Appearauce and disappearance of activity of enzymes have been followed at different stages of development and these have been equated with the switching o f specific protei n synthesis. However, in such studies one has to he able to distinguish conclusively between enzyme synthesis and er~zyme activation and also between cessation Of synthesis and inactivation. We have attempted to develop a more direct approach involving iucorporation of specific amino acids as markers. The amino acid comL~osition of all l~he proteins would not be the same and there might be some uroteins which would have disproportionately high amount of one or other amino acid. If the synthesis of such a protein is switched, the incorporation of the amino acid which is present in it in preponderance, would be expected to suffer a sharp change. We have used wheat embryo as the test system and have indeed found that the rate of incorporation o f some amino acids is not the same throughout the development of the embryo, but well defined spurts and declines are discernible. The different maxima of incorporation of individual amino acids could thus be utilised as markers of the switching of the synthesis of different proteins during ~he development of any tissue, which exhibit such a phenomenon. * Present addre~: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110051, India.
4~
The mefulne~ of the m~kem has been tested in defining the ~ t i o n a l activity of preform~ messenger RNA. MATERIALS AND METHODS
Materia~ See~ of wheat (IYitivum eestivum L. vat. Sonora 64)harvest~ f ~ m imgsted fields of the tmtitute weee reed [U-14C]giycine (10,3 Ci/mole), I~ [U-14C]alanine (33 Ci/mole), L-[Uo14C]valine(6.1 Cilmole), L-[U-z4ci~ cine (120C i/mole, L-[U-~4C]isoleucine (66Ci/mole), L-[U-Z4C]phenylal, anine (14 CJ/mole), L - [ G - 3 H ] ~ (]:500Ci/mole), L - [ G . ~ H ] h ~ e (I400 Ci/mole), L-[G-3H]atginine (516C i/mole), L-[,~',5'-3H]tyrosine (2400 Ci/mole and 5600 Ci/r~ole), [G-3H]uridine (6400 Ci/mole) and [U-"~C]chloreUa protein hydroly~te (42 Cilatom C) were obta/ned from the Bhabha Atomic Research Centre, Trombay, India. L-[G-3H]methionine (200 Ci/mole) and DL-[2-:4C]tyrosine (50 Ci/mole) wexe pro'chased from the Radiochemical Centre, Amersham, England. Actinomycin D was a generous gift from Merck, Sharpe and Dohme, Rahway, NJ., U~$.A. Chloramphanicol was a kind gLCtfrom Dey's Medical Stores, Calcutta, India. Germafion of seed¢ Seeds were germinated at 18°C as described earlier (Yadav et al., 1972). ChlorampberJicol (100 pg/ml) has been employed to emmre that the results would not be vitiated by bacterial contL~ination. Estgmatu~n of protein content Protein was determined by the me~od of Lowry et al. (1951). Bovine serum albumin was used as standani. Determination of radioacz~vf~y of amino acid in the free intemal pool o f embryo and that incorporated into proteins Ten embryos were dissected out from the germirating seeds and incubated at 18°C for 1 h in a medium which contained the following in 0.5 ml : 50 mM Tris.acetate buffer, pH 7,I, 0.1 ml; clfloramphenicoL 500 pg/ml, 0.1 ml and the radioactive amino acid (1.0 pCi). After the incubation, the embryos were picked up, washed in 50 ml ice water, blotted and then added to 5 ml ice-cold t~richloroacetic acid (5%) containh~ the same amino acid (1 mg/ml), but in nonradioactive form. The embryos were weshed again by five changes in similar chilled acid mixture and then heated for ~0 rain at 80°C in 3 ml acid mixture. It has been shown that this treatment with trich]oroacetic acid solu~flises
43 s
very negligible m o u n t of proteiws (Marchesi et xl., 1967). We have also confirmed this. The embryos were then homogenized in a Potter Elvehjem homogenizer. The p z o t e ~ were sedimented by centrifugation. An aliquot of the supernatant solution (1 ml) ~as counted for radioactivity with Bray's (1960) scintillation mixtu~ in the Packard Liquid Scintillation Spectr0meter, Model 3320. Counting efficiency for 3H was about 7.5% and that for x4C was about 20%. ~ would be a measure of the radioactivity of the free amino acid in the intenml pool of the tissue. The sedimented protein precipitate was washed twice by resuspension in trichloroacetic acid and recentrifu~gation. The proteins were then dissolved in 1 N NaOH, undissolved material removed by filtration through glass fibre filter and the proteins were reprecipi~,ated with trichloroacetic acid. The precipitate was collected on glass fibre filter (GF/C, W h a ~ u , ) , the ~flter was put in glass counting via!, dried at 105°C for 2 h and was counted for radioactivity with a toluene based scintillation mixtu~. In this case, counting efficiency for 3H was about 30% and that for '4C was about 80%. This would be a measure of the radioactivity of the amino acid incorporated into the proteins.
Determination of the proportion of tyrosine in labelled amino acids of radio. active proteins obtained after incubation of embryos with 14C-tyrosine [l+C]tyrosine label!ed +proteins ware obtained from wheat embryos as described in the precedingsection. The precipitated proteins were washed five times with trichloroacetic acid (5%) and then transferred to digestion tubes+ HCI (6N) was added, the tubes were evacuated and sealed and the proteins were h y d r o l y ~ i for 24 h a t 110°C. The hydrolysate was freed from HCI by repeated evaporation and addition of water and was finaliy taken up in isc~ propanol (10%). The tmd'~solved matters were removed by centrifugation and i suitable aliquot was q)otted on chromatography paper (Whatman no+ I). De~ending chromatography was done with the butane! layer after mix+ ing butanol, acetic acid and water in the proportion, 4 : 1 : 4. This solvent system was e h o ~ n because it would separate tyrosine from all other amino acids in an unidimansional run. HydrolTsates of nonradioactive proteins from wheat embryo were also spotted side by side vnd the position of tyrosine in the ehromatogram was determined by the ~pecific a-nitrose ~-naphtol reaction. Radioactivity in tyrosine was determined by cutting the relevant area from the paper and taking it in glass vials with the toluene based scintillation mixture (A). Efficiency of counting was ~5%. The hydrolysate of radioactive proteins was simultaneously spotted on separate chromatography paper discs, which had been prer.m ~ith the developing solvent. Radioactivity in these discs was determined (B). The proportion of tyrosine in labelled ~nino acids of radioactive proteins (A/B) thus determined would have to be corrected for recovery of amino acids. Nonlabellea proteins were obtained from wheat embryos at all stages of
44 germination, exactly as for [14C]tyrosine labelled proteins, except that t h e initial incubation was done without [~4C]tyrosine. These proteins were then hydrolysed in presence of added [l~C]tyTosine and l~e amino acids in an aliquot were separated chromatographically. Radioactivity i n t h e tyrosh~e region was determined (X). Total radioactivity present inthese aliquots was determined (Y) as described in the preceding paragraph. The corrected proportion (%) of tyrosine in labelled amino acids of radio. active proteins would then be given by AY BX
•
100
Ifmay be noted that this experiment can not be done with the [SH]tyrosine because of extensive tritium exchange during the hydrolysis step, Study o f the incorporation o f [SH]uridine into R N A
Ten embryos were incubated with [~H]uridine for I h as de~ribed in the preceding section. The embryos were then picked up and washed successively i n water (50 ml) and five changes of 5 ml unlabelled uridine solution (1 mg/ml). The embryos were homogenized with 0.2 N perchlo~c acid in a Potter-Elvehjem homogenizer. The precipitate was collected by cent~ifugation and washed twice by resuspension in fres~ 0.2 N perchloric acid and recentrfl~ugation. These operations were done at 0°C. The pellet was taken up in 0.5 N KOH and digested for 3 h at 37°C to hydrolyse RNA. Perchloric acid was added to make a final concentration of 0,2 N after neutmlisation of the KOH. The precipitate containing DNA, proteins and potassium perchlo. rate was discarded by centrifugation. The supernatant solution containing ribonucleotides was then neutralised with KOH, the potassium perchlorate centrifuged down and an aliquot (1 ml) of the solution was counted for radioactivity with Bray's (1960) scintillation mixture. S~udy o f the effect o f actinomycin D on R N A synthesis and protein synthesis Conditions for optimal inhibition of R N A synthesis by actinomycin D were first determined ~nd also used for inhibition of protein synthesis. Possibly because of the seed coat, little effect of actinomycin D was detected on
RNA synthesis even if the soaked whole seeds were treated with the drug (up to 60/~g/ml) 6 h before ~he addition of radioactive uridine. The embryos were therefore taken out from the germinating seeds several hours before the incubation and were alllowed to grow in sterile White's medium {Street et al., 1952) with added actinomycin D. The optimum dose of the drug was found to be 60/~g/:ml. It was also revealed that pretreatment of the embryos with actinomych~ D was necessary to obtain bett~er inhibition of RNA synthesis. A pretreatment period of 6 h was c h o ~ n for convenience, though
45 maximum effect could be obtained around 9 h. Chloramphenicol (100 ~g/ml) was added to the White's meclium to ensure that results were not vitiated by bacterial contamination. Ten embryos were incubated at 18°C with 0.5 ml medium containing radioactive uridine or the amino acid.
Determination o f the specific radioactivity o f amino acids in the free pool of wheat embryo ~
A measure of the apparent effective pool in the tissue has been obtained by using-an amino acid at two different specific radioactivities. The same amount of radioactivity has been employed and in one case unlabeUed amino acid has been used as diluent. This procedure would be proper, since the "over~U rate of protein synthesis is unlikely to be affected by the increase in concentration of a single amino acid. Incorporation of radioactivity into the proteins would be a function of the specific radioactivity of the amino acids inside the tissue. Thus Pr = K - - Fr Pl + FrlSPex where Pr denotes radioactivity (cpm) incorporated into a given amount of protein ha a given time. Fr r~resents radioactivity (cprn) of the amino acid in the free pool inside the tissue (the efficiency ol counting o~?radioactivity in Fr need not be the same as that in Pr), SPex is the specific radioactivity of the exogenous amino acid (cpm/nmole), P1 is the effective free amino acid pool of the tissue (nmole) and K is the proportio,'~ity constant for the particular amino acid at a given stage of development. The value of apparent effective endogenous amino acid pool can be expressed ~s: PI = (Frl Fr2-Pr2)/(Spe~:2) -- (Frl Fr2Prl )/(Spe~ l) Prl Fr2 ~ Pr:: Fr I
T h e values of Frl, Frz, Pr z and Pr z have been determined in two experiments using two different specific radioactivities (Spexl and Spcxz ) of the exogenous amino acid.
It is important to note that the value of apparent effective amino acid pool that would be obtained here may have no absolute significance and would be related to the total entry of labelled amino acid. On the other hand, all the labelled amino acid molecules that have entered into the tissue may not be available for protein synthesis. However, the value of specific radioactivity derived, would, nevertheless, be a true representation of the specific radioactivity of the amino acid involved in protein synthesis. It may also be noted that the specific radioactivity thus computed, would remain valid in spite of possible interconversion of free amino acids in l~he tissue.
F~ctior~tfon of l~b~It~ e~bryo,~tei~ sy #~te t ~ e ~ , h ~
.... :
Embryos were incub~ containing the followm nicol, 100 pg/ml, L-J3', lysate, I ~Ci. Six embry 5, 4, 3 and 2 embryos tively. The embryos wel with a mixture (0.2 ml) containing 8 M urea, 2% ~ i ~ m dodecyl sulphate (w/v), 5% mercaptoethanol (w/v) and 0:0525 M Tris~acetate, pH 6.8. The homogenate was incubated at 25 ~.2°C for 48 and was then centrifuged at 10~000 g. The supematant ~lution was diluted 1 . 1 (v/v) with 40% glycerol(v/v) and 0~2 ml u ~ for electrophoresis essentially as described by Flint eta]: (1975), The columns (6 mm diam) consisted of 1 cm stacking gel and 7 cm separating gel. The Stack~g gels contained 3% acrytamide, 0.08% N,N'-methylene bis-acrylarnide, 0i025% N,N,N',N'tetramethyl ethylene diarnine, 0.125 M Tris-chloride, p H 6i8 and 0.1% sodium dodecyl ~ulphate. The separating gelscontahled 15% acrylamide, 0.4% r r t N,N-methylene bis acrylamide, 0.025% N,N,N ,N-tetrxnet.yl ethylene diarnine, 0.375 M Tris-chloride, pH 8.8 and 0.1% s o d i ~ d o d e c y l sulphate. The gel preparations were polymerized chemically wi¢~ 0,03% ammonium persulphate. The electrode buffer, pH 8.3 cor~sisted c,f 0;025 M Tris, 0.192 M glycine and 0.1% sodium dodeeyl sulphate. Bro~ophenolblue (0,001%, w]v) was used as the tracking dye and electrophores~ w;ts c ~ out with 3 mA current per column, towards the anode. The gels were :sliced (1 mm thick) and 2 slices were treated in each capped scintillation vial for 48 h with 1% sodium dodecyl sulphate (0~5 ml) at 65°C. Radioactivitywas then determined with a mixt~xre of-Triton X-100 and toluene based scintillation mixture (3 : 1). Contribution from ~H and ~C was computed by using simultaneous equations from radioactivity in each individual pair of slices of gels in "which proteins labelled with only either [ ~H] tyrosine or [ ~C] chloreUa protein by. drolysate were e]ectrophoresed. RESULTS A N D DISCUSSION
Incorporation o[ amino acids into ~he proteins of wheat embryo Incorporation of labelled valine, isoleueine, pbenylalanine, lysine, histidine, methionhae, arginine, alanine, gIycine, tyzosbae and leucine into the proteins of wheat embryo has beer~ studied at different stages of seed germination. With each of these amino acids, except tyrosh~e and alanh~e, there was a distinct incorporation m a x i m u m at 86 to 48 h of gennh~tion. The patterns ~f incorporation were reproducible. Molecular quantity of incorporation of four amino acids has been determined on the bask of radioactivity incorporated into proteins and specific radioactivity of the amino acids in
47
Tyrosine
Glyclne 5(:
4G
t2
5o
~ ¢
z0
6
w
~o
Leucine
70
42
2~
~4
PERIOD OF GERMINATION | h r )
Fig. 1. Incorporation of amino acids into the proteins of wheat embryo at different stages of germination. Wheat embryos -were incubated with labelled amino acids as described in Methods. The radioactivity in the free internal pool and in the proteins were determined and the apparent effective pool was estimated. Specific radioactivity of the amino acid inside the t ~ u e was then derived and incorparation in terrn~ of molar quantity was calculated.
t h e i n ~ r n a l pool. T h e results are given in Fig. 1. Tihe i n c o r p o r a t i o n o f t h e a m i n o acids into t h e proteins was n o t u n i f o r m t h r o u g h o u t the stages o f germ i n a t i o n . Tyros~ae ~nco~oration was m a x i m u m around 12 h, but alanine was i n c o r p o r a t e d m o s t at 6 0 h. G l y c i n e and leucine were i n c o r p o r a t e d maxim a l l y a r o u n d 48 h. The~e were r e p r o d u c i b l e . We i n t e r p r e t this t o m e a n t h a t very early in germination, t h e synthesis o f some specific proteins, which are rich in t y r o s i n e , is switched on. This i,~ switched o f f gradually a f t e r 12 h, foIlowed by a burst of synthesisof other proteinsaround 48 h. At about 60 h, proteins rich in alanine are synthesised. T h e i n c o @ o r a t i o n o f t h e amino acids
48 a 24 br
'~&
4500
900
,oooth,.,
/Lo
....
000
,¢, 300
,,ooI'
~
120
,~
va
m
15000
30hr f
,ooi ioooo
~ 12000
", ,.
~ ,I "~ 9000
IW
'~'
~ ~,"
~,
'~' ,'
" ".
1800
1800 'l
";'
h 11 /', l~', ,l",
;'~
200 0 o
Z § 4000
0ooo
,~oo 5 ~
3000
~oo ~ ~_
800 z
'
0 2000
o 60
.
f -.,
4 0 0 a. Lu
4-
hr
'°°°°t !, ,ooo~
48ht
8000
3000~
"F - v y , , ~ /
3000
~
2400
8000 t
,,,oo
~oo~
,...oo
-., ,ooo~;,,,/
oo.
72
',~
/
~
!
Fractions
~
~! Ii200
, ,",, ,~ ;, ,~ ;',/ '/ ~ ;".~', ~'~ / ', ~n', ~,oo
v
t
~
hr
,,,~,
',,
.
9
.
.
.
~-~'
,,'
L'/
.
.
.
t~!
,,/v///~koo t'
,~,
t,oo
.
J2 15 ~8 2J 24 27
30
33-g6
Frocl,ons
Fig. 2,
at their respective peak positions, can therefore be used as markers of the switching of the synthesis of proteins in the developing tissue. The possibility of amino acid interconversion during incubation has been checked for tyrosine, since incorporation of this amino acid into embryo proteins changed most dramatically during germination. Almost 9 0 % of the radioactivity incorporated into proteins could be accounted for as tyrosine in embryo up to 36 h of germination (12h, 88%; 2 ~ h , 88%; 36 h, 87%), but this declined progressively thereafter (48 h, 59%;60 h, 49%; 72 h, 46%). If data are corrected accordingly, the drop in the curve for tyrosine in Fig. 1 would be even more dramatic.
49
~
l°°IA 9
.
b
0
f°°[i "1"
T
A
I
i
i
3.0,. ",,, ,,-
l"~%.'~,~,./',,,.,,.~
I
I
'
I
~
I
i
I
~ ,~
,
,t_.
i
i
~>~ ,',,...,' /
Froctlon5
Fire 2. Eleetrophoresis of proteins from, wheat embryo IabeiJed with [3Hlty. rosme" and [ JfC]chlorel[a protein hydrolysate. Embryos were incubated with a mlxtuIe of [3Hl~.Trosine and [ ~4C]chlorel|a protein hydrolysate. Proteins from w~_shedembryos were electrophozesed in acrylamide ge!s containing sodium dodecy! sulphate and radioactivity in ~he slices were determined as described in Methods. The fractJon~ have been numbered from bottom to top. (a) Radioactivity in fraction. (b) Ratio of radioactivity due to [3H]tyro. sine and [14C]chlorella protein hydrolysate: o, 12 h;$, 24 h;v, 36 h;v, 48 h;A, 60 h;A, 72h.
Change in the nature of proteins '.~vnthesized during embryo development The proposition p u t forward in tile preceding se¢:tion calls for a demonstration that during embryo development there is co~siderable change in the nature of proteins synthesized, in ~elation to their tyrosine content. Embryos were incubated with a mixture o:~ ['~H]tyrosine and [14C]chlorella protein hydrolysate, the labelled proteins were extracted with urea-SDS.mercaptoethanol and electrophoresed in SDS-acrylamide columns as described under Methods. Radioactivity in gel slice~ is pre~nt~d in Fig. 2a and the ratio of 3H/14C in Fig. 2b. It is evident that the distribution of radioactivity due to tyrosine and chloreUa protein hydrolysate differed considerably at the early stages but were more ,~h~ilar at the late stages of germination, implying that the tyrosine.rich proteins synthesized early were unique.
50
Usefuln~ preform Pro~: appears 1968; i~ early (D h~g the
actinom sis in w] were ~ul traasla~i ~yrvsjne The act~
tion fo~ Vy~o~ine
of it (around 21 to 27 h) needs new m R N A , The ~hibition by actinomycin D of the incorporationof glycine or ~ , however! ~ m e d robe considerable throughout the period of development s~died~ ~ggesthc~g that the synthesis of the related proteins was due mostly to the activity of n e w m R N A .
Tyrosin~ -
-
Glycim~
Alanine
i
5
~RiO0 oF 6E~M~NATIOU~hr~ Fig. 3. Effect of aefinomyeinD on the incorporationof tyr~ine,glyclneand alanineinto the proteinsof gerrainating Wheat e ~ b ~ o . Erab~o~ w ~ taken ou~ ~ h prio~ to ~he
amino acid (2 pCi/ml), w~ ~dded and the in~fibhti0h cc,htih~ 3or aa~,~her i h' Period~ of gemination ~fcr~ed ¢o in the f i g ~ rcl~r~nt ~h~ *il~ at which t ~ lab~|i~d a~i~o
~.cidhas bucn addad.
;
51 REFERENCES
~y~ a!Ai: ~ i Biochem.1,279-285(196o). ~ D,i SI SaHd and ~. Katchalski: Proc. Natl. Acad. ScL U.S.A. 60, 992--909 (1968). D ~ n s k a ~ M.i M. Tornaszewa~ki, Z. Grzelczak, E. Rejman and J. Buchowicz: Nature
~4,507-5o9 (19 73).
E~lingson, JIS.~ A. Telser and M. Su~sman: Bioch~_m.Biophys. Acta 244,388--395 (1971). F~knt, D., G.S. Ayers and S.K. Ries: Plan*~Physiol. 56, 381--384 (1975). Ihle, J.N. and L.S, Bure, III: J. Biol. Chem. 247, 5048--5055 (1972). Lowry, O.H., N.J. Rosebrough, A.L. Farr a~d R.J. Randall: J. Biol. Chem. 193, 265--276
(1951).
Marche~, S,L. and D. Kennel: J. Bae~HoL 93, 35~'--366 (1967). Marcus, A.: Syrup. Soc. Exp. Biol. 23,142--~60 (1969). Morris, J.]~. and A.A. Moscona: Develop. Biol. 25,420--444 (1971). S~reet, H.E. and S.M. McGregor: Ann. Bot., London 16, 185--205 (1952). Yadav, S,P:, V.P. Ahuja and H.K. Das: Ind. J. Bioehem. Biophys. 9, 350--351 (1972).
41
© Elsevier/North-Holland Sci,vntific Publishers Ltd.
INCORPORATION OF SPECIFIC AMINO ACIDS AS MARKERS OF CELL DIFFERENTIATION
S.P. YADAV and H.K. DAS * Division of Biochemistry, Indian Agricultural Research Institute, New Delhi-l l O012, India
Accepted 10 January, 1977 Incorporation of different amino acids into the proteins of wheat embryo has been found to be not uniform during the various stages of seed germination. It is suggested that incorporation maxima o f appropriate amino acids can be used as markers of switching of specific protein synthesis during embryo development. Such markers have been found suitable for defining the functional activity of preformed messenger RNA.
Cell differentiation is the ultimate result of switching on of the synthesis o f new proteins and also possibly switching off of some old ones. An experimental system would be suital)le for analysis, if these changes can be linked to some perceptible markers. !Jsing enzymes as markers is an accepted approach (Ellingson et al., 1971; Morris et al., 1971" Ihle et al. 1972). Appearauce and disappearance of activity of enzymes have been followed at different stages of development and these have been equated with the switching o f specific protei n synthesis. However, in such studies one has to he able to distinguish conclusively between enzyme synthesis and er~zyme activation and also between cessation Of synthesis and inactivation. We have attempted to develop a more direct approach involving iucorporation of specific amino acids as markers. The amino acid comL~osition of all l~he proteins would not be the same and there might be some uroteins which would have disproportionately high amount of one or other amino acid. If the synthesis of such a protein is switched, the incorporation of the amino acid which is present in it in preponderance, would be expected to suffer a sharp change. We have used wheat embryo as the test system and have indeed found that the rate of incorporation o f some amino acids is not the same throughout the development of the embryo, but well defined spurts and declines are discernible. The different maxima of incorporation of individual amino acids could thus be utilised as markers of the switching of the synthesis of different proteins during ~he development of any tissue, which exhibit such a phenomenon. * Present addre~: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110051, India.
4~
The mefulne~ of the m~kem has been tested in defining the ~ t i o n a l activity of preform~ messenger RNA. MATERIALS AND METHODS
Materia~ See~ of wheat (IYitivum eestivum L. vat. Sonora 64)harvest~ f ~ m imgsted fields of the tmtitute weee reed [U-14C]giycine (10,3 Ci/mole), I~ [U-14C]alanine (33 Ci/mole), L-[Uo14C]valine(6.1 Cilmole), L-[U-z4ci~ cine (120C i/mole, L-[U-~4C]isoleucine (66Ci/mole), L-[U-Z4C]phenylal, anine (14 CJ/mole), L - [ G - 3 H ] ~ (]:500Ci/mole), L - [ G . ~ H ] h ~ e (I400 Ci/mole), L-[G-3H]atginine (516C i/mole), L-[,~',5'-3H]tyrosine (2400 Ci/mole and 5600 Ci/r~ole), [G-3H]uridine (6400 Ci/mole) and [U-"~C]chloreUa protein hydroly~te (42 Cilatom C) were obta/ned from the Bhabha Atomic Research Centre, Trombay, India. L-[G-3H]methionine (200 Ci/mole) and DL-[2-:4C]tyrosine (50 Ci/mole) wexe pro'chased from the Radiochemical Centre, Amersham, England. Actinomycin D was a generous gift from Merck, Sharpe and Dohme, Rahway, NJ., U~$.A. Chloramphanicol was a kind gLCtfrom Dey's Medical Stores, Calcutta, India. Germafion of seed¢ Seeds were germinated at 18°C as described earlier (Yadav et al., 1972). ChlorampberJicol (100 pg/ml) has been employed to emmre that the results would not be vitiated by bacterial contL~ination. Estgmatu~n of protein content Protein was determined by the me~od of Lowry et al. (1951). Bovine serum albumin was used as standani. Determination of radioacz~vf~y of amino acid in the free intemal pool o f embryo and that incorporated into proteins Ten embryos were dissected out from the germirating seeds and incubated at 18°C for 1 h in a medium which contained the following in 0.5 ml : 50 mM Tris.acetate buffer, pH 7,I, 0.1 ml; clfloramphenicoL 500 pg/ml, 0.1 ml and the radioactive amino acid (1.0 pCi). After the incubation, the embryos were picked up, washed in 50 ml ice water, blotted and then added to 5 ml ice-cold t~richloroacetic acid (5%) containh~ the same amino acid (1 mg/ml), but in nonradioactive form. The embryos were weshed again by five changes in similar chilled acid mixture and then heated for ~0 rain at 80°C in 3 ml acid mixture. It has been shown that this treatment with trich]oroacetic acid solu~flises
43 s
very negligible m o u n t of proteiws (Marchesi et xl., 1967). We have also confirmed this. The embryos were then homogenized in a Potter Elvehjem homogenizer. The p z o t e ~ were sedimented by centrifugation. An aliquot of the supernatant solution (1 ml) ~as counted for radioactivity with Bray's (1960) scintillation mixtu~ in the Packard Liquid Scintillation Spectr0meter, Model 3320. Counting efficiency for 3H was about 7.5% and that for x4C was about 20%. ~ would be a measure of the radioactivity of the free amino acid in the intenml pool of the tissue. The sedimented protein precipitate was washed twice by resuspension in trichloroacetic acid and recentrifu~gation. The proteins were then dissolved in 1 N NaOH, undissolved material removed by filtration through glass fibre filter and the proteins were reprecipi~,ated with trichloroacetic acid. The precipitate was collected on glass fibre filter (GF/C, W h a ~ u , ) , the ~flter was put in glass counting via!, dried at 105°C for 2 h and was counted for radioactivity with a toluene based scintillation mixtu~. In this case, counting efficiency for 3H was about 30% and that for '4C was about 80%. This would be a measure of the radioactivity of the amino acid incorporated into the proteins.
Determination of the proportion of tyrosine in labelled amino acids of radio. active proteins obtained after incubation of embryos with 14C-tyrosine [l+C]tyrosine label!ed +proteins ware obtained from wheat embryos as described in the precedingsection. The precipitated proteins were washed five times with trichloroacetic acid (5%) and then transferred to digestion tubes+ HCI (6N) was added, the tubes were evacuated and sealed and the proteins were h y d r o l y ~ i for 24 h a t 110°C. The hydrolysate was freed from HCI by repeated evaporation and addition of water and was finaliy taken up in isc~ propanol (10%). The tmd'~solved matters were removed by centrifugation and i suitable aliquot was q)otted on chromatography paper (Whatman no+ I). De~ending chromatography was done with the butane! layer after mix+ ing butanol, acetic acid and water in the proportion, 4 : 1 : 4. This solvent system was e h o ~ n because it would separate tyrosine from all other amino acids in an unidimansional run. HydrolTsates of nonradioactive proteins from wheat embryo were also spotted side by side vnd the position of tyrosine in the ehromatogram was determined by the ~pecific a-nitrose ~-naphtol reaction. Radioactivity in tyrosine was determined by cutting the relevant area from the paper and taking it in glass vials with the toluene based scintillation mixture (A). Efficiency of counting was ~5%. The hydrolysate of radioactive proteins was simultaneously spotted on separate chromatography paper discs, which had been prer.m ~ith the developing solvent. Radioactivity in these discs was determined (B). The proportion of tyrosine in labelled ~nino acids of radioactive proteins (A/B) thus determined would have to be corrected for recovery of amino acids. Nonlabellea proteins were obtained from wheat embryos at all stages of
44 germination, exactly as for [14C]tyrosine labelled proteins, except that t h e initial incubation was done without [~4C]tyrosine. These proteins were then hydrolysed in presence of added [l~C]tyTosine and l~e amino acids in an aliquot were separated chromatographically. Radioactivity i n t h e tyrosh~e region was determined (X). Total radioactivity present inthese aliquots was determined (Y) as described in the preceding paragraph. The corrected proportion (%) of tyrosine in labelled amino acids of radio. active proteins would then be given by AY BX
•
100
Ifmay be noted that this experiment can not be done with the [SH]tyrosine because of extensive tritium exchange during the hydrolysis step, Study o f the incorporation o f [SH]uridine into R N A
Ten embryos were incubated with [~H]uridine for I h as de~ribed in the preceding section. The embryos were then picked up and washed successively i n water (50 ml) and five changes of 5 ml unlabelled uridine solution (1 mg/ml). The embryos were homogenized with 0.2 N perchlo~c acid in a Potter-Elvehjem homogenizer. The precipitate was collected by cent~ifugation and washed twice by resuspension in fres~ 0.2 N perchloric acid and recentrfl~ugation. These operations were done at 0°C. The pellet was taken up in 0.5 N KOH and digested for 3 h at 37°C to hydrolyse RNA. Perchloric acid was added to make a final concentration of 0,2 N after neutmlisation of the KOH. The precipitate containing DNA, proteins and potassium perchlo. rate was discarded by centrifugation. The supernatant solution containing ribonucleotides was then neutralised with KOH, the potassium perchlorate centrifuged down and an aliquot (1 ml) of the solution was counted for radioactivity with Bray's (1960) scintillation mixture. S~udy o f the effect o f actinomycin D on R N A synthesis and protein synthesis Conditions for optimal inhibition of R N A synthesis by actinomycin D were first determined ~nd also used for inhibition of protein synthesis. Possibly because of the seed coat, little effect of actinomycin D was detected on
RNA synthesis even if the soaked whole seeds were treated with the drug (up to 60/~g/ml) 6 h before ~he addition of radioactive uridine. The embryos were therefore taken out from the germinating seeds several hours before the incubation and were alllowed to grow in sterile White's medium {Street et al., 1952) with added actinomycin D. The optimum dose of the drug was found to be 60/~g/:ml. It was also revealed that pretreatment of the embryos with actinomych~ D was necessary to obtain bett~er inhibition of RNA synthesis. A pretreatment period of 6 h was c h o ~ n for convenience, though
45 maximum effect could be obtained around 9 h. Chloramphenicol (100 ~g/ml) was added to the White's meclium to ensure that results were not vitiated by bacterial contamination. Ten embryos were incubated at 18°C with 0.5 ml medium containing radioactive uridine or the amino acid.
Determination o f the specific radioactivity o f amino acids in the free pool of wheat embryo ~
A measure of the apparent effective pool in the tissue has been obtained by using-an amino acid at two different specific radioactivities. The same amount of radioactivity has been employed and in one case unlabeUed amino acid has been used as diluent. This procedure would be proper, since the "over~U rate of protein synthesis is unlikely to be affected by the increase in concentration of a single amino acid. Incorporation of radioactivity into the proteins would be a function of the specific radioactivity of the amino acids inside the tissue. Thus Pr = K - - Fr Pl + FrlSPex where Pr denotes radioactivity (cpm) incorporated into a given amount of protein ha a given time. Fr r~resents radioactivity (cprn) of the amino acid in the free pool inside the tissue (the efficiency ol counting o~?radioactivity in Fr need not be the same as that in Pr), SPex is the specific radioactivity of the exogenous amino acid (cpm/nmole), P1 is the effective free amino acid pool of the tissue (nmole) and K is the proportio,'~ity constant for the particular amino acid at a given stage of development. The value of apparent effective endogenous amino acid pool can be expressed ~s: PI = (Frl Fr2-Pr2)/(Spe~:2) -- (Frl Fr2Prl )/(Spe~ l) Prl Fr2 ~ Pr:: Fr I
T h e values of Frl, Frz, Pr z and Pr z have been determined in two experiments using two different specific radioactivities (Spexl and Spcxz ) of the exogenous amino acid.
It is important to note that the value of apparent effective amino acid pool that would be obtained here may have no absolute significance and would be related to the total entry of labelled amino acid. On the other hand, all the labelled amino acid molecules that have entered into the tissue may not be available for protein synthesis. However, the value of specific radioactivity derived, would, nevertheless, be a true representation of the specific radioactivity of the amino acid involved in protein synthesis. It may also be noted that the specific radioactivity thus computed, would remain valid in spite of possible interconversion of free amino acids in l~he tissue.
F~ctior~tfon of l~b~It~ e~bryo,~tei~ sy #~te t ~ e ~ , h ~
.... :
Embryos were incub~ containing the followm nicol, 100 pg/ml, L-J3', lysate, I ~Ci. Six embry 5, 4, 3 and 2 embryos tively. The embryos wel with a mixture (0.2 ml) containing 8 M urea, 2% ~ i ~ m dodecyl sulphate (w/v), 5% mercaptoethanol (w/v) and 0:0525 M Tris~acetate, pH 6.8. The homogenate was incubated at 25 ~.2°C for 48 and was then centrifuged at 10~000 g. The supematant ~lution was diluted 1 . 1 (v/v) with 40% glycerol(v/v) and 0~2 ml u ~ for electrophoresis essentially as described by Flint eta]: (1975), The columns (6 mm diam) consisted of 1 cm stacking gel and 7 cm separating gel. The Stack~g gels contained 3% acrytamide, 0.08% N,N'-methylene bis-acrylarnide, 0i025% N,N,N',N'tetramethyl ethylene diarnine, 0.125 M Tris-chloride, p H 6i8 and 0.1% sodium dodecyl ~ulphate. The separating gelscontahled 15% acrylamide, 0.4% r r t N,N-methylene bis acrylamide, 0.025% N,N,N ,N-tetrxnet.yl ethylene diarnine, 0.375 M Tris-chloride, pH 8.8 and 0.1% s o d i ~ d o d e c y l sulphate. The gel preparations were polymerized chemically wi¢~ 0,03% ammonium persulphate. The electrode buffer, pH 8.3 cor~sisted c,f 0;025 M Tris, 0.192 M glycine and 0.1% sodium dodeeyl sulphate. Bro~ophenolblue (0,001%, w]v) was used as the tracking dye and electrophores~ w;ts c ~ out with 3 mA current per column, towards the anode. The gels were :sliced (1 mm thick) and 2 slices were treated in each capped scintillation vial for 48 h with 1% sodium dodecyl sulphate (0~5 ml) at 65°C. Radioactivitywas then determined with a mixt~xre of-Triton X-100 and toluene based scintillation mixture (3 : 1). Contribution from ~H and ~C was computed by using simultaneous equations from radioactivity in each individual pair of slices of gels in "which proteins labelled with only either [ ~H] tyrosine or [ ~C] chloreUa protein by. drolysate were e]ectrophoresed. RESULTS A N D DISCUSSION
Incorporation o[ amino acids into ~he proteins of wheat embryo Incorporation of labelled valine, isoleueine, pbenylalanine, lysine, histidine, methionhae, arginine, alanine, gIycine, tyzosbae and leucine into the proteins of wheat embryo has beer~ studied at different stages of seed germination. With each of these amino acids, except tyrosh~e and alanh~e, there was a distinct incorporation m a x i m u m at 86 to 48 h of gennh~tion. The patterns ~f incorporation were reproducible. Molecular quantity of incorporation of four amino acids has been determined on the bask of radioactivity incorporated into proteins and specific radioactivity of the amino acids in
47
Tyrosine
Glyclne 5(:
4G
t2
5o
~ ¢
z0
6
w
~o
Leucine
70
42
2~
~4
PERIOD OF GERMINATION | h r )
Fig. 1. Incorporation of amino acids into the proteins of wheat embryo at different stages of germination. Wheat embryos -were incubated with labelled amino acids as described in Methods. The radioactivity in the free internal pool and in the proteins were determined and the apparent effective pool was estimated. Specific radioactivity of the amino acid inside the t ~ u e was then derived and incorparation in terrn~ of molar quantity was calculated.
t h e i n ~ r n a l pool. T h e results are given in Fig. 1. Tihe i n c o r p o r a t i o n o f t h e a m i n o acids into t h e proteins was n o t u n i f o r m t h r o u g h o u t the stages o f germ i n a t i o n . Tyros~ae ~nco~oration was m a x i m u m around 12 h, but alanine was i n c o r p o r a t e d m o s t at 6 0 h. G l y c i n e and leucine were i n c o r p o r a t e d maxim a l l y a r o u n d 48 h. The~e were r e p r o d u c i b l e . We i n t e r p r e t this t o m e a n t h a t very early in germination, t h e synthesis o f some specific proteins, which are rich in t y r o s i n e , is switched on. This i,~ switched o f f gradually a f t e r 12 h, foIlowed by a burst of synthesisof other proteinsaround 48 h. At about 60 h, proteins rich in alanine are synthesised. T h e i n c o @ o r a t i o n o f t h e amino acids
48 a 24 br
'~&
4500
900
,oooth,.,
/Lo
....
000
,¢, 300
,,ooI'
~
120
,~
va
m
15000
30hr f
,ooi ioooo
~ 12000
", ,.
~ ,I "~ 9000
IW
'~'
~ ~,"
~,
'~' ,'
" ".
1800
1800 'l
";'
h 11 /', l~', ,l",
;'~
200 0 o
Z § 4000
0ooo
,~oo 5 ~
3000
~oo ~ ~_
800 z
'
0 2000
o 60
.
f -.,
4 0 0 a. Lu
4-
hr
'°°°°t !, ,ooo~
48ht
8000
3000~
"F - v y , , ~ /
3000
~
2400
8000 t
,,,oo
~oo~
,...oo
-., ,ooo~;,,,/
oo.
72
',~
/
~
!
Fractions
~
~! Ii200
, ,",, ,~ ;, ,~ ;',/ '/ ~ ;".~', ~'~ / ', ~n', ~,oo
v
t
~
hr
,,,~,
',,
.
9
.
.
.
~-~'
,,'
L'/
.
.
.
t~!
,,/v///~koo t'
,~,
t,oo
.
J2 15 ~8 2J 24 27
30
33-g6
Frocl,ons
Fig. 2,
at their respective peak positions, can therefore be used as markers of the switching of the synthesis of proteins in the developing tissue. The possibility of amino acid interconversion during incubation has been checked for tyrosine, since incorporation of this amino acid into embryo proteins changed most dramatically during germination. Almost 9 0 % of the radioactivity incorporated into proteins could be accounted for as tyrosine in embryo up to 36 h of germination (12h, 88%; 2 ~ h , 88%; 36 h, 87%), but this declined progressively thereafter (48 h, 59%;60 h, 49%; 72 h, 46%). If data are corrected accordingly, the drop in the curve for tyrosine in Fig. 1 would be even more dramatic.
49
~
l°°IA 9
.
b
0
f°°[i "1"
T
A
I
i
i
3.0,. ",,, ,,-
l"~%.'~,~,./',,,.,,.~
I
I
'
I
~
I
i
I
~ ,~
,
,t_.
i
i
~>~ ,',,...,' /
Froctlon5
Fire 2. Eleetrophoresis of proteins from, wheat embryo IabeiJed with [3Hlty. rosme" and [ JfC]chlorel[a protein hydrolysate. Embryos were incubated with a mlxtuIe of [3Hl~.Trosine and [ ~4C]chlorel|a protein hydrolysate. Proteins from w~_shedembryos were electrophozesed in acrylamide ge!s containing sodium dodecy! sulphate and radioactivity in ~he slices were determined as described in Methods. The fractJon~ have been numbered from bottom to top. (a) Radioactivity in fraction. (b) Ratio of radioactivity due to [3H]tyro. sine and [14C]chlorella protein hydrolysate: o, 12 h;$, 24 h;v, 36 h;v, 48 h;A, 60 h;A, 72h.
Change in the nature of proteins '.~vnthesized during embryo development The proposition p u t forward in tile preceding se¢:tion calls for a demonstration that during embryo development there is co~siderable change in the nature of proteins synthesized, in ~elation to their tyrosine content. Embryos were incubated with a mixture o:~ ['~H]tyrosine and [14C]chlorella protein hydrolysate, the labelled proteins were extracted with urea-SDS.mercaptoethanol and electrophoresed in SDS-acrylamide columns as described under Methods. Radioactivity in gel slice~ is pre~nt~d in Fig. 2a and the ratio of 3H/14C in Fig. 2b. It is evident that the distribution of radioactivity due to tyrosine and chloreUa protein hydrolysate differed considerably at the early stages but were more ,~h~ilar at the late stages of germination, implying that the tyrosine.rich proteins synthesized early were unique.
50
Usefuln~ preform Pro~: appears 1968; i~ early (D h~g the
actinom sis in w] were ~ul traasla~i ~yrvsjne The act~
tion fo~ Vy~o~ine
of it (around 21 to 27 h) needs new m R N A , The ~hibition by actinomycin D of the incorporationof glycine or ~ , however! ~ m e d robe considerable throughout the period of development s~died~ ~ggesthc~g that the synthesis of the related proteins was due mostly to the activity of n e w m R N A .
Tyrosin~ -
-
Glycim~
Alanine
i
5
~RiO0 oF 6E~M~NATIOU~hr~ Fig. 3. Effect of aefinomyeinD on the incorporationof tyr~ine,glyclneand alanineinto the proteinsof gerrainating Wheat e ~ b ~ o . Erab~o~ w ~ taken ou~ ~ h prio~ to ~he
amino acid (2 pCi/ml), w~ ~dded and the in~fibhti0h cc,htih~ 3or aa~,~her i h' Period~ of gemination ~fcr~ed ¢o in the f i g ~ rcl~r~nt ~h~ *il~ at which t ~ lab~|i~d a~i~o
~.cidhas bucn addad.
;
51 REFERENCES
~y~ a!Ai: ~ i Biochem.1,279-285(196o). ~ D,i SI SaHd and ~. Katchalski: Proc. Natl. Acad. ScL U.S.A. 60, 992--909 (1968). D ~ n s k a ~ M.i M. Tornaszewa~ki, Z. Grzelczak, E. Rejman and J. Buchowicz: Nature
~4,507-5o9 (19 73).
E~lingson, JIS.~ A. Telser and M. Su~sman: Bioch~_m.Biophys. Acta 244,388--395 (1971). F~knt, D., G.S. Ayers and S.K. Ries: Plan*~Physiol. 56, 381--384 (1975). Ihle, J.N. and L.S, Bure, III: J. Biol. Chem. 247, 5048--5055 (1972). Lowry, O.H., N.J. Rosebrough, A.L. Farr a~d R.J. Randall: J. Biol. Chem. 193, 265--276
(1951).
Marche~, S,L. and D. Kennel: J. Bae~HoL 93, 35~'--366 (1967). Marcus, A.: Syrup. Soc. Exp. Biol. 23,142--~60 (1969). Morris, J.]~. and A.A. Moscona: Develop. Biol. 25,420--444 (1971). S~reet, H.E. and S.M. McGregor: Ann. Bot., London 16, 185--205 (1952). Yadav, S,P:, V.P. Ahuja and H.K. Das: Ind. J. Bioehem. Biophys. 9, 350--351 (1972).
