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Labelling of nucleotide pools in sea urchin eggs
Experimental Cell Research 57 (1969) 71-73
LABELLING
OF NUCLEOTIDE
POOLS IN SEA URCHIN EGGS
F. R. MACKINTOSH1 Department
and E. BELL
of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139, USA
SUMMARY Nucleotide triphosphate pools were compared in unfertilized and fertilized sea urchin eggs and shown to be similar. In labelling experiments it was found that essentially all of the ATP and GTP of the unfertilized egg is turning over and is available for use.
We have recently proposed [6] that peptide chain initiation is limiting for protein synthesis in sea urchin eggs. Hultin [4] made a similar proposal based on the observation that exposure of sea urchin embryos to inhibitors of energy metabolism caused disaggregation of polyribosomes. Disaggregation of polyribosomes in the presence of inhibitors of energy metabolism may be a general phenomenon in higher cells [5] and suggests that the peptide chain initiation step may be particularly sensitive to the level of nucleotide triphosphates or some other compound whose concentration drops rapidly when energy metabolism is inhibited. The direct involvement of high energy phosphate compounds (GTP) in chain initiation in bacterial systems is well established [3]. It is known [2, 7, 8, 91 that sea urchin eggs have large ATP pools, so we considered the possibility that the pools of ATP and GTP might be sequesteredin some way and unavailable for use in protein synthesis. Our results suggestthat the nucleotide triphosphates of the egg are not sequesteredand are available for use. MATERIALS AND METHODS Gametes were obtained and handled as described elsewhere 161.Extracts for nucleotide pool analysis were prepared by grinding 1 vol of eggs in two vol of 0.5 N 1 Present address: Mt Herman School, Mt Herman, Mass. 01354, USA.
HClO,, centrifuging the homogenate, and re-extracting the pellet in the same way. The pooled supernatants from these two extractions were diluted tenfold and applied directly to the columns. The nucleotide triphosphate pools were loaded on to Dowex-1 (formate) columns (5 cm x 0.5 cm) and analyzed by eluting the column with a linear gradient formed from 300 ml each of 3 M formic acid and 3 M formic acid: 1 M ammonium formate. Fractions were collected with a Technicon Time/Flow fraction collector and the optical density of the fractions measured in a Zeiss snectrophotometer. Radioactivity was measured using -Bray’s Solution and a Packard Tri-Carb scintillation counter. or by drying samples on planchets for counting in a Tracerlab low background gas flow counter. Nucleotide standards were obtained from Sigma and Pabst.
RESULTS AND DISCUSSION Since some limitation on energy metabolism has been implicated as a possible mechanism for repression of the unfertilized egg (see p. 72), it seemed desirable to measure nucleotide triphosphate pools before and after fertilization to seeif there were any indications of energy limitation. Our results are in agreement with those of Yanagisawa & Isono [S, 91, that unfertilized eggs have at least as much ATP and GTP as fertilized eggs (fig. 1). In fact, when eggs were pulse-labelled for 10 min with 32POq,unfertilized eggs incorporated significant radioactivity into ATP. Unfertilized eggs which were maintained under CO,-free conditions incorporated still more radioactivity, and fertilized eggs incorporated even more. Exptl Cell Res 57
72
F. R. Mackintosh & E. Bell b' l20000
d; I
0.6 -
) I
0.4 -
I I
02 I
’
b.
ATP 06
04 I/
0.2
20
40
60
00
100
L 120
i
Fig. 1.
Fig. 3.
Abscissa: ml effluent; ordinate: absorbance 260 mp. Fig. 1. A perchloric acid extract of 0.1 ml of Strongylocenfrotus eggs (a) or 0.2 ml of Arbaciu eggs (b), either fertilized (-) or unfertilized (------) was eluted from Dowex-1 (formate) with a 300 ml gradient of 0.1 M ammonium formate in 3 M formic acid. A standard column (c) containing 3 OD units of ATP, 1.5 of GTP, 2 of CTP, and 2 of miscellaneous nucleoside diphosphates and nucleotides was eluted from Dowex-1 (formate) in the same way. Identification of the peaks in this column was based both on the amount of material recovered in each peak and its position compared to peaks in other cqlumns (1) used as standards. The numbers in these peaks give the recovery of OD in each. A radioactive UTP standard was also included and found to elute with the late half of the GTP peak (at about lo@-105 ml). Ordinate: (right) cpm. (c, d) Arbucia eggs which were stimulated by 5 h of CO, removal (d) or fertilized (c) (------) or unfertilized controls (c) (. . . * .) were pulse-labelled for 10 min with 82PO;2 (10 ,&ml). Fig. 2. (a, b) Arbucia eggs were labelled for 6 h with SaPO;2 (3 PC/ml). Some were washed and fertilized (a) and both these and the unfertilized (b) were extracted with perchloric acid and the extracts eluted from Dowex-1 (formate) as usual. The similarity of the patterns of radioactivity (-----) indicates no significant increase in specific activity of ATP (or GTP) after fertilization, which would have resulted if a sequestered ATP (or GTP) pool were made available. Actual specific activities at the ATP peak were 465 (unfertilized) and 473 (fertilized) cpm/ OD x 10e8. Total cpm on the column were 8950 (unfertilized), 9105 (CO, removal) and 14,206 (fertilized). Most of this radioactivity is found in the off-scale peak eluting at about 10 ml, which must represent PO;‘. Abscissa: ml effluent; ordinate: absorbance, 260 rnp. Fig. 3. Unfertilized Arbacia eggs were extracted with perchloric acid after 0 (-) and the extracts eluted from Dowex-1 (formate) as usual.
A possibility that seemed reasonable mechanism for limiting energy supply in
as a
the unfertilized eggs, in spite of the large pools of nucleotide triphosphates, was that part of this pool might be sequesteredin some way in unExptl Cell Res 57
3 (-----) h exposure to anoxia,
fertilized eggs. To test this, eggs were labelled for an extended period of time (6 h) with 92POp, washed, and some fertilized. All were prepared for column analysis as usual. The lack of any significant increase in the specific activity of
Labelling of nucleotide pools
13
ATP and GTP in the first hour after fertilization (fig. 2) suggeststhat all the ATP and GTP of the unfertilized egg is turning over and has attained equilibrium with the internal phosphate pool of the egg. That the ATP pool is in fact largely available for use, and is not merely turning over by exchange, was demonstrated by measurement of ATP levels after a period of anoxia (fig. 3). The strong depression of ATP level shows that at least 70 % of the egg’s ATP is available for use under anaerobic conditions.
1. Cohn. W E. Chromatoeranhv. 3rd edn (ed E Heftmann) p. 554. Reinhold, New York (1963): 2. Epel, D, Biochem biophys res comm 17 (1964) 62. 3. Hershey, J W B & Thach, R E, Proc natl acad sci Wash 57 (1967) 759. 4. H&in, T:Develop biol 10 (1964) 305. 5. Lin. C Y & Kev. J L. J mol biol 26 (1967) 237. 6. MaeKintosh, F ‘R &‘Bell, E, J mol biol 41 (1969). In press. 7. Monroy, A, Chemistry and physiology of fertilization, p. 88. Rinehart & Winston, New York, Chicago, San Francisco, Toronto, London (1965). Yanagisawa, T & Isono, N, Embryologia 9 (1966) 170. f : - Ibid 9 (1966) 184.
Supported in part by grant no. GB-7312 from the National Science Foundation.
Received February 19, 1969
REFERENCES .,
.
-I
Exptl Cell Res 57
LABELLING
OF NUCLEOTIDE
POOLS IN SEA URCHIN EGGS
F. R. MACKINTOSH1 Department
and E. BELL
of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139, USA
SUMMARY Nucleotide triphosphate pools were compared in unfertilized and fertilized sea urchin eggs and shown to be similar. In labelling experiments it was found that essentially all of the ATP and GTP of the unfertilized egg is turning over and is available for use.
We have recently proposed [6] that peptide chain initiation is limiting for protein synthesis in sea urchin eggs. Hultin [4] made a similar proposal based on the observation that exposure of sea urchin embryos to inhibitors of energy metabolism caused disaggregation of polyribosomes. Disaggregation of polyribosomes in the presence of inhibitors of energy metabolism may be a general phenomenon in higher cells [5] and suggests that the peptide chain initiation step may be particularly sensitive to the level of nucleotide triphosphates or some other compound whose concentration drops rapidly when energy metabolism is inhibited. The direct involvement of high energy phosphate compounds (GTP) in chain initiation in bacterial systems is well established [3]. It is known [2, 7, 8, 91 that sea urchin eggs have large ATP pools, so we considered the possibility that the pools of ATP and GTP might be sequesteredin some way and unavailable for use in protein synthesis. Our results suggestthat the nucleotide triphosphates of the egg are not sequesteredand are available for use. MATERIALS AND METHODS Gametes were obtained and handled as described elsewhere 161.Extracts for nucleotide pool analysis were prepared by grinding 1 vol of eggs in two vol of 0.5 N 1 Present address: Mt Herman School, Mt Herman, Mass. 01354, USA.
HClO,, centrifuging the homogenate, and re-extracting the pellet in the same way. The pooled supernatants from these two extractions were diluted tenfold and applied directly to the columns. The nucleotide triphosphate pools were loaded on to Dowex-1 (formate) columns (5 cm x 0.5 cm) and analyzed by eluting the column with a linear gradient formed from 300 ml each of 3 M formic acid and 3 M formic acid: 1 M ammonium formate. Fractions were collected with a Technicon Time/Flow fraction collector and the optical density of the fractions measured in a Zeiss snectrophotometer. Radioactivity was measured using -Bray’s Solution and a Packard Tri-Carb scintillation counter. or by drying samples on planchets for counting in a Tracerlab low background gas flow counter. Nucleotide standards were obtained from Sigma and Pabst.
RESULTS AND DISCUSSION Since some limitation on energy metabolism has been implicated as a possible mechanism for repression of the unfertilized egg (see p. 72), it seemed desirable to measure nucleotide triphosphate pools before and after fertilization to seeif there were any indications of energy limitation. Our results are in agreement with those of Yanagisawa & Isono [S, 91, that unfertilized eggs have at least as much ATP and GTP as fertilized eggs (fig. 1). In fact, when eggs were pulse-labelled for 10 min with 32POq,unfertilized eggs incorporated significant radioactivity into ATP. Unfertilized eggs which were maintained under CO,-free conditions incorporated still more radioactivity, and fertilized eggs incorporated even more. Exptl Cell Res 57
72
F. R. Mackintosh & E. Bell b' l20000
d; I
0.6 -
) I
0.4 -
I I
02 I
’
b.
ATP 06
04 I/
0.2
20
40
60
00
100
L 120
i
Fig. 1.
Fig. 3.
Abscissa: ml effluent; ordinate: absorbance 260 mp. Fig. 1. A perchloric acid extract of 0.1 ml of Strongylocenfrotus eggs (a) or 0.2 ml of Arbaciu eggs (b), either fertilized (-) or unfertilized (------) was eluted from Dowex-1 (formate) with a 300 ml gradient of 0.1 M ammonium formate in 3 M formic acid. A standard column (c) containing 3 OD units of ATP, 1.5 of GTP, 2 of CTP, and 2 of miscellaneous nucleoside diphosphates and nucleotides was eluted from Dowex-1 (formate) in the same way. Identification of the peaks in this column was based both on the amount of material recovered in each peak and its position compared to peaks in other cqlumns (1) used as standards. The numbers in these peaks give the recovery of OD in each. A radioactive UTP standard was also included and found to elute with the late half of the GTP peak (at about lo@-105 ml). Ordinate: (right) cpm. (c, d) Arbucia eggs which were stimulated by 5 h of CO, removal (d) or fertilized (c) (------) or unfertilized controls (c) (. . . * .) were pulse-labelled for 10 min with 82PO;2 (10 ,&ml). Fig. 2. (a, b) Arbucia eggs were labelled for 6 h with SaPO;2 (3 PC/ml). Some were washed and fertilized (a) and both these and the unfertilized (b) were extracted with perchloric acid and the extracts eluted from Dowex-1 (formate) as usual. The similarity of the patterns of radioactivity (-----) indicates no significant increase in specific activity of ATP (or GTP) after fertilization, which would have resulted if a sequestered ATP (or GTP) pool were made available. Actual specific activities at the ATP peak were 465 (unfertilized) and 473 (fertilized) cpm/ OD x 10e8. Total cpm on the column were 8950 (unfertilized), 9105 (CO, removal) and 14,206 (fertilized). Most of this radioactivity is found in the off-scale peak eluting at about 10 ml, which must represent PO;‘. Abscissa: ml effluent; ordinate: absorbance, 260 rnp. Fig. 3. Unfertilized Arbacia eggs were extracted with perchloric acid after 0 (-) and the extracts eluted from Dowex-1 (formate) as usual.
A possibility that seemed reasonable mechanism for limiting energy supply in
as a
the unfertilized eggs, in spite of the large pools of nucleotide triphosphates, was that part of this pool might be sequesteredin some way in unExptl Cell Res 57
3 (-----) h exposure to anoxia,
fertilized eggs. To test this, eggs were labelled for an extended period of time (6 h) with 92POp, washed, and some fertilized. All were prepared for column analysis as usual. The lack of any significant increase in the specific activity of
Labelling of nucleotide pools
13
ATP and GTP in the first hour after fertilization (fig. 2) suggeststhat all the ATP and GTP of the unfertilized egg is turning over and has attained equilibrium with the internal phosphate pool of the egg. That the ATP pool is in fact largely available for use, and is not merely turning over by exchange, was demonstrated by measurement of ATP levels after a period of anoxia (fig. 3). The strong depression of ATP level shows that at least 70 % of the egg’s ATP is available for use under anaerobic conditions.
1. Cohn. W E. Chromatoeranhv. 3rd edn (ed E Heftmann) p. 554. Reinhold, New York (1963): 2. Epel, D, Biochem biophys res comm 17 (1964) 62. 3. Hershey, J W B & Thach, R E, Proc natl acad sci Wash 57 (1967) 759. 4. H&in, T:Develop biol 10 (1964) 305. 5. Lin. C Y & Kev. J L. J mol biol 26 (1967) 237. 6. MaeKintosh, F ‘R &‘Bell, E, J mol biol 41 (1969). In press. 7. Monroy, A, Chemistry and physiology of fertilization, p. 88. Rinehart & Winston, New York, Chicago, San Francisco, Toronto, London (1965). Yanagisawa, T & Isono, N, Embryologia 9 (1966) 170. f : - Ibid 9 (1966) 184.
Supported in part by grant no. GB-7312 from the National Science Foundation.
Received February 19, 1969
REFERENCES .,
.
-I
Exptl Cell Res 57