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Two methods for determining the water content of the giant amoeba Pelomyxa carolinensis
Effect of X-radiation
on nuclear nucleotide
content
6. KILLANDER, D., Unpublished data. 7. KILLANDER, D., RIBBING, C., RINGERTZ, N. R. and RICHARDS, B. hr., Expfl. Cell Research 27, 63 (1962). 8. KLEIN, G. and FORSSBERG, A., Espfl. Cell Research 6,211 (1954). 9. MITCHELL, J. S., Bril. J. Expfl. Pafhol. 23, 285 (1942). 10. ORD, hf. G. and STOCKEN, L. h., in Advances in Radiobiology, p. 65. HERESY, C. G., FORSSBERG, A. and .\BBATT, J. D., (eds). Oliver and Boyd Ltd., Edinburgh, 1957. 11. -Biochim. et Biophys. dcta 29, 201 (1958). 12. RICHMOND, J. E., ORD, M. G. and STOCKEN, L. A., Biochem. J. 66, 123 (1957). 13. RINGERTZ, N. R., Acla C’nion Intern. Confre le Cancer 16, 1153 (1960). 14. SHERMAN, F. G. and ALMEIDA, A. B., in Advances in Radiobiologg, p. 49. HEVESY, C. G., FORSSBERG, A. and ABBATT, J. D., (eds). Oliver and Boyd Ltd., Edinburgh, 1957.
TWO
METHODS THE
FOR
GIANT
DETERMINING
WATER
PELOMYXA
AMOEBA J.
Department
THE
of Zoology, Received
CONTENT
OF
CAROLINENSIS
RIDDLE
The
University,
Bristol,
England
March 19, 1962
DURING a recent
study of the membrane potential of the giant amoeba, Pelomyxa [6], it was necessary to measure the water content of the cell so that determinations of the internal potassium could be expressed as concentrations. Two methods were evolved, both employing the Cartesian diver-balance. ISOTOPIC L.&BELLING METHOD WITH D,O.-If the cell water is exchanged for a known concentration of heavy water, then the resulting increase in the reduced weight should enable the volume of the cell water to be calculated, assuming that it is all exchangeable. Lavtrup and Pigon [3] used both D,O and H,018 in a study of water exchange in Pelomyxa. In their theoretical treatment they mention the possibility of calculating the water content from their theoretical data, but do not go on to derive any such information. Prescott and Zeuthen [5] studied diffusion and filtration rates of water across a variety of cell membranes, including that of Pelomyxa, by means of the diver-balance and D,O: they conclude that the water content of the frog egg is 75-80 per cent. -4 similar value for amphibian eggs was arrived at by Lovtrup [2]. The experimental technique in the present work was essentially the same as that of Lovtrup and Pigon. A group of three animals was starved in Pringsheim medium for 2-1 hr and then weighed on a diver-balance in the same medium. They were then transferred in the minimum amount of medium to a watch-glass containing Pringsheim medium prepared with 5 per cent v/v D,O, and thence to a second diver equilibrated in this labeled medium. The pressure in the diver vessel was adjusted constantly to maintain equilibrium and readings made every minute for the first quarter of an hour and every 5 min thereafter, until no further increase in reduced weight was recorded. The temperature of the diver vessels was maintained at 22”C, and the
carolinensis
?“-621801
Experimental
Cell Research
27
,I. Riddlr specific gravity of the labeled medium measured with respect to distilled water at the same temperature, by means of a 2 ml pyknometer. The reduced weight of the animals at the end of the experiment was taken to be R.W.,=,; strictly, the approach to the final value is asymptotic, but it was quite clear from the data that the eschange process was virtually complete in the finite
Fig.
l.--Graph
of a typical
experiment
showing the -increase
time of the experiment. A graph was then plotted of log,, (R.W.,=, -R.\V.,=,) versus time in the labeled medium (t). Such a graph is linear for the greater part, and a linear extrapolation was then used to calculate the reduced weight at the moment of transfer to the labeled medium. As may be seen from the accompanying graph (Fig. l), there is a marked departure from linearity during the first few minutes. A similar effect was noted by Prescott and Zeuthen, who attributed it to a layer of unlabeled water slipping off the animals into the cup of the diver during the initial minutes of exchange. ,4 further likely cause in the present work is failure to bring the labeled medium in the watch-glass to exactly the same temperature as the diver-vessel. There were several occasions in which a linear graph was not obtained; these results were discarded. Theory and method of calculation.-Consider the total mass of the animal to be composedof two fractions: w, the weight of the intracellular water (pug);d, the dry weight of the protoplasm (Fig). L.et the specific gravity of the labeled medium be g. Let the total volume of the animals be u (m/d.) Therefore, R.\V.t=o 7 d -t LO- ug and
R.M-.&,
= cl + wg ~ vg
Subtracting (I) from (II), (R.\l-.t=, - R.\%7.,=0)= w (g - 1j l~~=R.~~.~=,-R.~~.~=~;(g-l).
i.e. Experimental
(Ml
Resenrch
27
(1) (11)
327
Water content of the giant amoeba
The total volume of the animals may readily be found from the preliminary weighing in unlabeled medium (S.G. =l) from the relationship: u =R.W./(e -l), where e is the density of the animals. In the present work, e was determined by weighing a group of six animals, and then accurately measuring their individual volumes by the “compression” method of Lumsden and Robinson [4]. -4sHolter and Zeuthen [l] have shown. the density of Pelomyxa remains remarkably constant during a period of starvation. It was found unnecessary to make repeated density measurements in connection with the water content determinations, and a mean value of 1.018 g/ml was used. Table I shows the results obtained for the water content. TABLE Vol.
of
Vol.
animals, Espt.
no.
D. Mean
11 :.; water,
v/v
of
water,
P1
D. 8 D. 9 D. 10
I.
Pl
No.
of
animals
161.5
137.5
3
191.5
158.0
327.0 553.0
481.0
3 5 5
= 83.9
265.0
7. (Std.
Dev.
:b Water (by vol.)
85.1 82.5
81.0 57.0
= 2.5 pb).
DIRECT confpmIsoN OF WET AND DRY WEIGHT.If the weight of an organism is accurately determined and the organism then dried to constant weight, comparison of these two measurementswill obviously enable the water content to be calculated. It is usually difficult to get an accurate measure of the wet weight of an aquatic organism; usual methods are blotting the organism to remove adhering water and then weighing in air, or elsetransferring the organism to a weighed vessel containing a known weight of water and then weighing the entire assembly. Clearly, neither of thesemethods is appropriate for protozoa: it would be impracticable to dry the animal, and the likely errors would in any casebe of comparable magnitude with the weight of the animal itself. However, it is a simple matter to derive the wet weight of any organism that can be weighed on a diver-balance, since the desired quantity is related to the reduced weight by the following equation:
m=---R.W. I- l/e’ where m is the weight of the organism in air (j(g) (i.e. the wet weight) and e is the density of the organism, referred to a density of 1 for the flotation medium (g/ml). In practice, about 30 Pelomyxa were washed in clean Pringsheim medium and then weighed on a diver-balance; the wet weight was then calculated. They were then rapidly washed in distilled water and transferred to a small glasspan. This consisted of a 3 mm square of a No. 0 coverslip, to one corner of which had been fused a glass-fibre hook, and which had been accurately weighed on a quartz torsion balance. E+perimentul
Cell
Research
27
The
pan and contents were then dried to constant weight at 100 C: and the rlrh of the animals so found. The water content was calculated by difference and expressed as a percentage of the wet weight. From three such rlcterminations, a mean value of RO.:I per cent w/w was obtained (Std. De\,. :( per cent). This corresponds to 92.0 per cent v/v. From the above results it is seen that the two methods show a considerable discrepancy, the reason for which is not known. There is little doubt that the second method is preferable as a technique, requiring far less manipulation than the heavywater method. r\ further and more serious objection to the use of heavy water is the need for extrapolating the graph, since a very small error in the position of the is line would cause a large error in the reading at zero time. At the present stage, there is little point in speculating whether the different results yielded by the two methods have any biological significance. Shortage of time precluded any further investigation of this; in the subsequent work on potassium distribution, a figure of 90 per cent \V/W was taken as sufficiently accurate for the purpose. weight
I am grateful to Dr. J. X. Kitching, F.R.S., for advice and criticism of this work, and to the Department of Scientific and Industrial Research for the provision of a maintenance grant. REFERENCES 1. HOLTER,
H. and ZEUTHEN, E., Corn@. Rend. True. Cnrlsberg Ser. Chim. 26, 277 S., J. Esptl. Zool. 145, 139 (1960). S. and PIGON, A.? Compt. Rend. Tmo. Carlsberg Ser. Chim. 28, 1 (1951). 4. LUMS~EN,~. E.and ROBINSON,C.V., Ibid.28,358 (1953). 5. PRESCOTT, D. RI. and ZEUTHEN, E., A& Physiol. Stand. 28, 77 (1953). 6. RIDDLE, J., Espfl. Cell Research 26, 158 (1962).
(1918).
2. LOVTRUP, 3. LBVTRUP,
DISTRIBUTION
OF PROTEOLYTIC
FRACTIONS
FROM
NON-FERTILIZED
EGGS OF THE SEA URCHIN G. LUNDBLAD National
Bacteriological Laboratory. Stockholm, Sweden, and The for Experimental Biology, University of Stockholm,
BEsums cathepsin egg with
II three different the pH-optima
1 This investigation was supported Council and from the Swedish Canrer at Stazione Zoologica, Naples. Experimenlnl
AND
PARACENTROTUS
Cell Research
27
IN PROTEIN FERTILIZED LIVIDUS’
and .J. RUNNSTRijM
Received
sea urchin
ENZYMES
March
Wenner-Gren Sweden
Institute
20, 1962
proteolytic enzymes were demonstrated in the (cf. [3, 41). The) 6.7, -7.0 and 7.X respectively
bv grants from the Swedish Soc‘ietg. The experimental part
Natural Sciences Research of the work was carried out
on nuclear nucleotide
content
6. KILLANDER, D., Unpublished data. 7. KILLANDER, D., RIBBING, C., RINGERTZ, N. R. and RICHARDS, B. hr., Expfl. Cell Research 27, 63 (1962). 8. KLEIN, G. and FORSSBERG, A., Espfl. Cell Research 6,211 (1954). 9. MITCHELL, J. S., Bril. J. Expfl. Pafhol. 23, 285 (1942). 10. ORD, hf. G. and STOCKEN, L. h., in Advances in Radiobiology, p. 65. HERESY, C. G., FORSSBERG, A. and .\BBATT, J. D., (eds). Oliver and Boyd Ltd., Edinburgh, 1957. 11. -Biochim. et Biophys. dcta 29, 201 (1958). 12. RICHMOND, J. E., ORD, M. G. and STOCKEN, L. A., Biochem. J. 66, 123 (1957). 13. RINGERTZ, N. R., Acla C’nion Intern. Confre le Cancer 16, 1153 (1960). 14. SHERMAN, F. G. and ALMEIDA, A. B., in Advances in Radiobiologg, p. 49. HEVESY, C. G., FORSSBERG, A. and ABBATT, J. D., (eds). Oliver and Boyd Ltd., Edinburgh, 1957.
TWO
METHODS THE
FOR
GIANT
DETERMINING
WATER
PELOMYXA
AMOEBA J.
Department
THE
of Zoology, Received
CONTENT
OF
CAROLINENSIS
RIDDLE
The
University,
Bristol,
England
March 19, 1962
DURING a recent
study of the membrane potential of the giant amoeba, Pelomyxa [6], it was necessary to measure the water content of the cell so that determinations of the internal potassium could be expressed as concentrations. Two methods were evolved, both employing the Cartesian diver-balance. ISOTOPIC L.&BELLING METHOD WITH D,O.-If the cell water is exchanged for a known concentration of heavy water, then the resulting increase in the reduced weight should enable the volume of the cell water to be calculated, assuming that it is all exchangeable. Lavtrup and Pigon [3] used both D,O and H,018 in a study of water exchange in Pelomyxa. In their theoretical treatment they mention the possibility of calculating the water content from their theoretical data, but do not go on to derive any such information. Prescott and Zeuthen [5] studied diffusion and filtration rates of water across a variety of cell membranes, including that of Pelomyxa, by means of the diver-balance and D,O: they conclude that the water content of the frog egg is 75-80 per cent. -4 similar value for amphibian eggs was arrived at by Lovtrup [2]. The experimental technique in the present work was essentially the same as that of Lovtrup and Pigon. A group of three animals was starved in Pringsheim medium for 2-1 hr and then weighed on a diver-balance in the same medium. They were then transferred in the minimum amount of medium to a watch-glass containing Pringsheim medium prepared with 5 per cent v/v D,O, and thence to a second diver equilibrated in this labeled medium. The pressure in the diver vessel was adjusted constantly to maintain equilibrium and readings made every minute for the first quarter of an hour and every 5 min thereafter, until no further increase in reduced weight was recorded. The temperature of the diver vessels was maintained at 22”C, and the
carolinensis
?“-621801
Experimental
Cell Research
27
,I. Riddlr specific gravity of the labeled medium measured with respect to distilled water at the same temperature, by means of a 2 ml pyknometer. The reduced weight of the animals at the end of the experiment was taken to be R.W.,=,; strictly, the approach to the final value is asymptotic, but it was quite clear from the data that the eschange process was virtually complete in the finite
Fig.
l.--Graph
of a typical
experiment
showing the -increase
time of the experiment. A graph was then plotted of log,, (R.W.,=, -R.\V.,=,) versus time in the labeled medium (t). Such a graph is linear for the greater part, and a linear extrapolation was then used to calculate the reduced weight at the moment of transfer to the labeled medium. As may be seen from the accompanying graph (Fig. l), there is a marked departure from linearity during the first few minutes. A similar effect was noted by Prescott and Zeuthen, who attributed it to a layer of unlabeled water slipping off the animals into the cup of the diver during the initial minutes of exchange. ,4 further likely cause in the present work is failure to bring the labeled medium in the watch-glass to exactly the same temperature as the diver-vessel. There were several occasions in which a linear graph was not obtained; these results were discarded. Theory and method of calculation.-Consider the total mass of the animal to be composedof two fractions: w, the weight of the intracellular water (pug);d, the dry weight of the protoplasm (Fig). L.et the specific gravity of the labeled medium be g. Let the total volume of the animals be u (m/d.) Therefore, R.\V.t=o 7 d -t LO- ug and
R.M-.&,
= cl + wg ~ vg
Subtracting (I) from (II), (R.\l-.t=, - R.\%7.,=0)= w (g - 1j l~~=R.~~.~=,-R.~~.~=~;(g-l).
i.e. Experimental
(Ml
Resenrch
27
(1) (11)
327
Water content of the giant amoeba
The total volume of the animals may readily be found from the preliminary weighing in unlabeled medium (S.G. =l) from the relationship: u =R.W./(e -l), where e is the density of the animals. In the present work, e was determined by weighing a group of six animals, and then accurately measuring their individual volumes by the “compression” method of Lumsden and Robinson [4]. -4sHolter and Zeuthen [l] have shown. the density of Pelomyxa remains remarkably constant during a period of starvation. It was found unnecessary to make repeated density measurements in connection with the water content determinations, and a mean value of 1.018 g/ml was used. Table I shows the results obtained for the water content. TABLE Vol.
of
Vol.
animals, Espt.
no.
D. Mean
11 :.; water,
v/v
of
water,
P1
D. 8 D. 9 D. 10
I.
Pl
No.
of
animals
161.5
137.5
3
191.5
158.0
327.0 553.0
481.0
3 5 5
= 83.9
265.0
7. (Std.
Dev.
:b Water (by vol.)
85.1 82.5
81.0 57.0
= 2.5 pb).
DIRECT confpmIsoN OF WET AND DRY WEIGHT.If the weight of an organism is accurately determined and the organism then dried to constant weight, comparison of these two measurementswill obviously enable the water content to be calculated. It is usually difficult to get an accurate measure of the wet weight of an aquatic organism; usual methods are blotting the organism to remove adhering water and then weighing in air, or elsetransferring the organism to a weighed vessel containing a known weight of water and then weighing the entire assembly. Clearly, neither of thesemethods is appropriate for protozoa: it would be impracticable to dry the animal, and the likely errors would in any casebe of comparable magnitude with the weight of the animal itself. However, it is a simple matter to derive the wet weight of any organism that can be weighed on a diver-balance, since the desired quantity is related to the reduced weight by the following equation:
m=---R.W. I- l/e’ where m is the weight of the organism in air (j(g) (i.e. the wet weight) and e is the density of the organism, referred to a density of 1 for the flotation medium (g/ml). In practice, about 30 Pelomyxa were washed in clean Pringsheim medium and then weighed on a diver-balance; the wet weight was then calculated. They were then rapidly washed in distilled water and transferred to a small glasspan. This consisted of a 3 mm square of a No. 0 coverslip, to one corner of which had been fused a glass-fibre hook, and which had been accurately weighed on a quartz torsion balance. E+perimentul
Cell
Research
27
The
pan and contents were then dried to constant weight at 100 C: and the rlrh of the animals so found. The water content was calculated by difference and expressed as a percentage of the wet weight. From three such rlcterminations, a mean value of RO.:I per cent w/w was obtained (Std. De\,. :( per cent). This corresponds to 92.0 per cent v/v. From the above results it is seen that the two methods show a considerable discrepancy, the reason for which is not known. There is little doubt that the second method is preferable as a technique, requiring far less manipulation than the heavywater method. r\ further and more serious objection to the use of heavy water is the need for extrapolating the graph, since a very small error in the position of the is line would cause a large error in the reading at zero time. At the present stage, there is little point in speculating whether the different results yielded by the two methods have any biological significance. Shortage of time precluded any further investigation of this; in the subsequent work on potassium distribution, a figure of 90 per cent \V/W was taken as sufficiently accurate for the purpose. weight
I am grateful to Dr. J. X. Kitching, F.R.S., for advice and criticism of this work, and to the Department of Scientific and Industrial Research for the provision of a maintenance grant. REFERENCES 1. HOLTER,
H. and ZEUTHEN, E., Corn@. Rend. True. Cnrlsberg Ser. Chim. 26, 277 S., J. Esptl. Zool. 145, 139 (1960). S. and PIGON, A.? Compt. Rend. Tmo. Carlsberg Ser. Chim. 28, 1 (1951). 4. LUMS~EN,~. E.and ROBINSON,C.V., Ibid.28,358 (1953). 5. PRESCOTT, D. RI. and ZEUTHEN, E., A& Physiol. Stand. 28, 77 (1953). 6. RIDDLE, J., Espfl. Cell Research 26, 158 (1962).
(1918).
2. LOVTRUP, 3. LBVTRUP,
DISTRIBUTION
OF PROTEOLYTIC
FRACTIONS
FROM
NON-FERTILIZED
EGGS OF THE SEA URCHIN G. LUNDBLAD National
Bacteriological Laboratory. Stockholm, Sweden, and The for Experimental Biology, University of Stockholm,
BEsums cathepsin egg with
II three different the pH-optima
1 This investigation was supported Council and from the Swedish Canrer at Stazione Zoologica, Naples. Experimenlnl
AND
PARACENTROTUS
Cell Research
27
IN PROTEIN FERTILIZED LIVIDUS’
and .J. RUNNSTRijM
Received
sea urchin
ENZYMES
March
Wenner-Gren Sweden
Institute
20, 1962
proteolytic enzymes were demonstrated in the (cf. [3, 41). The) 6.7, -7.0 and 7.X respectively
bv grants from the Swedish Soc‘ietg. The experimental part
Natural Sciences Research of the work was carried out