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The metabolism of pentose phosphate in sea urchin sperm and eggs
734
F. Ghiretti and
V. D’Amelio
archenteron rudiment elongates to about I/& to 1/8 its final length. This phase of invagination is probably autonomous, i.e. essentially dependent on factors residing in the vegetal region itself. Invagination can thus be attained in an isolated vegetal fragment of a blastula [3]. After this phase of invagination the archenteron tip will more or less remain on the same level for some time, or slowly advance in the animal direction. During the second phase of invagination the process occurs at a considerably higher rate than during the first phase and is here caused by pseudopodia emitted by the archenteron tip and attaching themselves to the ectoderm. The archenteron tip is pulled in the animal direction by contractions of these pseudopodia. The role of the pseudopodia is evident from the fact that their appearance closely corresponds to the increased rate of invagination, and also that a break of the pseudopodia can be correlated with a retardation of invagination. Furthermore, a bend of the archenteron is preceded by an eccentric formation of the pseudopodia in the same direction. Sooner or later the pseudopodia forming cells themselves are extracted from the archenteron tip owing to the contractions of the pseudopodia, which may be explained by the fact that the intestine cannot respond to the contractions at a sufficient rate. The cells liberated are now called secondary mesenchyme cells. The connections between these cells and the archenteron tip may be completely or partially interrupted during extraction. In such cases the invagination ceases until new pseudopodia are formed. A detailed description with further data on the technical device for this study and on results obtained will be presented in a later publication [2]. After our paper [2] had been submitted for publication in this journal, an important paper on the mechanism of invagination [l] appeared. The data presented therein on the contractile elements connecting the archenteron tip with the ectoderm are also in good agreement with our own observations. REFERENCES 1. DAN, K. and OKAZAKI, K., Biol. Bull. 110, 29 (1956). 2. GUSTAFSON, T. and KINNANDER, H., Exptl. Cell Research (In press). 3. MOORE, A. R. and BURT, A. S., J. Exptl. Zoot. 82, 159 (1939).
THE
METABOLISM
OF PENTOSE PHOSPHATE SPERM AND EGGS F. GHIRETTI
Department
of Physiology,
IN SEA URCHIN
and V. D’AMELIOI &a&one Zoologica, Nales, Italy.
Received February
11, 19.56
T HE
mechanism of stepwise oxidation of glucose through the glucose-6-phosphate dehydrogenase shunt has recently been elucidated by the investigations of Racker
1 Fellow of the Centro di Biologia de1 Consiglio Istituto di Anatomia Comparata, Palermo. Experimental
Cell Research 10
Nazionale
delle Ricerche.
Present
address:
Penfose phosphafe in sea urchin sperm
735
and of Horecker. Several steps of this pathway have been found in a great variety of plants, microorganisms and higher vertebrates (see for ref. [1, 2, 4, 111). The existence of an operating phosphorylative oxidation pathway of carbohydrates in sea urchin eggs was first suggested by Runnstrom [13, 141 and later indicated by the work of orstrom and Lindberg [lo] and Lindberg and Ernster [S]. A hexokinase which phosphorylates glucose and to a less extent mannose and galactose was found
Fig. 1 (left). Breakdown of ribose-5-phosphate by cell-free extracts of Arbacia sperm (1) and eggs (2) as determined by the orcinol reaction. Abscissa: Min. of incubation. Ordinate: y of pentose. Fig. 2 (righf). Formation of ketopentoses and ketohexoses by cell-free extracts of Arbacia sperm as indicated by the cystein-carhazole reaction. (1) Zero time; (2) after 15 min.; (3) after 30 min.; (4) after 60 min.; (5) after 90 min. Abscissa: Wavelength. Ordinate: Optical density.
in unfertilized and fertilized eggs by Krahl ef al. [6]. The same authors have recently studied the dehydrogenation of glucose-6-phosphate and of phosphogluconate by the corresponding TPN depending enzymes in Arbacia eggs; they suggest that the glucose&phosphate dehydrogenase system may be the major pathway for utilization of carbohydrates in sea urchin eggs [7]. There is no information available concerning the existence of some steps of the glucoseS-phosphate dehydrogenase shunt in sea urchin sperm. In this note evidence is given for the presence in sea urchin eggs and sperm of an active enzymatic system utilizing pentosed-phosphate. Fresh sperm and eggs of Arbacia lixula and of Paracen!rotus lividus were used. The jelly layer of the eggs was removed by treating one volume of egg suspension with one volume of 0.005N HCl in sea water. The eggs were homogenized with O.OlM phosphate buffer pH 7.2 in a tissue grinder and, after standing for two hours in the cold, the thick paste was centrifuged at 12,000 rpm. Sperm extracts were prepared by crushing the frozen cells in the press described by Hughes [5]. The paste was diluted with 5 vol. of O.OlM phosphate buffer pH 7.2 and extracted in the cold for two hours. The viscous suspension was centrifuged in the cold at 12,000 rpm. The Ba salt of ribose-5-phosphate was converted into the K salt before use. Total nitrogen in the cell-free extracts was determined in a micro-Kjeldahl apparatus and Experimental
Cell Research 10
736
F. Ghiretti and TABLE
Synthesis
of fructose
from ribose-5-phosphate
Time min. 0 15 30 60 90
V. D’Amelio I by cell-free
PM Ribose-5-phokphate disappeared 0 0.22 0.33 0.42 0.50
extracts
of Paracentrotus
sperm.
,uM Fructose formed 0 0.06 0.13 0.18 0.16
the protein concentration was calculated by using a factor of 6.2. Fructose was determined calorimetrically by the method of Roe [12]. Ketohexoses, ketopentoses and trioses were estimated by the method of Dische and Borenfreund [3]. The presence of ketohexoses and of ketopentoses is indicated by the development of a purple color after a few minutes. Ketohexoses have a maximum absorption at 560 rnp and ketopentoses at 540 rnp. If trioses are present, a blue color with a maximum absorption at 660 rnp develops at the end of 24 hrs. Ribose was determined by the modified orcinol reaction of Mejbaum [9] using 0.033 per cent of FeCl, and a 30 min. period of heating. The same reaction was used for the detection of sedoheptulose, which has a maximum absorption at 580 m,u, and for ribulose-6phosphate which has a small peak at 540 rnp together with a large one at 660 rnp, the latter being that of aldo-pentoses. All spectrophotometric measurements were made with a Beckman Model DU spectrophotometer. Cell-free extracts of sperm and eggs were incubated at 35°C with IO PM of ribose5-phosphate in O.OlM phosphate buffer pH 7.2 in presence of 0.05M MgCI,. At intervals samples of the mixture were taken, deproteinized with TCA 10 per cent and analysed for pentoses and hexoses. Addition of ribose-5-phosphate to cell-free extracts of Arbacia and Puracentrotus eggs and sperm caused a rapid disappearance of this pentose as shown by the orcinol reaction measured at 660 rnp (Fig. 1). At the same time there was a definite increase in light absorption at 580 rnp, the absorption peak of heptulose, which is an indication of the presence of transketolase. The appearance of ribulose-&phosphate formed by phosphopentose isomerase was demonstrated by the peak at 540 rnp in the cystein carbazole reaction. The ketopentose appeared after 15 min. and increased in concentration up to 60 min. to be replaced later by fructose as indicated by the shifting of the peak to 560 rnp (Fig. 2). The quantitative formation of fructose as determined with Roe’s reaction is reported in Table I. REFERENCES 1. COHEN, S. $:, in Chemical Pathways of Metabolism. Vol. 1, p. 173. D. M. Greenberg, Press Inc. Pub]., New York, 1954. 2. DICKENS, F., Rapports 3’ Congres Intern. Biochim. 1955. 3. DISCHE, Z. and BORENFREUND, E., J. BioZ. Chem. 192, 583 (1951). Experimental
Cell Research 10
Academic
Pentose phosphate in sea urchin sperm
737
4. GUNSALUS, I. C., HORECKER, B. L., and WOOD, W. A., Bact. Rev. 19, 79 (1955). 5. HUGHES, D. E., Brit. J. Bxpll. Pathol. 32, 197 (1951). 6. KRAHL, M. E., KELTCH, A. K., WALTERS, C. P., and CLOWES,G. H. A., Biol. Bull. 105, 377
(1953). J. Gen. Physiol. 38, 431 (1955). LINDBERO, 0. and ERNSTER, L., Biochim. et Biophys. Acta 2, 471 (1948). MEJBAUM, W., Z. Physiol. Chem. 258, 117 (1939). ORSTRBM,A. and LINDBERG, O., Enzymologia 8, 67 (1940). RACKER, E., Advances in Enzymol. 15, 141 (1954). ROE, J. H., J. Biol. Chem. 107, 15 (1934). RUNNSTR~M, J., Biochem. Z. 258, 257 (1933). Advances in Enzymol. 9, 241 (1949).
7. 8. 9.
10. 11. 12. 13. 14.
THE RELATION
OF DNA CONTENT TO CELL SIZE IN ASPERGIUUS G. ISHITANI,
K. UCHIDA,
andY.
Tke Institute of Applied Microbiology, University
IKEDA
of Tokyo, Tokyo, Japan
Received March 10, 1956
As
is usually
the
case
in other
organisms,
the
DNA
content
of diploid nuclei of in Aspergillus and the DNA content per conidium are the same in haploids and diploids [4]. The number of nuclei in the diploid conidium is, however, half that in the haploid and thus the constancy of the DNA-cytoplasmic ratio is maintained (Table I). The strains of Asp. nidulans, Asp. niger and Penicillium chrysogenum studied by Pontecorvo and his coworkers [6, 7, 81 have uninucleate conidia and, consequently, the DNA-cytoplasmic ratio in diploids cannot be maintained, as it is in Asp. sojae, by a reduction in nuclear number. Rather, it is maintained by an increase in the amount of cytoplasm and hence in the size of the spores which have about double
Aspergillus nidulans is about twice that of haploid nuclei [3]. Similarly, sojae the same relation holds. In this organism the size of the conidia
TABLE I Interrelations
between DNA, cell volume and nuclear number in the conidia of haploid and diploid Asp. sojae.
(P3)
DNA per conidium P8 ( x 10-9
Number of nuclei per conidium
DNA per nucleus P8 ( x 10-Y
135 128
3.71 3.73
4.22 2.21
0.88 1.69
Conidial Average
Haploidr Diploid*
volume
1 The haploid strains studied were Wild type and Yellow leucineless mutant (y leu). e The diploid strains were Green prototroph (y leu/al his) and Yellow prototrophic segregant (from y leu/al his). Experimental Cell Research 10
F. Ghiretti and
V. D’Amelio
archenteron rudiment elongates to about I/& to 1/8 its final length. This phase of invagination is probably autonomous, i.e. essentially dependent on factors residing in the vegetal region itself. Invagination can thus be attained in an isolated vegetal fragment of a blastula [3]. After this phase of invagination the archenteron tip will more or less remain on the same level for some time, or slowly advance in the animal direction. During the second phase of invagination the process occurs at a considerably higher rate than during the first phase and is here caused by pseudopodia emitted by the archenteron tip and attaching themselves to the ectoderm. The archenteron tip is pulled in the animal direction by contractions of these pseudopodia. The role of the pseudopodia is evident from the fact that their appearance closely corresponds to the increased rate of invagination, and also that a break of the pseudopodia can be correlated with a retardation of invagination. Furthermore, a bend of the archenteron is preceded by an eccentric formation of the pseudopodia in the same direction. Sooner or later the pseudopodia forming cells themselves are extracted from the archenteron tip owing to the contractions of the pseudopodia, which may be explained by the fact that the intestine cannot respond to the contractions at a sufficient rate. The cells liberated are now called secondary mesenchyme cells. The connections between these cells and the archenteron tip may be completely or partially interrupted during extraction. In such cases the invagination ceases until new pseudopodia are formed. A detailed description with further data on the technical device for this study and on results obtained will be presented in a later publication [2]. After our paper [2] had been submitted for publication in this journal, an important paper on the mechanism of invagination [l] appeared. The data presented therein on the contractile elements connecting the archenteron tip with the ectoderm are also in good agreement with our own observations. REFERENCES 1. DAN, K. and OKAZAKI, K., Biol. Bull. 110, 29 (1956). 2. GUSTAFSON, T. and KINNANDER, H., Exptl. Cell Research (In press). 3. MOORE, A. R. and BURT, A. S., J. Exptl. Zoot. 82, 159 (1939).
THE
METABOLISM
OF PENTOSE PHOSPHATE SPERM AND EGGS F. GHIRETTI
Department
of Physiology,
IN SEA URCHIN
and V. D’AMELIOI &a&one Zoologica, Nales, Italy.
Received February
11, 19.56
T HE
mechanism of stepwise oxidation of glucose through the glucose-6-phosphate dehydrogenase shunt has recently been elucidated by the investigations of Racker
1 Fellow of the Centro di Biologia de1 Consiglio Istituto di Anatomia Comparata, Palermo. Experimental
Cell Research 10
Nazionale
delle Ricerche.
Present
address:
Penfose phosphafe in sea urchin sperm
735
and of Horecker. Several steps of this pathway have been found in a great variety of plants, microorganisms and higher vertebrates (see for ref. [1, 2, 4, 111). The existence of an operating phosphorylative oxidation pathway of carbohydrates in sea urchin eggs was first suggested by Runnstrom [13, 141 and later indicated by the work of orstrom and Lindberg [lo] and Lindberg and Ernster [S]. A hexokinase which phosphorylates glucose and to a less extent mannose and galactose was found
Fig. 1 (left). Breakdown of ribose-5-phosphate by cell-free extracts of Arbacia sperm (1) and eggs (2) as determined by the orcinol reaction. Abscissa: Min. of incubation. Ordinate: y of pentose. Fig. 2 (righf). Formation of ketopentoses and ketohexoses by cell-free extracts of Arbacia sperm as indicated by the cystein-carhazole reaction. (1) Zero time; (2) after 15 min.; (3) after 30 min.; (4) after 60 min.; (5) after 90 min. Abscissa: Wavelength. Ordinate: Optical density.
in unfertilized and fertilized eggs by Krahl ef al. [6]. The same authors have recently studied the dehydrogenation of glucose-6-phosphate and of phosphogluconate by the corresponding TPN depending enzymes in Arbacia eggs; they suggest that the glucose&phosphate dehydrogenase system may be the major pathway for utilization of carbohydrates in sea urchin eggs [7]. There is no information available concerning the existence of some steps of the glucoseS-phosphate dehydrogenase shunt in sea urchin sperm. In this note evidence is given for the presence in sea urchin eggs and sperm of an active enzymatic system utilizing pentosed-phosphate. Fresh sperm and eggs of Arbacia lixula and of Paracen!rotus lividus were used. The jelly layer of the eggs was removed by treating one volume of egg suspension with one volume of 0.005N HCl in sea water. The eggs were homogenized with O.OlM phosphate buffer pH 7.2 in a tissue grinder and, after standing for two hours in the cold, the thick paste was centrifuged at 12,000 rpm. Sperm extracts were prepared by crushing the frozen cells in the press described by Hughes [5]. The paste was diluted with 5 vol. of O.OlM phosphate buffer pH 7.2 and extracted in the cold for two hours. The viscous suspension was centrifuged in the cold at 12,000 rpm. The Ba salt of ribose-5-phosphate was converted into the K salt before use. Total nitrogen in the cell-free extracts was determined in a micro-Kjeldahl apparatus and Experimental
Cell Research 10
736
F. Ghiretti and TABLE
Synthesis
of fructose
from ribose-5-phosphate
Time min. 0 15 30 60 90
V. D’Amelio I by cell-free
PM Ribose-5-phokphate disappeared 0 0.22 0.33 0.42 0.50
extracts
of Paracentrotus
sperm.
,uM Fructose formed 0 0.06 0.13 0.18 0.16
the protein concentration was calculated by using a factor of 6.2. Fructose was determined calorimetrically by the method of Roe [12]. Ketohexoses, ketopentoses and trioses were estimated by the method of Dische and Borenfreund [3]. The presence of ketohexoses and of ketopentoses is indicated by the development of a purple color after a few minutes. Ketohexoses have a maximum absorption at 560 rnp and ketopentoses at 540 rnp. If trioses are present, a blue color with a maximum absorption at 660 rnp develops at the end of 24 hrs. Ribose was determined by the modified orcinol reaction of Mejbaum [9] using 0.033 per cent of FeCl, and a 30 min. period of heating. The same reaction was used for the detection of sedoheptulose, which has a maximum absorption at 580 m,u, and for ribulose-6phosphate which has a small peak at 540 rnp together with a large one at 660 rnp, the latter being that of aldo-pentoses. All spectrophotometric measurements were made with a Beckman Model DU spectrophotometer. Cell-free extracts of sperm and eggs were incubated at 35°C with IO PM of ribose5-phosphate in O.OlM phosphate buffer pH 7.2 in presence of 0.05M MgCI,. At intervals samples of the mixture were taken, deproteinized with TCA 10 per cent and analysed for pentoses and hexoses. Addition of ribose-5-phosphate to cell-free extracts of Arbacia and Puracentrotus eggs and sperm caused a rapid disappearance of this pentose as shown by the orcinol reaction measured at 660 rnp (Fig. 1). At the same time there was a definite increase in light absorption at 580 rnp, the absorption peak of heptulose, which is an indication of the presence of transketolase. The appearance of ribulose-&phosphate formed by phosphopentose isomerase was demonstrated by the peak at 540 rnp in the cystein carbazole reaction. The ketopentose appeared after 15 min. and increased in concentration up to 60 min. to be replaced later by fructose as indicated by the shifting of the peak to 560 rnp (Fig. 2). The quantitative formation of fructose as determined with Roe’s reaction is reported in Table I. REFERENCES 1. COHEN, S. $:, in Chemical Pathways of Metabolism. Vol. 1, p. 173. D. M. Greenberg, Press Inc. Pub]., New York, 1954. 2. DICKENS, F., Rapports 3’ Congres Intern. Biochim. 1955. 3. DISCHE, Z. and BORENFREUND, E., J. BioZ. Chem. 192, 583 (1951). Experimental
Cell Research 10
Academic
Pentose phosphate in sea urchin sperm
737
4. GUNSALUS, I. C., HORECKER, B. L., and WOOD, W. A., Bact. Rev. 19, 79 (1955). 5. HUGHES, D. E., Brit. J. Bxpll. Pathol. 32, 197 (1951). 6. KRAHL, M. E., KELTCH, A. K., WALTERS, C. P., and CLOWES,G. H. A., Biol. Bull. 105, 377
(1953). J. Gen. Physiol. 38, 431 (1955). LINDBERO, 0. and ERNSTER, L., Biochim. et Biophys. Acta 2, 471 (1948). MEJBAUM, W., Z. Physiol. Chem. 258, 117 (1939). ORSTRBM,A. and LINDBERG, O., Enzymologia 8, 67 (1940). RACKER, E., Advances in Enzymol. 15, 141 (1954). ROE, J. H., J. Biol. Chem. 107, 15 (1934). RUNNSTR~M, J., Biochem. Z. 258, 257 (1933). Advances in Enzymol. 9, 241 (1949).
7. 8. 9.
10. 11. 12. 13. 14.
THE RELATION
OF DNA CONTENT TO CELL SIZE IN ASPERGIUUS G. ISHITANI,
K. UCHIDA,
andY.
Tke Institute of Applied Microbiology, University
IKEDA
of Tokyo, Tokyo, Japan
Received March 10, 1956
As
is usually
the
case
in other
organisms,
the
DNA
content
of diploid nuclei of in Aspergillus and the DNA content per conidium are the same in haploids and diploids [4]. The number of nuclei in the diploid conidium is, however, half that in the haploid and thus the constancy of the DNA-cytoplasmic ratio is maintained (Table I). The strains of Asp. nidulans, Asp. niger and Penicillium chrysogenum studied by Pontecorvo and his coworkers [6, 7, 81 have uninucleate conidia and, consequently, the DNA-cytoplasmic ratio in diploids cannot be maintained, as it is in Asp. sojae, by a reduction in nuclear number. Rather, it is maintained by an increase in the amount of cytoplasm and hence in the size of the spores which have about double
Aspergillus nidulans is about twice that of haploid nuclei [3]. Similarly, sojae the same relation holds. In this organism the size of the conidia
TABLE I Interrelations
between DNA, cell volume and nuclear number in the conidia of haploid and diploid Asp. sojae.
(P3)
DNA per conidium P8 ( x 10-9
Number of nuclei per conidium
DNA per nucleus P8 ( x 10-Y
135 128
3.71 3.73
4.22 2.21
0.88 1.69
Conidial Average
Haploidr Diploid*
volume
1 The haploid strains studied were Wild type and Yellow leucineless mutant (y leu). e The diploid strains were Green prototroph (y leu/al his) and Yellow prototrophic segregant (from y leu/al his). Experimental Cell Research 10